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Neuroprotective Effects of Inhibiting Poly(ADP-Ribose) Synthetase on Focal Cerebral Ischemia in Rats
Abstract
Poly(adenosine 5'-diphosphoribose) synthetase (PARS) has been described as an important candidate for mediation of neurotoxicity by nitric oxide. In the current study, we demonstrate for the first time that in vivo administration of a potent PARS inhibitor, 3,4-dihydro 5-[4-1(1-piperidinyl) butoxy]-1(2H)-isoquinolinone, leads to a significant reduction of infarct volume in a focal cerebral ischemia model in the rat. Focal cerebral ischemia was produced by cauterization of the right distal middle cerebral artery (MCA) with bilateral temporary common carotid artery occlusion for 90 minutes. 3,4-Dihydro 5[4-(1-piperidinyl) butoxy]-1(2H)-isoquinolinone was dissolved in dimethyl sulfoxide and injected intraperitoneally. Animals were treated 2 hours before MCA occlusion (control, n = 14; 5 mg/kg, n = 7; 10 mg/kg, n = 7; 20 mg/kg, n = 7; 40 mg/kg, n = 7), and 2 hours after MCA occlusion (same doses as before treatment). Twenty-four hours after MCA occlusion, the total infarct volume was measured using 2,3,5-triphenyltetrazolium chloride. Inhibition of PARS leads to a significant decrease in the damaged volume in the 5 mg/kg-treated group (106.7 +/- 23.2 mm3; mean +/- SD, P < 0.002), the 10 mg/kg-treated group (76.4 +/- 16.8 mm3, P < 0.001), and the 20 mg/kg-treated group (110.2 +/- 42.0 mm3, P < 0.02) compared with the control group (165.2 +/- 34.0 mm3). The substantial reduction in infarct volume indicates that the activation of PARS may play an important role in the pathogenesis of brain damage in cerebral ischemia through intracellular energy depletion.
... A few studies using LDF (Alkayed et al., 1998;Takahashi et al., 1997) or conventional DCT (Culver et al., 2003) have monitored CBF reductions in rats with acute cerebral artery occlusions. Previously using LDF, Alkayed et al. and Takahashi et al. found that CBF levels were reduced during acute MCAO from 100% baseline to 18.9 AE 1.4% in 22 rats and 20.8 AE 7.7% in 5 rats, respectively (Alkayed et al., 1998;Takahashi et al., 1997). ...
... A few studies using LDF (Alkayed et al., 1998;Takahashi et al., 1997) or conventional DCT (Culver et al., 2003) have monitored CBF reductions in rats with acute cerebral artery occlusions. Previously using LDF, Alkayed et al. and Takahashi et al. found that CBF levels were reduced during acute MCAO from 100% baseline to 18.9 AE 1.4% in 22 rats and 20.8 AE 7.7% in 5 rats, respectively (Alkayed et al., 1998;Takahashi et al., 1997). Using a convetional DCT system, Culver et al. found that CBF levels were reduced from 100% baseline to 42 AE 4% in 5 rats during acute MCAO (Culver et al., 2003). ...
... In the rat model of permanent distal MCAO plus temporary bilateral carotid occlusion, the PARP inhibitor DPQ reduced infarct volume by 54% when administered as a pre-treatment (96) and by 40% when administered at 30 min after MCAO (97). Likewise, MP-124 administered at 5 min of MCAO reduced infarct volume by 42% in the same model (88). ...
Parthanatos is a cell death signaling pathway in which excessive oxidative damage to DNA leads to over-activation of poly(ADP-ribose) polymerase (PARP). PARP then generates the formation of large poly(ADP-ribose) polymers that induce the release of apoptosis-inducing factor from the outer mitochondrial membrane. In the cytosol, apoptosis-inducing factor forms a complex with macrophage migration inhibitory factor that translocates into the nucleus where it degrades DNA and produces cell death. In a review of the literature, we identified 24 publications from 13 laboratories that support a role for parthanatos in young male mice and rats subjected to transient and permanent middle cerebral artery occlusion (MCAO). Investigators base their conclusions on the use of nine different PARP inhibitors (19 studies) or PARP1-null mice (7 studies). Several studies indicate a therapeutic window of 4–6 h after MCAO. In young female rats, two studies using two different PARP inhibitors from two labs support a role for parthanatos, whereas two studies from one lab do not support a role in young female PARP1-null mice. In addition to parthanatos, a body of literature indicates that PARP inhibitors can reduce neuroinflammation by interfering with NF-κB transcription, suppressing matrix metaloproteinase-9 release, and limiting blood-brain barrier damage and hemorrhagic transformation. Overall, most of the literature strongly supports the scientific premise that a PARP inhibitor is neuroprotective, even when most did not report behavior outcomes or address the issue of randomization and treatment concealment. Several third-generation PARP inhibitors entered clinical oncology trials without major adverse effects and could be repurposed for stroke. Evaluation in aged animals or animals with comorbidities will be important before moving into clinical stroke trials.
... Gene disruption or silencing of PARP significantly reduced infarct size, attenuated neurotoxicity, protected the neurovascular unit, and improved the neurological outcomes in experimental stroke animal models [85][86][87]. Pharmacological inhibition of PARP yielded similar neuroprotective effects to the genetic interventions [86,[88][89][90][91]. Furthermore, PARP inhibitors revealed to reduce hemorrhagic transformation in ischemic stroke with t-PA treatment [92][93][94][95]. ...
Oxidative/nitrosative stress and neuroinflammation are critical pathological processes in cerebral ischemia-reperfusion injury, and their intimate interactions mediate neuronal damage, blood-brain barrier (BBB) damage and hemorrhagic transformation (HT) during ischemic stroke. We review current progress towards understanding the interactions of oxidative/nitrosative stress and inflammatory responses in ischemic brain injury. The interactions between reactive oxygen species (ROS)/reactive nitrogen species (RNS) and innate immune receptors such as TLR2/4, NOD-like receptor, RAGE, and scavenger receptors are crucial pathological mechanisms that amplify brain damage during cerebral ischemic brain injury. Furthermore, we review the current progress of omics and systematic biology approaches for studying complex network regulations related to oxidative/nitrosative stress and inflammation in the pathology of ischemic stroke. Targeting oxidative/nitrosative stress and neuroinflammation could be a promising therapeutic strategy for ischemic stroke treatment.
We then review recent advances in discovering compounds from medicinal herbs with the bioactivities of simultaneously regulating oxidative/nitrosative stress and pro-inflammatory molecules for minimizing ischemic brain injury. These compounds include sesamin, baicalin, salvianolic acid A, 6-paradol, silymarin, apocynin, 3H-1,2-Dithiole-3-thione, (−)-epicatechin, rutin, Dl-3-N-butylphthalide, and naringin. We finally summarize recent developments of the omics and systematic biology approaches for exploring the molecular mechanisms and active compounds of Traditional Chinese Medicine (TCM) formulae with the properties of antioxidant and anti-inflammation for neuroprotection. The comprehensive omics and systematic biology approaches provide powerful tools for exploring therapeutic principles of TCM formulae and developing precision medicine for stroke treatment.
... PARPs catalyze ADP ribose unit from NAD+ to target protein which include histones and transcription factors. Some studies have detected the applications of PARP inhibitors (e.g., olaparib and veliparib) after stroke, which found that they inhibit the activation of microglia and improve the survival rate of neurons (143,144). Studies have shown that the application of histone deacetylase (HDAC) inhibitors-including trichostatin A and sodium butyrate-suppressed microglia activation and suppresses inflammatory markers in the ischemic brain, which also supports the beneficial effect of targeting inflammatory response (145). Mammalian target of rapamycin (mTOR) is a known immune response regulation factor. ...
After ischemic stroke, the integrity of the blood-brain barrier is compromised. Peripheral immune cells, including neutrophils, T cells, B cells, dendritic cells, and macrophages, infiltrate into the ischemic brain tissue and play an important role in regulating the progression of ischemic brain injury. In this review, we will discuss the role of different immune cells after stroke in the secondary inflammatory reaction and focus on the phenotypes and functions of macrophages in ischemic stroke, as well as briefly introduce the anti-ischemic stroke therapy targeting macrophages.
... (7) Potential anti-apoptotic characteristics of agmatine reduce cell death . (8) Agmatine interferes with intracellular-signaling pathways by inhibiting ADP ribosylation of proteins, a process implicated in neuronal injury following cerebral ischemia in rats (Takahashi et al., 1997;Moss et al., 1983;Laing et al., 2011). (9) Agmatine is a regulator of the polyamine pathway via formation of putrescine, a precursor for biosynthesis of polyamines, which are essentially involved in the response to cellular injury and in neuroprotection (Oble et al., 2004). ...
Agmatine, an endogenous polyamine in CNS, is derived from arginine by dearboxylation. Like polyamines, agmatine has been studied for its neuroprotetive effects. At present, a large body of experimental evidences has been gathered that demonstrate the neuroprotective effects of agmatine. The neuroprotective effects have been observed in various CNS cell lines and animal models against the excitotocity, oxidative damage, corticosteroidid induced neurotoxicity, ischemic/hypoxic or oxygen-glucose deprivation toxicity, spinal cord injury and traumatic brain injury. The studies have been extended to rescue of retinal ganglion cells from toxicities. The mechanistic studies suggest that neuroprotection offered by agmatine can be assigned to its multimolecular biological effects. These include its action as glutamatergic receptor antagonist, α2-adrenoceptor agonist, imidazoline binding site ligand, NOS inhibitor, ADP ribosylation inhibitor, and blocker of ATP-sensitive potassium and voltage-gated calcium channels, anti-apoptotic and antioxidant. Its action as regulator for polyamine synthesis, insulin release assists the neuroprotection.
The cumulative evidences of preclinical studies support the possible use of agmatine as an agent for neuronal damage and neurodegenerative diseases. However, it will be hasty to assert and promote agmatine as a novel therapeutic agent for neuroprotection. The review is focused on the role of agmatine in different types and mechanisms of neural injuries. The aspects of concern like dose range, pharmacokinetics of exogenous agmatine, levels of endogenous agmatine during events of injury etc. has to be addressed.
... In support of PARP-1 mediated regulation of the central nervous system in disease it has been shown that PARP-1 overactivation leads to neuronal degeneration in Drosophila [43]. PARP-1 activation has also been linked to Alzheimer's (AD), Parkinson's (PD) and ischemic stroke [19,31,51,71,72,79], and the use of PARP-1/2 inhibitors is beneficial to mouse models of these diseases [2,20,25,30,87,105,109,115]. These data indicate that, despite dampening the DNA damage response, PARP-1/2 inhibition provides improved neuronal integrity and function in these animal models of disease. ...
Amyotrophic lateral sclerosis (ALS) is a devastating and fatal motor neuron disease. Diagnosis typically occurs in the fifth decade of life and the disease progresses rapidly leading to death within ~ 2-5 years of symptomatic onset. There is no cure, and the few available treatments offer only a modest extension in patient survival. A protein central to ALS is the nuclear RNA/DNA-binding protein, TDP-43. In > 95% of ALS patients, TDP-43 is cleared from the nucleus and forms phosphorylated protein aggregates in the cytoplasm of affected neurons and glia. We recently defined that poly(ADP-ribose) (PAR) activity regulates TDP-43-associated toxicity. PAR is a posttranslational modification that is attached to target proteins by PAR polymerases (PARPs). PARP-1 and PARP-2 are the major enzymes that are active in the nucleus. Here, we uncovered that the motor neurons of the ALS spinal cord were associated with elevated nuclear PAR, suggesting elevated PARP activity. Veliparib, a small-molecule inhibitor of nuclear PARP-1/2, mitigated the formation of cytoplasmic TDP-43 aggregates in mammalian cells. In primary spinal-cord cultures from rat, Veliparib also inhibited TDP-43-associated neuronal death. These studies uncover that PAR activity is misregulated in the ALS spinal cord, and a small-molecular inhibitor of PARP-1/2 activity may have therapeutic potential in the treatment of ALS and related disorders associated with abnormal TDP-43 homeostasis.
... and transcriptional factors. Several studies have examined the effects of PARP inhibitors after stroke and have found that they suppress microglial activation and improve neuronal survival (Hamby et al., 2007;Kauppinen et al., 2009;Takahashi et al., 1997). ...
Ischemic stroke is a devastating and debilitating medical condition with limited therapeutic options. However, accumulating evidence indicates a central role of inflammation in all aspects of stroke including its initiation, the progression of injury, and recovery or wound healing. A central target of inflammation is disruption of the blood brain barrier or neurovascular unit. Here we discuss recent developments in identifying potential molecular targets and immunomodulatory approaches to preserve or protect barrier function and limit infarct damage and functional impairment. These include blocking harmful inflammatory signaling in endothelial cells, microglia/macrophages, or Th17/γδ T cells with biologics, third generation epoxyeicosatrienoic acid (EET) analogs with extended half-life, and miRNA antagomirs. Complementary beneficial pathways may be enhanced by miRNA mimetics or hyperbaric oxygenation. These immunomodulatory approaches could be used to greatly expand the therapeutic window for thrombolytic treatment with tissue plasminogen activator (t-PA). Moreover, nanoparticle technology allows for the selective targeting of endothelial cells for delivery of DNA/RNA oligonucleotides and neuroprotective drugs. In addition, although likely detrimental to the progression of ischemic stroke by inducing inflammation, oxidative stress, and neuronal cell death, 20-HETE may also reduce susceptibility of onset of ischemic stroke by maintaining autoregulation of cerebral blood flow. Although the interaction between inflammation and stroke is multifaceted, a better understanding of the mechanisms behind the pro-inflammatory state at all stages will hopefully help in developing novel immunomodulatory approaches to improve mortality and functional outcome of those inflicted with ischemic stroke.
... Understanding how the performance of the BER system contributes to AD development would allow us to discover molecular markers of this disease. It has already been shown that the BER enzyme PARP1 is an effective marker of biochemical changes in the brain associated with AD [16,17] . Overactivation of PARP1 is associated with higher energy usage, which is manifested by an increased consumption of NAD+, which in turn may lead to apoptotic cell death. ...
Many clinical studies have shown that oxidative stress pathways and the efficiency of the oxidative DNA damage base excision repair (BER) system are associated with the pathogenesis of Alzheimer's disease (AD). Reduced BER efficiency may result from polymorphisms of BER-related genes. In the present study, we examine whether single nucleotide polymorphisms (SNPs) of BER genes are associated with increased risk of AD.
SNP genotyping was carried out on DNA isolated from peripheral blood mononuclear cells obtained from 120 patients with AD and 110 healthy volunteers. Samples were genotyped for the presence of BER-related SNPs, i.e. XRCC1-rs1799782, rs25487; MUTYH-rs3219489, and PARP1-rs1136410.
We found a positive association between AD risk and the presence of G/A genotype variant of the XRCC1 rs25487 polymorphism [odds ratio (OR) = 3.762, 95% CI: 1.793-7.891]. The presence of the A/A genotype of this polymorphism reduced the risk of AD (OR = 0.485, 95% CI: 0.271-0.870). In cases of the PARP1 gene rs1136410 polymorphism, we observed that the T/C variant increases (OR = 4.159, 95% CI: 1.978-8.745) while the T/T variant reduces risk (OR = 0.240, 95% CI: 0.114-0.556) of AD.
We conclude that BER gene polymorphisms may play an important role in the etiology of AD. Diagnosing the presence or absence of particular genetic variants may be an important marker of AD. Further research on a larger population is needed. There is also a need to examine polymorphisms of other BER in the context of AD risk. © 2015 S. Karger AG, Basel.
... While both enzymes have previously characterized roles in DNA repair, cell death and global genomic stability, studies evaluating the effects of combination therapy that utilizes the inhibition of both PARP-1 and PARG in chemotherapy are lacking. Here, we utilized the PARP-1-specific inhibitors 3,4-dihydro-5[4-(1-piperindinyl) butoxy]-1(2H)-isoquinoline (DPQ) (36), N-(6-oxo-5,6dihydrophenanthridin-2-yl)-(N,N-dimethylamino)acetamide (PJ34) (37) and 2-((R)-2-methylpyrrolidin-2-yl)-1H-benzimidazole-4-carboxamide (ABT-888; Veliparib) (22), and the molecular genetic deletion or RNAi knockdown of PARG to determine the effects of combination PARP-1 and PARG inhibition. Our results demonstrate that the knockdown/inhibition of each PAR metabolic enzyme alone enhances chemotherapeutic effectiveness, but combination therapy does not lead to synergistic levels of cell death in HeLa cells. ...
The genome-protecting role of poly(ADP-ribose) (PAR) has identified PAR polymerase-1 (PARP-1) and PAR glycohydrolase (PARG), two enzymes responsible for the synthesis and hydrolysis of PAR, as chemotherapeutic targets. Each has been previously individually evaluated in chemotherapy, but the effects of combination PARP-1 and PARG inhibition in cancer cells are not known. Here we determined the effects of the inhibition of PARP-1 and the absence or RNAi knockdown of PARG on PAR synthesis, cell death after chemotherapy and long-term viability. Using three experimental/clinical PARP-1 inhibitors in PARG-null cells, we show decreased levels of PAR and increased short‑term and long‑term viability with each inhibitor, with the exception of DPQ. Treatment with the experimental chemotherapeutic agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), led to increased cell death in PARG-null cells, but decreased cell death when pretreated with each PARP-1 inhibitor. Similar results were observed in MNNG-treated HeLa cells, where RNAi knockdown of PARG or pretreatment with ABT-888 led to increased HeLa cell death, whereas combination PARG RNAi knockdown + ABT-888 failed to produce increased cell death. The results demonstrate the ability of the PARP-1 inhibitors to decrease PAR levels, maintain viability and decrease PAR-mediated cell death after chemotherapeutic treatment in the absence of PARG. Further, the results demonstrate that the combination of PARP-1 and PARG inhibition in chemotherapy does not produce increased HeLa cell death. Thus, the results indicate that inhibiting both PARP-1 and PARG, which both are chemotherapeutic targets that increase cancer cell death, does not lead to synergistic cell death in HeLa cells. Therefore, strategies that target PAR metabolism for the improved treatment of cancer may be required to target PARP-1 and PARG individually in order to optimize cancer cell death.
... 27 A potent PARP inhibitor, 3,4-dihydro-5-[4-1(1-piperidynil) buthoxy]-1(2H)-isoquinolinone (DPQ), prevented neural damage following vascular stroke in rats. 28 Post-treatment with DPQ diminished poly(ADP-ribose) production in the ischemic region in a rat model of focal stroke, 29 indicating that DPQ exerted its neuroprotective effect in vivo by PARP inhibition. Neuroprotective effects of several novel PARP inhibitors in mouse and rat experimental models of cerebral ischemia have recently been reported, 30,31 although selectively for PARP-1 over PARP-2 may be difficult to achieve. ...
Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear enzyme that is activated primarily by DNA damage. Upon activation, the enzyme hydrolyzes NAD+ to nicotinamide and transfers ADP ribose units to a variety of nuclear proteins, including histones and PARP-1 itself. This process is important in facilitating DNA repair. However, excessive activation of PARP-1 can lead to significant decrements in NAD+, and ATP depletion, and cell death (suicide hypothesis). In response to cellular damage by oxygen radicals or excitotoxicity, a rapid and strong activation of PARP-1 occurs in neurons. Excessive PARP-1 activation is implicated in a variety of insults, including cerebral and cardiac ischemia, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism, traumatic spinal cord injury, and streptozotocin-induced diabetes. The use of PARP inhibitors has, therefore, been proposed as a protective therapy in decreasing excitotoxic neuronal cell death, as well as ischemic and other tissue damage. Excitotoxic brain lesions initially result in the primary destruction of brain parenchyma and subsequently in secondary damage of neighboring neurons hours after the insult. This secondary damage of initially surviving neurons accounts for most of the volume of the infarcted area and the loss of brain function after a stroke. One major component of secondary neuronal damage is the migration of macrophages and microglial cells toward the sites of injury, where they produce large quantities of toxic cytokines and oxygen radicals. Recent evidence indicates that this microglial migration is strongly controlled in living brain tissue by expression of the integrin CD11a, which is regulated in turn by PARP-1, proposing that PARP-1 downregulation may, therefore, be a promising strategy in protecting neurons from this secondary damage, as well. Studies demonstrating an important role for PARP-1 in the regulation of gene transcription have further increased the intricacy of poly(ADP-ribosyl)ation in the control of cell homeostasis and challenge the notion that energy collapse is the sole mechanism by which poly(ADP-ribose) formation contributes to cell death. The hypothesis that PARPs might regulate cell fate as essential modulators of death and survival transcriptional programs is discussed with relation to nuclear factor κB and p53.
... Because PARP activation utilizes NAD ϩ , extensive activity may deplete NAD ϩ (Schanraufstatter et al., 1986) and, in fact, be detrimental to cellular survival following an insult to the brain (Eliasson et al., 1997;Endres et al., 1997;Takahashi et al., 1997;Lo et al., 1998). The lack of PARP activity at later time points following TBI may be protective against energy depletion given the increased energy consumption following TBI (Yoshino et al., 1991). ...
The activation of poly(ADP-ribose) polymerase, a DNA base excision repair enzyme, is indicative of DNA damage. This enzyme also undergoes site-specific proteolysis during apoptosis. Because both DNA fragmentation and apoptosis are known to occur following experimental brain injury, we investigated the effect of lateral fluid percussion brain injury on poly(ADP-ribose) polymerase activity and cleavage. Male Sprague-Dawley rats (n = 52) were anesthetized, subjected to fluid percussion brain injury of moderate severity (2.5-2.8 atm), and killed at 30 min, 2 h, 6 h, 24 h, 3 days, or 7 days postinjury. Genomic DNA from injured cortex at 24 h, but not at 30 min, was both fragmented and able to stimulate exogenous poly(ADP-ribose) polymerase. Endogenous poly(ADP-ribose) polymerase activity, however, was enhanced in the injured cortex at 30 min but subsequently returned to baseline levels. Slight fragmentation of poly(ADP-ribose) polymerase was detected in the injured cortex in the first 3 days following injury, but significant cleavage was detected at 7 days postinjury. Taken together, these data suggest that poly(ADP-ribose) polymerase-mediated DNA repair is initiated in the acute posttraumatic period but that subsequent poly(ADP-ribose) polymerase activation does not occur, possibly owing to delayed apoptosis-associated proteolysis, which may impair the repair of damaged DNA.
... Additionally, Endres and colleagues (Endres et al., 1997) showed that 3AB reduced the infarcted region after ischemia-reperfusion injury to the same extend as PARP-1 knockout. Also, a report showed that PARP inhibition in a rat model of focal cerebral ischemia reduced the size of the infarcted area (Takahashi et al., 1997). Genetic deletion of nNOS prevented PAR formation after ischemia, and peroxynitrite, but not NO-donors, were effective in stimulating PARP activity in glioma cells (Endres et al., 1998a). ...
In this Chapter, we review the evidence suggesting that the family of poly(ADP-ribose) polymerases (PARPs) is involved in
regulation of the aging process. First, as genotoxic stress, mainly produced by reactive oxygen species, is believed to be
the major driving force of cellular aging, the importance of mechanisms that counteract it or revert its consequences is quiet
obvious. A pivotal pathway for eliminating oxidative DNA damage, spontaneously formed abasic sites, or DNA single strand breaks
is DNA base-excision repair and its activity is facilitated by PARP-1 and PARP-2. In line with these observations, the capacity
of mononuclear blood cells to synthesize poly(ADP-ribose), largely reflecting PARP-1 activity, is positively correlated with
the life span of the donor species in mammalians. Second, maintenance of telomere length is very important for replicating
cells to avoid cellular senescence and replicative crisis. Two important regulators are the poly(ADP-ribose) polymerases tankyrase-1
and tankyrase-2, which inhibit via modification of TRF-1 its negative influence on telomerase activity. Third, the interaction
of PARP-1 with proteins important in preventing premature aging and retarding age-related disease like the Werner syndrome
protein (WRN) further supports the importance of PARP-1 in this process. Forth, as several PARPs are components of the mitotic
apparatus with apparent regulatory function, they counteract genomic instability also on this level of defence, i.e., beyond
DNA repair. Fifth, by interacting with important cell cycle regulators such as p53, PARPs and probably their product, poly(ADP-ribose),
take an active part in DNA damage surveillance and regulation of cell division. In conclusion, PARP proteins are crucial players
in the cellular responses to various kinds of impaired functionality of genomic DNA, as they represent versatile tools to
fight all kinds of threats to genomic integrity, thus keeping in check aging-related dysfunction and disease. In doing so,
they might be importanthelpers to keep the speed of the aging process low and may also contribute to attaining “healthy aging”,
i.e., a state of being old, yet free from major age-related disease or disability.
... In particular, Dawson and her group report that brain infarct is 80% smaller in PARPknockout mice compared to wild type 22 h after 2 h MCAO. Likewise, oxygen and glucose deprivation (OGD)-induced neuronal death is highly reduced in mixed cortical cultures from PARP-1-knockout mice with respect to that occurring in cortical cultures from wild-type counterparts [152,153]. Numerous studies showing the neuroprotective effects of chemical inhibitors of PARP-1 in in vitro and in vivo models of brain ischemia have been reported (Table 1). For instance, the PARP-1 inhibitor 3-AB affords significant neuroprotection when preinjected into mice and rats subjected to transient [151,154] or permanent [155] brain ischemia. ...
Poly(ADP-ribose) polymerases (PARPs) are defined as cell signaling enzymes that catalyze the transfer of ADP-ribose units from NAD+ to a number of acceptor proteins. PARP-1, the best characterized member of the PARP family, which currently comprises 18 members, is an abundant nuclear enzyme implicated in cellular responses to DNA injury provoked by genotoxic stress. PARP is involved in DNA repair and transcriptional regulation and is now recognized as a key regulator of cell survival and cell death as well as a master component of a number of transcription factors involved in tumor development and inflammation. PARP-1 is essential to the repair of DNA single-strand breaks via the base excision repair pathway. Inhibitors of PARP-1 have been shown to enhance the cytotoxic effects of ionizing radiation and DNA-damaging chemotherapy agents, such as the methylating agents and topoisomerase I inhibitors. There are currently at least five PARP inhibitors in clinical trial development. Recent in vitro and in vivo evidence suggests that PARP inhibitors could be used not only as chemo/radiotherapy sensitizers, but also as single agents to selectively kill cancers defective in DNA repair, specifically cancers with mutations in the breast cancer-associated genes (BRCA1 and BRCA2). PARP becomes activated in response to oxidative DNA damage and depletes cellular energy pools, thus leading to cellular dysfunction in various tissues. The activation of PARP may also induce various cell death processes and promotes an inflammatory response associated with multiple organ failure. Inhibition of PARP activity is protective in a wide range of inflammatory and ischemia–reperfusion-associated diseases, including cardiovascular diseases, diabetes, rheumatoid arthritis, endotoxic shock, and stroke. The aim of this review is to overview the emerging data in the literature showing the role of PARP in the pathogenesis of cancer and inflammatory diseases and unravel the solid body of literature that supports the view that PARP is an important target for therapeutic intervention in critical illness.
Differential evolution of apoptosis, programmed necrosis, and autophagy, parthanatos is a form of cell death mediated by poly(ADP-ribose) polymerase 1 (PARP1), which is caused by DNA damage. PARP1 hyper-activation stimulates apoptosis-inducing factor (AIF) nucleus translocation, and accelerates nicotinamide adenine dinucleotide (NAD+) and adenosine triphosphate (ATP) depletion, leading to DNA fragmentation. The mechanisms of parthanatos mainly include DNA damage, PARP1 hyper-activation, PAR accumulation, NAD+ and ATP depletion, and AIF nucleus translocation. Now, it is reported that parthanatos widely exists in different diseases (tumors, retinal diseases, neurological diseases, diabetes, renal diseases, cardiovascular diseases, ischemia-reperfusion injury...). Excessive or defective parthanatos contributes to pathological cell damage; therefore, parthanatos is critical in the therapy and prevention of many diseases. In this work, the hallmarks and molecular mechanisms of parthanatos and its related disorders are summarized. The questions raised by the recent findings are also presented. Further understanding of parthanatos will provide a new treatment option for associated conditions.
The catastrophe of the ongoing COVID-19 pandemic is caused by Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2). The respiratory system appears to be ground zero in the majority of the patients. However, many other organs can get infected by cytokines, chemokines and other mediators released in response to the presence of the virus. The neurotropism by the SARS-CoV-2 is established beyond doubt. In addition to non-specific symptoms, the symptoms specific to central and/or peripheral nervous system diseases as well as neuromuscular diseases have been observed in numerous clinical cases. These observations and the experiences with other coronavirus infections earlier and flu pandemics raise concerns not only about the neurological effects in active disease but also about the long-term effects generated by the infection, immune and inflammatory functions. The knowledge of biological actions of agmatine in the backdrop of physiological events instigated by invading SARS-CoV-2 and host’s response, especially in neural events, focuses on the possible overlaps of biomolecular pathways at a number of instances. This is not surprising since the factors stimulated during SARS-CoV-2 infection are the disease-generating neuroinflammatory components altered by agmatine. Hence, we hypothesize the possible beneficial role of agmatine in SARS-CoV-2 infection. Based on a narrative review of the literature, agmatine can be proposed as a plausible beneficial candidate for supporting treatment of SARS-CoV-2 infection and for addressing post-infection neurological complications.
Cell death is a key feature of neurological diseases, including stroke and neurodegenerative disorders. Studies in a variety of ischemic/hypoxic mouse models demonstrate that poly(ADP‐ribose) polymerase 1 (PARP‐1)‐dependent cell death, also named PARthanatos, plays a pivotal role in ischemic neuronal cell death and disease progress. PARthanatos has its unique triggers, processors, and executors that convey a highly orchestrated and programmed signaling cascade. In addition to its role in gene transcription, DNA damage repair, and energy homeostasis through PARylation of its various targets, PARP‐1 activation in neuron and glia attributes to brain damage following ischemia/reperfusion. Pharmacological inhibition or genetic deletion of PARP‐1 reduces infarct volume, eliminates inflammation, and improves recovery of neurological functions in stroke. Here, we reviewed the role of PARP‐1 and PARthanatos in stroke and their therapeutic potential.
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Cerebral ischemia is one of the major complications of diabetes. Eighty-four percent of diabetics die from either heart disease or stroke (both cause cerebral ischemia). Diabetes-induced oxidative stress is one of the factors responsible for the secondary complications of diabetes, even after diabetes treatment. Oxidative stress also causes an increase in cerebral ischemic damage, resulting in the activation of necrotic and apoptotic pathways. In this chapter, we provide a literature review on how cerebral ischemia-induced oxidative stress is exacerbated in diabetics. We also provide correlates of how this increased oxidative stress may be responsible for increased cerebral ischemia damage in diabetics.
A central and causative feature of age-related neurodegenerative disease is the deposition of misfolded proteins in the brain. To devise novel approaches to treatment, regulatory pathways that modulate these aggregation-prone proteins must be defined. One such pathway is post-translational modification by the addition of poly(ADP-ribose) (PAR), which promotes protein recruitment and localization in several cellular contexts. Mounting evidence implicates PAR in seeding the abnormal localization and accumulation of proteins that are causative of neurodegenerative disease. Inhibitors of PAR polymerase (PARP) activity have been developed as cancer therapeutics, raising the possibility that they could be used to treat neurodegenerative disease. We focus on pathways regulated by PAR in neurodegenerative disease, with emphasis on amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD).
The hypothesis that cerebral ischemia may recognize an excitoxic pathophysiological component is supported by a number of consolidated arguments, including the neuroprotective activity of glutamate receptor antagonists in experimental animal models and the use of anti-excitotoxic agents in clinical trials for stroke. Despite the dramatic results in the preclinical setting, phase III clinical trials with neuroprotective drugs have been generally unsuccessful so far. Several complicating variables have been put forward to explain this discrepancy, including population heterogeneity, morphological and functional differences between human and animal brain, and side-effects of the tested compounds that prevent reaching effective plasma concentrations. Fine-tuning in the design of clinical trials, the use of imaging techniques for the evaluation of human brain injury, and the development of more appropriate experimental animal models are among the strategies that need to be utilized in future clinical studies. Also, drugs with a better therapeutic index and aimed at alternative targets in the excitotoxic cascade appear to be required. In our laboratory, we have recently investigated two alternative mechanisms promoted by extracellular glutamate that lead to post-ischemic neuronal damage: (1) the stimulation of mGlu1 receptors and (2) the overactivation of poly(ADP-ribose) polymerase.
Ischemia-induced mechanisms of brain injury are a final common pathway for a variety of acute brain insults. Age at the time of brain insult may also have significant bearing on severity of injury induced. For example, for many years it has been known that, in comparison with adult animals, neonatal animals display a reduced sensitivity to ischemia-induced damage (Fazekas et al. 1941; Adolph 1948; Adolph 1971) and a different distribution of neuropathological sequelae (Brierley et al. 1984). A number of properties of brain tissue may account for these ontogeny-related differences, and in the laboratory, the rat has been used to elucidate potential mechanisms, since its neurochemistry over the first 28-postnatal days has similarities with changes in humans over the first few years of life (Clarke et al. 1970; Benjamins et al. 1981; Clark et al. 1989). This review will therefore focus on the experimental work about ischemia-induced ionic failure and its interrelation with excitatory amino acid- and oxidant-related injury in the developing brain.
The complete global brain ischemia of cardiac arrest (CA), potentially reversible by cardiopulmonary-cerebral resuscitation (CPCR) [1,2] is the most common cause of sudden coma and death [1,3]. Sudden CA kills about 400,000 persons each year in the U.S. In addition, in the over 100,000 accidental deaths each year, coma occurs as a result of trauma, intoxication, asphyxiation, severe shock, or other insults. This talk focused on CPCR research results by our teams, and mentioned only some of the important contributions made by others, most of which have been reviewed [4–7]. Epidemiologic studies suggest that the chance for conscious survival of normothermic CAdecreases by about 10% for every minute of normothermic complete global brain ischemia (no-flow) [3].
A potential critical role of mitochondrial dysfunction in neurodegenerative diseases is becoming increasingly compelling. Mitochondrial dysfunction leads to a number of deleterious consequences for the cell including impaired calcium buffering, generation of free radicals, activation of nitric oxide synthase, activation of the mitochondrial permeability transition, and secondary excitotoxicity (Beal 1992, 1995). This can lead to both apoptotic and necrotic cell death depending on the severity of the insult. Neurodegenerative diseases have widely disparate etiologies but may share mitochondrial dysfunction as a final common pathway.
The major excitatory transmitter in the mammalian central nervous system is glutamate, which exerts its signaling actions through the stimulation of ionotropic and metabotropic receptors (Watkins et al. 1981; Mayer and Westbrook 1987; Nakanishi and Masu 1994). Under pathological conditions, glutamate receptor overactivation can trigger neuronal death, a phenomenon known as excitotoxicity (Lucas and Newhouse 1957; Olney 1969). Incentive for developing practical methods for blocking excitotoxicity arises from its implication in several acute and chronic neurological disease states. While recent clinical trials aimed at blocking excitotoxicity in stroke patients have been disappointing, there are several plausible reasons for these trial failures, including specific study design issues, treatment side effects, and a need to achieve concurrent block of parallel injury pathways. In our view, the case for antiexcitotoxic approaches in stroke remains open, and there are other possible disease targets yet to be explored. Ongoing delineation of the cellular and molecular underpinnings of excitotoxicity has led to the progressive unveiling of countermeasures, aimed at attenuating presynaptic glutamate release, postsynaptic receptor activation, the movement or action of cation second messengers, or downstream intracellular injury cascades. The excitotoxicity concept itself may need to be expanded, to encompass the death of oligodendrocytes as well as neurons, and ionic derangements besides Ca2+ overload.
This article provides the first extensive documentation of the dose response features of pre- and postconditioning. Pre- and postconditioning studies with rigorous study designs, using multiple doses/concentrations along with refined dose/concentration spacing strategies, often display hormetic dose/concentration response relationships with considerable generality across biological model, inducing (i.e., conditioning) agent, challenging dose treatment, endpoint, and mechanism. Pre- and postconditioning hormesis dose/concentration-response relationships are reported for 154 diverse conditioning agents, affecting more than 550 dose/concentration responses, across a broad range of biological models and endpoints. The quantitative features of the pre- and postconditioning-induced protective responses are modest, typically being 30-60% greater than control values at maximum, findings that are consistent with a large body (>10,000) of hormetic dose/concentration responses not related to pre- and postconditioning. Regardless of the biological model, inducing agent, endpoint or mechanism, the quantitative features of hormetic dose/concentration responses are similar, suggesting that the magnitude of response is a measure of biological plasticity. This paper also provides the first documentation that hormetic effects account for preconditioning, induced early (1-3h) and delayed (12-72h) windows of protection. These findings indicate that pre- and postconditioning are specific types of hormesis.
Cerebral ischemia is one of the major complications of diabetes. Eighty four percent of diabetics die from either heart disease or stroke (both cause cerebral ischemia). Diabetes-induced oxidative stress is one of the factors responsible for the secondary complications of diabetes. Oxidative stress also plays a key role in cerebral ischemic damage. In this chapter we provide a literature review of how cerebral ischemia-induced oxidative stress is exacerbated in diabetics. We also provide correlates of how this increased oxidative stress may be responsible for increased cerebral ischemia damage in diabetics.
Ischemic stroke triggers an inflammatory reaction in the affected area, which progresses for days to weeks after the onset of symptoms. There is evidence that selected aspects of such inflammatory processes contribute to the progression of ischemic brain injury, leading to worsening of the tissue damage and exacerbation of neurologic deficits. Therefore, interventions aimed at suppressing postischemic inflammation offer attractive therapeutic strategies for human stroke, with a potentially wide therapeutic window. A large body of work has addressed the inflammatory process in the postischemic brain. 1 2 3 4 5 In this chapter, we review the basic cellular and molecular features of postischemic inflammation, focusing on recent advances and insights on the potential mechanisms by which such inflammation influences stroke outcome. We then analyze the potential therapeutic implications of modulators of specific inflammatory targets from the perspective of near-future translational approaches.
Despite being studied for over 200 years, the posture, locomotion, paleobiology, and phylogeny of pterosaurs are still poorly understood due to lack of well-preserved, three-dimensional specimens. In this paper we investigate the posture, locomotion, and paleoecology of pterosaurs based on anatomy and biomechanics: how they walked, how they flew, and how they lived. We want to understand how evolution has adjusted their skeletal structures and movements to maximize performance. The limb joints of an exquisite skeleton of the Cretaceous pterodactyl Anhanguera piscator are analyzed to estimate the range of movement during terrestrial and aerial locomotion. On land, pterosaurs were quadrupedal knuckle walkers with laterally directed digitigrade manus and forwardly directed plantigrade pes. From this position, pterosaurs could stand on their rear legs and run bipedally in an upright posture for a short distance during takeoff and landing. Pterosaurs evolved two basic wing planforms over time: the basal "rhampho-rhynchoids" had broad wings in bat-like fashion where the patagium was attached to the ankle; in pterodactyloids, the wings became narrow and the patagium was anchored near the knee joint. The wingtips appear to have been more rounded to avoid stalling. The actinofibrils in the membrane would confer some stiffness to the wing to maintain a flatter camber, preventing it from billowing and tearing during flight. They would also facilitate the folding of the wing when not in use. The flight performance of pterosaurs is investigated using ten genera in a wide size spectrum during their 160 million years of evolution, where the body mass ranges from 0.015 kg to 70 kg and the wingspan from 0.4 m to 10.4 m. Thus the largest pterosaur in our study weighs about 4700 times more than the smallest species, and the longest wingspan is 25 times the shortest. Helicopter momentum stream tube theory has been adapted to estimate the scaling of aerial locomotion of pterosaurs and to minimize the complexities of animal physiology. The aerodynamic data were calculated using the two computer programs ANFLTPWR (animal flight power) and ANFLTSIM (animal flight simulation). Pterosaur wings were long and narrow, similar to those of seabirds, with high aspect ratios. However, they had relatively low wing loadings and low cruising speeds compared to seabirds with similar masses. Gliding performance, deduced from the polar curves, indicates that smaller pterosaurs such as Eudimorphodon, Pterodactylus, Rhamphorhynchus, and Dorygnathus had lower gliding airspeeds, with a gliding angle close to 4°. The giant Cretaceous pterodactyloids such as Pteranodon and Quetzalcoatlus were excellent soarers comparable to the albatross, human-powered planes, and sailplanes, with a gliding angle between 1° and 2°. The cruising speed for best gliding depends on size, increasing proportionally to mass and wing loading, from as low as 4 m/s for Eudimorphodon to 16 m/s for Quetzalcoatlus. The power curves, displaying maximum and minimum level flight speeds, show three different styles of flight. In the four smaller genera (Mass < 0.3 kg) such as Eudimorphodon, Pterodactylus, Rhamphorhynchus, and Dorygnathus, the available aerobic power (Pa) exceeds the required power at zero speed, and they were evidently capable of hovering flight. Tapejara, Nyctosaurus, Dsungaripterus, Anhanguera, and Pteranodon appear to be capable of steady level flight at aerobic power, but within a limited speed range. The sustained power output of giant pterodactyloids such as Quetzalcoatlus was not enough for continuous level flapping flight; however, they could improve their flying performance if they flew in formation. Apparently, extended flight for large pterodactyloids was by soaring; they flapped normally when taking off or landing. Takeoff from the ground was initiated by running and hopping. Although small pterosaurs apparently had sufficient available power for running takeoff from the ground or from the water, larger pterodactyloids such as Pteranodon and Quetzalcoatlus were limited in their takeoff capabilities and were unable to take off with maximum aerobic power. They needed short bursts of anaerobic power to take off from the ground with a headwind of 5 m/s. For Quetzalcoatlus, a takeoff from a 10° downward slope would be helpful especially when it ventured inland. As their running speed increased, low-amplitude flapping was used to accelerate to take off; pterosaurs leaped into the air and flapped their wings for flight. The long axis rotation of the humerus in the upstroke position would have been useful during takeoff and landing. The function of the cranial crest may have been linked to thermoregulation, sexual display, and species recognition. The large head of pterodactyloids was probably downturned during flight and was used as a steering device for turning the body. The ecology of pterosaurs was similar to those of modern seabirds, spending much time in coastal areas for feeding. Small and medium-size pterosaurs probably foraged by plunge diving like modern pelicans. Large pterodactyloids were probably active waders or surface riders during feeding, using their feet to propel while folding their wings sidewise. Arising as small animals in the Triassic, pterosaurs exhibit long-term phyletic trends toward increasing body size during the Cretaceous, but the trend is erratic. They became extinct at the end of the Cretaceous along with dinosaurs and other organisms when multiple asteroids crashed into the Earth, accompanied by the spectacular Deccan volcanism that had devastating effects on the ecology.
Overactivation of the N-methyl-d-aspartate receptor (NMDAR) after cerebral ischemia is a crucial reason for neuron death. Although NMDAR antagonists have exhibited neuroprotective effects in animal models, it is disappointing that several severe side effects have occurred in patients. NMDAR is a heteromer containing 2 obligate N-methyl-D-aspartate receptor 1 (GluN1) subunits and a variety of GluN2 and GluN3 subunits. The GluN2 subunit, which contributes specifically to neuron death after stroke, has been studied extensively. An opposing action of the GluN2A and GluN2B subunits in mediating cell death and cell survival was observed.1,2 The results indicate that the GluN2A subunit produces prosurvival activity, whereas the GluN2B subunit leads to a prodeath signal. However, von Engelhardt et al3 found that the GluN2A subunit can also mediate NMDA-dependent toxicity in DIV21 cultures. This paradox may have resulted because the pharmacological approach used to study subunit composition was not flawless.4 In view of this limitation and according to the methods of molecular biology, Martel et al5 demonstrated that the C-terminal domains of GluN2B promote neuronal death more efficiently than those of GluN2A in cerebral ischemia.5 In short, NMDARs containing GluN2B are more lethal than those containing GluN2A. Prodeath signaling pathways mediated by neuronal nitric oxide synthase (nNOS), death-associated protein kinase 1 (DAPK1), phosphatase and tensin homolog located on chromosome 10 (PTEN), and calcium/calmodulin-dependent protein kinase II (CaMKII) have been linked to GluN2B activation. Therapeutic targets based on these signaling pathways of the GluN2B carboxyl terminus (C terminus) will be introduced in this review.
### GluN2B–nNOS Signaling Pathway
The GluN2B–nNOS signaling pathway, which plays an important role in neuron death, is the most widely studied GluN2B pathway (Figure 1).
Figure 1.
The GluN2B–nNOS signaling pathway. Based on the PDZ domains, postsynaptic density-95 (PSD-95) assembles GluN2B and nNOS into a macromolecular complex. After …
Experimental studies indicate that overactivation of the DNA repair protein poly(ADP-ribose) polymerase (PARP) in response to oxidative damage to DNA can cause cell death due to depletion of NAD+. Oxidative damage to DNA and other macromolecules has been reported to be increased in the brains of patients with Alzheimer's disease. In the present study we sought evidence of PARP activation in Alzheimer's disease by immunostaining sections of frontal and temporal lobe from autopsy material of 20 patients and 10 controls, both for PARP itself and for its end-product, poly(ADP-ribose). All of the brains had previously been subjected to detailed neuropathological examination to confirm the diagnosis of Alzheimer's disease or, in the controls, to exclude Alzheimer's disease-type pathology. Double immunolabelling for poly(ADP-ribose) and microtubuleassociated protein 2 (MAP2), glial fibrillary-acidic protein (GFAP), CD68, Aβ-protein or tau was used to assess the identity of the cells with poly(ADP-ribose) accumulation and their relationship to plaques and neurofibrillary tangles. Both PARP- and poly(ADPribose)-immunolabelled cells were detected in a much higher proportion of Alzheimer's disease (20 out of 20) brains than of control brains (5 out of 10) ( P = 0.0018). Double-immunolabelling for poly(ADP-ribose) and markers of neuronal, astrocytic and microglial differentiation (MAP2, GFAP and CD68, respectively) showed many of the cells containing poly(ADP-ribose) to be neurons. Most of these were small pyramidal neurons in cortical laminae 3 and 5. A few of the cells containing poly(ADP-ribose) were astrocytes. No poly(ADP-ribose) accumulation was detected in microglia. Doubleimmunolabelling for poly(ADP-ribose) and tau or Aβ-protein indicated that the cells with accumulation of poly(ADP-ribose) did not contain tangles and relatively few occurred within plaques. Our findings indicate that there is enhanced PARP activity in Alzheimer's disease and suggest that pharmacological interventions aimed at inhibiting PARP may have a role in slowing the progression of the disease.
An ischemic stroke is the sudden and permanent death of brain cells that occurs when the flow of blood to a part of the brain is blocked and oxygen cannot be delivered to the brain. Oxidative stress (OS) is a potential contributor to the pathophysiological consequences of stroke. On the basis of experimental models, there is ample evidence for enhanced free-radical production in the brain after stroke. Furthermore, reactive oxygen species (ROS) generation after stroke is a prolonged process. It is assumed that oxidative stress (OS) contributes to the initiation and development of stroke via different interrelated mechanisms: excitotoxicity resulting in cellular enzyme activation and ROS generation; mitochondrial dysfunction accompanied by excessive radical production; inflammation leading to leukocyte priming and activation; activation and oxidative damage of endothelium resulting in reduced bioavailability of nitric oxide; and lipid peroxidation of plasma and cellular components. Some studies demonstrate the protective role of natural and synthetic antioxidants in experimental stroke models-for example, the supplementation of vitamins C and E reduces the infarct size and severity of neurological damage after permanent ischemia/reperfusion in rats.
Astrocytes are now known to be involved in the most integrated functions of the central nervous system. These functions are not only necessary for the normally working brain but are also critically involved in many pathological conditions, including stroke. Astrocytes may contribute to damage by propagating spreading depression or by sending proapoptotic signals to otherwise healthy tissue via gap junction channels. Astrocytes may also inhibit regeneration by participating in formation of the glial scar. On the other hand, astrocytes are important in neuronal antioxidant defense and secrete growth factors, which probably provide neuroprotection in the acute phase, as well as promoting neurogenesis and regeneration in the chronic phase after injury. A detailed understanding of the astrocytic response, as well as the timing and location of the changes, is necessary to develop effective treatment strategies for stroke patients.
Cells die by a variety of mechanisms. Terminally differentiated cells such as neurones die in a variety of disorders, in part, via parthanatos, a process dependent on the activity of poly (ADP-ribose)-polymerase (PARP). Parthanatos does not require the mediation of caspases for its execution, but is clearly mechanistically dependent on the nuclear translocation of the mitochondrial-associated apoptosis-inducing factor (AIF). The nuclear translocation of this otherwise beneficial mitochondrial protein, occasioned by poly (ADP-ribose) (PAR) produced through PARP overactivation, causes large-scale DNA fragmentation and chromatin condensation, leading to cell death. This review describes the multistep course of parthanatos and its dependence on PAR signalling and nuclear AIF translocation. The review also discusses potential targets in the parthanatos cascade as promising avenues for the development of novel, disease-modifying, therapeutic agents.
This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8.
Ischemic stroke is caused by critical reductions in blood flow to a part of brain or spinal cord. Microglia are the resident immune cells of the central nervous system, and they respond to stroke by assuming an activated phenotype that releases cytotoxic cytokines, reactive oxygen species, proteases, and other factors. This acute, innate immune response may be teleologically adapted to limit infection, but in stroke this response can exacerbate injury by further damaging or killing nearby neurons and other cell types and by recruiting infiltration of circulating cytotoxic immune cells. The microglial response requires hours to days to fully develop, and this time interval presents a clinically accessible time window for initiating therapy. Redundancy in cytotoxic microglial responses suggest that the most effective therapeutic approach may be to target the global gene expression changes involved in microglial activation. Several classes of drugs can do this, including histone deacetylase inhibitors, minocycline and other PARP inhibitors, corticosteroids, and inhibitors of TNFα and scavenger receptor signaling. Here we review the pre-clinical studies in which these drugs have been used to suppress microglial activation after stroke. We also review recent advances in the understanding of sex differences in the CNS inflammatory response, as these differences are likely to influence the efficacy of drugs targeting post-stroke brain inflammation.
We have been studying the occurrence of low velocity detonations (LVI) in several liquid explosives. A hypothesis based on shock wave interactions and Mach reflections was proposed to explain the initiation and propagation of LVD. Using the card gap test we found general experimental agreement with this hypothesis. However, certain anomalous effects were noted. In addition, we developed a method to study the internal wave structure of a shocked liquid. This technique was applied to a liquid that sustains LVD and to one that does not. A comparison of these liquids shows that the reaction zone in a liquid explosive undergoing LVD is consistent with the Mach zone hypothesis.
The anomalous effects noted during gap testing were subjected to a photographic study using a high speed framing camera. The results of this study showed that mechanisms other than that explainable by the Mach zone hypothesis were also responsible for LVD initiation. These were wave reflections from witness plates for high sound speed-high strength confinement and donor air shock initiation for lead (low sound speed-low strength) confinement. We conclude that there appears to be no unique mechanism for LVD and that each mechanism proposed so far explains some of the observations.
Poly (ADP-ribose) polymerase (PARP-1) is a nuclear enzyme that facilitates DNA repair and may be important in neuronal cell death in a variety of diseases including cardiac arrest/cardiopulmonary resuscitation (CPR) and stroke. This chapter briefly reviews the characteristics of PARP, its activation, and its role in both focal (stroke) and global (cardiac arrest/CPR) cerebral ischemia. We demonstrate PARP deficiency to be effective in reducing neuronal reperfusion injury after stroke and cardiac arrest/CPR in mouse models. These data suggest a selective role of PARP-1 in glutamate excitotoxicity and that strategies of inhibiting PARP-1 may offer substantial neuroprotection in focal and global cerebral ischemia.
Background : Nuclear enzyme poly (ADP-ribose) polymerase (PARP) activated by DNA damage participates in DNA repair. However, overactivation of PARP could be an important pathogenic mechanism of ischemic cell death. We investigated the protective effect of an inhibitor of PARP, 3-aminobenzamide (3-AB), against ischemia/reperfusion injury in ischemic stroke model. Methods :Occlusion of left middle cerebral artery (MCA) was done by intraluminal filament technique in 24 rats weighing from 315 g to 358 g, and reperfusion was done at 2 hours after occlusion. To evaluate the effect of PARP inhibitor in ischemic stroke, 3-AB was administered to 12 rats (3-AB group) 10 minutes before artificial occlusion of left MCA. Infarct area was confirmed by using 2,3,5-triphenyltetrazolium chloride stain. The immunoreactivities of poly (ADP-ribose) reflecting activity of enzyme PARP and activated caspase-3 were compared in infarct, peri-infarct and normal zones in 3-AB group and 12 controls. Results :The volume of infarction was decreased about 34% in 3-AB group compared with controls. In 3-AB group, immunoreactivities of PAR were significantly reduced in ischemic regions, especially peri-infarct zone, but those of activated caspase-3 were significantly increased in same region. Conclusions : These results suggest that treatment of PARP inhibitor can reduce the infarct volume by converting necrotic cell death into apoptosis. PARP inhibition can be another potential neuroprotective strategy in ischemic stroke.J Korean Neurol Assoc 21(6):634q641, 2003
Poly(ADP-ribose) polymerase (PARP), or poly-(ADP-ribose) synthetase, is a nuclear enzyme that consumes NAD when activated by DNA damage. The role of PARP in the pathogenesis of traumatic brain injury (TBI) is unknown. Using a controlled cortical impact (CCI) model of TBI and mice deficient in PARP, the authors studied the effect of PARP on functional and histologic outcome after CCI using two protocols. In protocol 1, naïve mice (n = 7 +/+, n = 6 −/−) were evaluated for motor and memory acquisition before CCI. Mice were then subjected to severe CCI and killed at 24 hours for immunohistochemical detection of nitrated tyrosine, an indicator of peroxynitrite formation. Motor and memory performance did not differ between naïve PARP +/+ and −/− mice. Both groups showed nitrotyrosine staining in the contusion, suggesting that peroxynitrite is produced in contused brain. In protocol 2, mice (PARP +/+, n = 8; PARP −/−, n = 10) subjected to CCI were tested for motor and memory function, and contusion volume was determined by image analysis. PARP −/− mice demonstrated improved motor and memory function after CCI versus PARP +/+ mice (P < 0.05). However, contusion volume was not different between groups. The results suggest a detrimental effect of PARP on functional outcome after TBI.Keywords: Brain injury; Poly(ADP-ribose) polymerase; Controlled cortical impact; Mice
Poly(ADP-ribose) polymerase (PARP) is one of the most abundant nuclear enzymes. When it is activated by DNA strand breaks, PARP synthesises poly(ADP-ribose) by using nicotinamide adenine dinucleotide (NAD) as substrate. Early PARP research focused primarily on its role in facilitating structural changes of chromosomal DNA, such as during DNA repair, gene expression, DNA replication, DNA rearrangement, sister chromatid exchange, differentiation and mutagenesis. The recent discovery of PARP as a substrate for caspases has led to an intensive search for possible PARP involvement in the apoptosis pathway. Although the exact physiologic functions of PARP still remain to be firmly established, it has long been known that PARP activation can cause a rapid depletion of cellular NAD and ATP. This is mainly due to the high turnover rate of poly(ADP-ribose), which is degraded by poly(ADP-ribose) glycohydrolase. Thus, it has been proposed as a 'suicidal hypothesis' that PARP activation contributes to the energy failure that leads to cell death elicited by massive DNA damage. Indeed, there is accumulating evidence to suggest that activation of PARP by free radical damaged DNA plays a pivotal role in mediating ischaemia/reperfusion injuries. PARP emerges as a novel target to treat such injuries. The strategy of targeting PARP inhibition is validated by the findings that PARP deficient mice are extremely resistant to both cerebral and myocardial ischaemia. PARP inhibitors have demonstrated remarkable efficacy in reducing the infarct volumes of cerebral focal ischaemia and regional heart ischaemia. They also exhibit strong anti-inflammatory activities in rodent models of inflammation related injuries, e.g., septic shock, arthritis and Type I diabetes. Therefore, even at an early stage of drug discovery, PARP inhibitors have shown great potential for treating a broad spectrum of diseases.
Poly(ADP-ribose) polymerase (PARP) is a DNA-binding protein that is activated by nicks in the DNA molecule. It regulates the activity of various enzymes, including itself, that are involved in the control of DNA metabolism. Evidence obtained with both benzamide and isoquinolinone PARP inhibitors and the PARP-1(-/-) phenotype, clearly indicate that PARP plays an important role in NO/ROS-induced cell damage during inflammation, ischaemia and neurodegeneration. PARP is involved in the maintenance of genomic stability and PARP inhibition may also potentiate the cytotoxic action of agents used in cancer therapy. Benzamides, although not very potent (IC50 ~ 20 – 50 μM) PARP inhibitors, have been widely used to probe PARP functions, because of their lack of toxicity both in vitro and in vivo, even at high doses. In the early 1990s, a new class of very potent PARP inhibitors (i.e., at least 100-fold more potent thatn benzamide), the dihydroisoquinolinones, benzamide derivatives with the carbamoyl group constrained into the antiorientation, was discovered. At the same time, a large structure–activity surevey identified over 13 chemical classes of PARP inhibitors, the most potent calss sharing a common structural feature, the presence of a carbonyl group built into a polyaromatic heterocyclic skeleton or a carbamoyl group attached to an aromatic ring. Recently, a better knowledge of the PARP catalytic domain and the use of its crystal structure have led to the design and synthesis of the tricyclic lactam indoles, active at low nanomolar concentrations, and with favourable physical properties and in vivo characteristics. In the last few years the interest in PARP as a therapeutic target has been rapidly growing. This article reviews the patents filed for new PARP inhibitors over the last three years, up to February 2002, and their development status.
Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA-binding protein, which is primarily activated by nicks in the DNA molecule. It regulates the activity of various enzymes, including itself, and those involved in the control of DNA metabolism. Upon binding to DNA breaks, activated PARP cleaves NAD
+ into nicotinamide and ADP-ribose and polymerizes the latter on nuclear acceptor proteins including histones, transcription factors, and PARP itself. Poly(ADP-ribosylation) contributes to DNA repair and to the maintenance of genomic stability. Evidence obtained with pharmacological PARP inhibitors of various structural classes, as well as animals lacking the PARP-1 enzyme indicate that PARP plays an important role in cerebral ischemia/reperfusion, stroke, neurotrauma, neuroinjury, and neurodegeneration. Overactivation of PARP consumes NAD
+ and ATP culminating in cell dysfunction and necrosis. PARP activation can also act as a signal that initiates cell death programs, for instance through apoptosis-inducing factor (AIF) translocation. PARP has also been shown to associate with and regulate the function of several transcription factors. Of special interest is the enhancement by PARP of nuclear factor (NF)-κB-mediated transcription, which plays a central role in the expression of inflammatory cytokines, chemokines, adhesion molecules, and inflammatory mediators. Via this mechanism, PARP is involved in the upregulation of numerous proinflammatory genes that play a pathogenetic role in the later stage of central nervous system (CNS) diseases. Here, we review the roles of PARP in DNA damage signaling and cell death and summarize the pathogenetic role of PARP in neuroinflammation and neuroinjury.
Poly(ADP-ribosyl)ation is the post-translational modification of proteins operated by poly(ADP-ribose) polymerases (PARPs).
PARPs are enzymes that are able to catalyze the transfer of ADP-ribose units from nicotinamide adenine dinucleotide (NAD)
to target proteins and are particularly abundant in cell nuclei, where they play a key role in the maintenance of homeostasis.
Poly(ADP-ribosyl)ation significantly affects protein functioning because of the high negative charge and steric hindrance
conferred by the chains of poly(ADP-ribose) (PAR). PARP-1 is the founding member and the most commonly studied of these enzymes
and shows the highest poly(ADP-ribosyl)ating activity. Sequences encoding novel PARPs have been identified and, overall, the
PARP superfamily is a growing family of enzymes with numerous members with roles that are yet to be identified (Ame et al.
2004; Smith 2001).
Neuronal cell death is a critical physiological process necessary for the normal development of the CNS, as well as essential
for removing dysfunctional cells after injury or other various pathological conditions. However, excessive or inappropriate
neuronal cell loss is also a hallmark of acute or chronic neurodegeneration (Eldadah and Faden 2000; Snider et al. 1999; Graeber
and Moran 2002; Honig and Rosenberg 2000). Many cell death effector pathways are common to both physiological and pathophysiological
processes. Elucidating such mechanisms and initiating signals is essential for understanding neurodegeneration and designing
effective therapies.
Neuronal injury resulting from glutamate receptor-mediated excitotoxicity has been implicated in a wide spectrum of neurological
disorders. Following dramatic results in the preclinical setting, anti-excitotoxic neuroprotective agents have been used in
clinical trials for stroke and head injury, but the results have generally been unsuccessful. Hence, alternative targets in
the excitotoxic cascade appear to be required. Poly(ADP-ribosyl)ation has been linked to the pathogenesis of numerous disorders
of the CNS, including excitotoxicity and ischemic injury. A presumed cascade of glutamate receptor activation leading to excessive
free radical formation, DNA damage and then overactivation of PARP-1 is based on studies with drugs that block these various
steps. Along this classical view, experiments in our laboratory have shown that the intracellular depletion of ATP and NAD
induced by PARP-1 overactivation leads to necrotic cell death in ischemic and excitotoxic models and that PARP-1 inhibitors
are protective against necrotic but not apoptotic neuronal death. Therefore, it appears reasonable to propose PARP-1 inhibitors
as useful therapeutic agents in pathological brain conditions where necrosis predominates.
Poly(ADP-ribose) polymerases (PARPs) are enzymes that catalyze the transfer of ADP-ribose units from β-nicotinamide adenine dinucleotide (NAD(+)) to acceptor proteins. PARP-1 is responsible for more than 90 % of protein poly-ADP-ribosylation in the brain and may play a role as a molecular switch for cell survival and death. The functional roles of PARP-1 are largely mediated by its activation after binding to damaged DNA. Upon binding, PARP-1 activity increases rapidly and cleaves NAD(+) into ADP-ribose and nicotinamide. Increased activity of PARP-1 can promote DNA repair and its interaction with several transcription factors, whereas hyperactivation of PARP-1 can result in poly(ADP-ribose) accumulation and depletion of NAD(+) and ATP which may lead to caspase independent apoptotic or necrotic cell death, respectively. Excessive PARP-1 activity has been implicated in the pathogenesis of numerous clinical conditions such as stroke, myocardial infarction, inflammation, diabetes, and neurodegenerative disorders. Therefore, it is not surprising that the search for PARP-1 inhibitors with specific therapeutic uses (e.g., brain ischemia, cancer) has been an active area of research. Beyond medicinal uses, naturally occurring PARP-1 inhibitors may also offer a unique preventative means at attenuating chronic inflammatory diseases through dietary supplementation. This possibility has prompted research for specific, naturally occurring inhibitors of PARP-1. Though fewer investigations focus on identifying endogenous inhibitors/modulators of PARP-1 activity in vivo, these activities are very important for better understanding the complex regulation of this enzyme and the potential long-term benefits of supplementation with PARP-1 inhibitors. With this in mind, the focus of this article will be on providing a state-of-the-science review on endogenous and naturally occurring compounds that inhibit PARP-1.
Poly(ADP-ribose) polymerase (PARP) can initiate an energy-consuming and inefficient repair cycle following cerebral ischemia/reperfusion by transferring ADP ribose units to nuclear proteins eventually leading to cellular dysfunction and neuronal death. 3-Aminobenzamide (3-AB) is a selective inhibitor of PARP that can significantly reduce brain damage after focal ischemia in rats and displays a low toxicity in vivo. The goals of this study were to determine if inhibiting PARP with 3-AB has a long-term neuroprotective effect and if functional outcome improves in rats following focal ischemia and treatment with 3-AB. Focal ischemia was induced by a 2-h occlusion of the middle cerebral artery (MCA), using an intraluminal filament. Motor functions were evaluated from 5 to 28 days after reperfusion in four groups of rats: stroke without treatment; stroke treated with 3-AB at doses of 15 mg/kg, stroke treated with 3-AB at doses of 55 mg/kg; and the non-ischemic control rats. Functional behaviors were tested by a series of motor function tasks (foot placing, parallel bar crossing, rope and ladder climbing), as well as a neurological examination. Infarct volume of stroke brain in the same rat was determined by Nissl staining 28 days after surgery. Comparison of the untreated stroke group (n=11) and the treated stroke groups indicates that impairment of motor function was significantly (P<0.001) reduced by administration of 3-AB at doses of 15 mg/kg (n=9) or 55 mg/kg (n=10). Neurological outcome was also improved significantly (P<0.001). Infarct volume was significantly (P<0.01) reduced in both treated groups. Long-term neuroprotection following ischemia/reperfusion injury to the brain can be obtained by administration of a PARP inhibitor. The motor tests employed in this study can be used as sensitive, objective and reproducible measurements of functional impairment in rats following an ischemic stroke.
An accurate, reproducible method for determining the infarct volumes of gray matter structures is presented for use with presently available image analysis systems. Areas of stained sections with optical densities above that of a threshold value are automatically recognized and measured. This eliminates the potential error and bias inherent in manually delineating infarcted regions. Moreover, the volume of surviving normal gray matter is determined rather than that of the infarct. This approach minimizes the error that is introduced by edema, which distorts and enlarges the infarcted tissue and surrounding white matter.Keywords: Brain; Infarction; Nissl stain; Succinate dehydrogenase
Nitric oxide (NO) mediates several biological actions, including relaxation of blood vessels, cytotoxicity of activated macrophages, and formation of cGMP by activation of glutamate receptors in cerebellar slices. Nitric oxide synthase (EC 1.14.23.-) immunoreactivity is colocalized with nicotinamide adenine di-nucleotide phosphate diaphorase in neurons that are uniquely resistant to toxic insults. We show that the nitric oxide synthase inhibitors, N omega-nitro-L-arginine (EC50 = 20 microM) and N omega-monomethyl-L-arginine (EC50 = 170 microM), prevent neurotoxicity elicited by N-methyl-D-aspartate and related excitatory amino acids. This effect is competitively reversed by L-arginine. Depletion of the culture medium of arginine by arginase or arginine-free growth medium completely attenuates N-methyl-D-aspartate toxicity. Sodium nitroprusside, which spontaneously releases NO, produces dose-dependent cell death that parallels cGMP formation. Hemoglobin, which complexes NO, prevents neurotoxic effects of both N-methyl-D-aspartate and sodium nitroprusside. These data establish that NO mediates the neurotoxicity of glutamate.
Superoxide dismutase and catalase enzymatically scavenge superoxide and hydrogen peroxide, respectively. Conjugation of polyethylene glycol to superoxide dismutase (PEG-SOD) or catalase (PEG-CAT) prolongs the circulatory half-life of the native enzymes and enhances their intracellular access. We studied the protective effect of these free radical scavengers on ischemic brain injury using a rat model of focal cerebral ischemia, which is suitable for therapeutic trials. Intravenous administration of PEG-SOD (10,000 U/kg) and PEG-CAT (10,000 U/kg) before ischemia reduced the infarct volume (treatment, 139 +/- 9 mm3, means +/- SE, N = 38; placebo, 182 +/- 8 mm3, n = 37, P less than 0.002). This finding supports the concept that superoxide and hydrogen peroxide contribute to brain injury following focal cerebral ischemia.
Nitric oxide, which mediates influences of numerous neurotransmitters and modulators on vascular smooth muscle and leukocytes, can be formed in the brain from arginine by an enzymatic activity that stoichiometrically generates citrulline. We show that glutamate and related amino acids, such as N-methyl-D-aspartate, markedly stimulate arginine--citrulline transformation in cerebellar slices stoichiometrically with enhancement of cGMP levels. N omega-monomethyl-L-arginine blocks the augmentation both of citrulline and cGMP with identical potencies. Arginine competitively reverses both effects of N omega-monomethyl-L-arginine with the same potencies. Hemoglobin, which complexes nitric oxide, prevents the stimulation by N-methyl-D-aspartate of cGMP levels, and superoxide dismutase, which elevates nitric oxide levels, increases cGMP formation. These data establish that nitric oxide mediates the stimulation by glutamate of cGMP formation.
H2O2, in concentrations achieved in the proximity of stimulated leukocytes, induces injury and lysis of target cells. This may be an important aspect of inflammatory injury of tissues. Cell lysis in two target cells, the murine macrophage-like tumor cell line P388D1 and human peripheral lymphocytes, was found to be associated with activation of poly(ADP-ribose) polymerase (EC 2.4.2.30), a nuclear enzyme. This enzyme is activated under various conditions of DNA damage. Poly(ADP-ribose) polymerase utilizes nicotinamide adenine dinucleotide (NAD) as substrate and has been previously shown to consume NAD during exposure of cells to oxidants that was associated with inhibition of glycolysis, a decrease in cellular ATP, and cell death. In the current studies, inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide, nicotinamide, or theophylline in cells exposed to lethal concentrations of H2O2 prevented the sequence of events that eventually led to cell lysis--i.e., the decrease in NAD, followed by depletion of ATP, influx of extracellular Ca2+, actin polymerization and, finally, cell death. DNA damage, the initial stimulus for poly(ADP-ribose) polymerase activation, occurred despite the inhibition of this enzyme. Cells exposed to oxidant in the presence of the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide failed to demonstrate repair of DNA strand breaks.
Poly(ADP-ribose) polymerase (PARP) plays an important role in a number of cellular processes including DNA repair. Since poly(ADP-ribosyl)ation occurs in response to radiation- or drug-induced DNA damage, inhibitors of the enzyme may enhance the antitumour activity of radiotherapy or cytotoxic drug treatment. In this review the development of PARP inhibitors is discussed, and structure-activity relationships amongst inhibitors of the enzyme are presented. Studies to date regarding the in vitro and in vivo activity of PARP inhibitors, as resistance modifying agents in cancer therapy, are also overviewed.
In addition to mediating several physiological functions, nitric oxide (NO) has been implicated in the cytotoxicities observed following activation of macrophages or excess stimulation of neurons by glutamate. We extend our previous observations of glutamate-stimulated, NO-mediated neurotoxicity in primary cultures of rat fetal cortical, striatal, and hippocampal neurons. Neurotoxicity elicited by either NMDA or sodium nitroprusside (SNP) exhibits a similar concentration-effect relationship and time course. The concentration-effect curve of NMDA-induced neurotoxicity is shifted to the right in the presence of nitro-L-arginine and farther to the right in arginine-free media. The rank order of potency of several NO synthase (NOS) inhibitors in preventing neurotoxicity is the same as the rank order of these compounds in inhibiting NOS, and this inhibition is stereospecific. NMDA neurotoxicity is also prevented by flavoprotein inhibitors and calmodulin inhibitors, fitting with the roles of flavoproteins and calmodulin as NOS regulators. 8-Bromo-cGMP and guanylyl cyclase inhibitors do not affect neurotoxicity, while superoxide dismutase attenuates neurotoxicity. NOS neurons appear to be the source of neurotoxic NO in culture, as lesions of these neurons with 20 microM quisqualate diminish subsequent NMDA neurotoxicity. Moreover, NMDA neurotoxicity develops over time in culture coincident with the expression of NOS. Immunohistochemical localization of NOS in cultures and intact brain demonstrates widespread distribution of the cell processes suggesting that NOS neurons contact the majority of cortical neurons and so could mediate widespread neurotoxicity.
Activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) is an early response of cells exposed to DNA-damaging compounds such as nitric oxide (NO) or reactive oxygen intermediates (ROI). Excessive poly-(ADP-ribose) formation by PARP has been assumed to deplete cellular NAD+ pools and to induce the death of several cell types, including the loss of insulin-producing islet cells in type I diabetes. In the present study we used cells from mice with a disrupted and thus inactivated PARP gene to provide direct evidence for a causal relationship between PARP activation, NAD+ depletion, and cell death. We found that mutant islet cells do not show NAD+ depletion after exposure to DNA-damaging radicals and are more resistant to the toxicity of both NO and ROI. These findings directly prove that PARP activation is responsible for most of the loss of NAD+ following such treatment. The ADP-ribosylation inhibitor 3-aminobenzamide partially protected islet cells with intact PARP gene but not mutant cells from lysis following either NO or ROI treatment. Hence the protective action of 3-aminobenzamide must be due to inhibition of PARP and does not result from its other pharmacological properties such as oxygen radical scavenging. Finally, the use of mutant cells an alternative pathway of cell death was discovered which does not require PARP activation and NAD+ depletion. In conclusion, the data prove the causal relationship of PARP activation and subsequent islet cell death and demonstrate the existence of an alternative pathway of cell death independent of PARP activation and NAD+ depletion.
The free radicals nitric oxide and superoxide anion react to form peroxynitrite (ONOO-), a highly toxic oxidant species. In vivo formation of ONOO- has been demonstrated in shock and inflammation. Herein we provide evidence that cytotoxicity in cells exposed to ONOO- is mediated by DNA strand breakage and the subsequent activation of the DNA repair enzyme poly(ADP ribose) synthetase (PARS). Exposure to ONOO- (100 microM to 1 mM) inhibited mitochondrial respiration in cultured J774 macrophages and in rat aortic smooth muscle cells. The loss of cellular respiration was rapid, peaking 1-3 h after ONOO- exposure, and reversible, with recovery after a period of 6-24 h. The inhibition of mitochondrial respiration was paralleled by a dose-dependent increase in DNA strand breakage, reaching its maximum at 20-30 min after exposure to ONOO-. We observed a dose-dependent increase in the activity of PARS in cells exposed to ONOO-. Inhibitors of PARS such as 3-aminobenzamide (1 mM) prevented the inhibition of cellular respiration in cells exposed to ONOO-. Activation of PARS by ONOO--mediated DNA strand breakage resulted in a significant decrease in intracellular energy stores, as reflected by a decline of intracellular NAD+ and ATP content. 3-Aminobenzamide prevented the loss of NAD+ and ATP in cells exposed to ONOO-. In contrast, impairment of cellular respiration by the addition of the nitric oxide donors S-nitroso-N-acetyl-DL-penicillamine or diethyltriamine nitric oxide complex, was not associated with the development of DNA strand breaks, in concentrations up to 1 mM, and was largely refractory to PARS inhibition. Our results suggest that DNA damage and activation of PARS, an energy-consuming futile repair cycle, play a central role in ONOO--mediated cellular injury.
Reperfusion of the ischemic myocardium results in the generation of oxygen-derived free radicals, NO, and presumably peroxynitrite. These, in turn, may cause strand breaks in DNA, which activate the nuclear enzyme poly(ADP ribose) synthetase (PARS). This results in a rapid depletion of intracellular NAD and ATP. When this reaction is excessive, there is ultimately cell death. Here we demonstrate that 3-aminobenzamide (and several other, chemically distinct, inhibitors of PARS activity) reduces the infarct size caused by ischemia and reperfusion of the heart or skeletal muscle of the rabbit. Inhibition of PARS activity also attenuates the myocardial dysfunction caused by global ischemia and reperfusion in the isolated, perfused heart of the rabbit. In skeletal muscle, inhibition of the activity of neuronal NO synthase reduces infarct size, indicating that the formation of NO contributes to the activation of PARS there. There is no significant neuronal NO synthase activity in the heart, and hence NO synthase inhibitors did not reduce myocardial infarct size. Thus, activation of PARS contributes to the cell death caused by ischemia-reperfusion, and PARS inhibitors may constitute a novel therapy for ischemia-reperfusion injury.
The objective of this study was to determine whether nitric oxide (NO) is responsible for the vascular smooth muscle relaxation elicited by endothelium-derived relaxing factor (EDRF). EDRF is an unstable humoral substance released from artery and vein that mediates the action of endothelium-dependent vasodilators. NO is an unstable endothelium-independent vasodilator that is released from vasodilator drugs such as nitroprusside and glyceryl trinitrate. We have repeatedly observed that the actions of NO on vascular smooth muscle closely resemble those of EDRF. In the present study the vascular effects of EDRF released from perfused bovine intrapulmonary artery and vein were compared with the effects of NO delivered by superfusion over endothelium-denuded arterial and venous strips arranged in a cascade. EDRF was indistinguishable from NO in that both were labile (t1/2 = 3-5 sec), inactivated by pyrogallol or superoxide anion, stabilized by superoxide dismutase, and inhibited by oxyhemoglobin or potassium. Both EDRF and NO produced comparable increases in cyclic GMP accumulation in artery and vein, and this cyclic GMP accumulation was inhibited by pyrogallol, oxyhemoglobin, potassium, and methylene blue. EDRF was identified chemically as NO, or a labile nitroso species, by two procedures. First, like NO, EDRF released from freshly isolated aortic endothelial cells reacted with hemoglobin to yield nitrosylhemoglobin. Second, EDRF and NO each similarly promoted the diazotization of sulfanilic acid and yielded the same reaction product after coupling with N-(1-naphthyl)-ethylenediamine. Thus, EDRF released from artery and vein possesses identical biological and chemical properties as NO.
Nitric oxide (NO) mediates several biological actions, including relaxation of blood vessels, cytotoxicity of activated macrophages, and formation of cGMP by activation of glutamate receptors in cerebellar slices. Nitric oxide synthase (EC 1.14.23.-) immunoreactivity is colocalized with nicotinamide adenine di-nucleotide phosphate diaphorase in neurons that are uniquely resistant to toxic insults. We show that the nitric oxide synthase inhibitors, N omega-nitro-L-arginine (EC50 = 20 microM) and N omega-monomethyl-L-arginine (EC50 = 170 microM), prevent neurotoxicity elicited by N-methyl-D-aspartate and related excitatory amino acids. This effect is competitively reversed by L-arginine. Depletion of the culture medium of arginine by arginase or arginine-free growth medium completely attenuates N-methyl-D-aspartate toxicity. Sodium nitroprusside, which spontaneously releases NO, produces dose-dependent cell death that parallels cGMP formation. Hemoglobin, which complexes NO, prevents neurotoxic effects of both N-methyl-D-aspartate and sodium nitroprusside. These data establish that NO mediates the neurotoxicity of glutamate.
We investigated the effect of nitric oxide (NO) upon CA1 neurons of the hippocampal slice. NO was given via perfusate without oxygen and with glucose concentration increased to 10 mM to prevent hypoxic injury. Exposure to NO for 10 min produced severe neuronal injury, with CA1 orthodromic and antidromic population spike regaining only 3 +/- 3% and 9 +/- 3% of initial amplitude after 1 h recovery. Hypoxic controls in contrast, showed orthodromic and antidromic recovery of 98 +/- 5% and 93 +/- 7%. Good protection from NO-induced injury was seen with 10 mM nicotinamide, an inhibitor of poly-ADP-ribosylation, with CA1 PS recovering to 116 +/- 10% orthodromically, and 96 +/- 4% antidromically. Protection was also seen with 3-aminobenzamide, another poly-ADP-ribosylation inhibitor, suggesting that poly-ADP-ribosylation may play an imoportant role in NO-Mediated neuronal injury.
A number of roles have been ascribed to the nuclear ADP-ribosyl transferase, including involvement in DNA repair, cellular differentiation, transformation and gene rearrangements and transpositions. In this brief article we concentrate on the unifying concept that the transferase is fundamentally involved in regulating cellular metabolism in response to DNA damage through modulation of cellular NAD+ pools. The ultimate contribution of the enzyme under conditions of extreme DNA damage may be to cause cell death.
Endothelium-derived relaxing factor (EDRF) is a labile humoral agent which mediates the action of some vasodilators. Nitrovasodilators, which may act by releasing nitric oxide (NO), mimic the effect of EDRF and it has recently been suggested by Furchgott that EDRF may be NO. We have examined this suggestion by studying the release of EDRF and NO from endothelial cells in culture. No was determined as the chemiluminescent product of its reaction with ozone. The biological activity of EDRF and of NO was measured by bioassay. The relaxation of the bioassay tissues induced by EDRF was indistinguishable from that induced by NO. Both substances were equally unstable. Bradykinin caused concentration-dependent release of NO from the cells in amounts sufficient to account for the biological activity of EDRF. The relaxations induced by EDRF and NO were inhibited by haemoglobin and enhanced by superoxide dismutase to a similar degree. Thus NO released from endothelial cells is indistinguishable from EDRF in terms of biological activity, stability, and susceptibility to an inhibitor and to a potentiator. We suggest that EDRF and NO are identical.
The abundant nuclear enzyme poly(ADP-ribose) polymerase catalyses the synthesis of poly(ADP-ribose) from nicotinamide adenine dinucleotide (NAD+). This protein has an N-terminal DNA-binding domain containing two zinc-fingers, which is linked to the C-terminal NAD(+)-binding domain by a short region containing several glutamic acid residues that are sites of auto-poly(ADP-ribosyl)ation. The intracellular production of poly(ADP-ribose) is induced by agents that generate strand interruptions in DNA. The branched homopolymer chains may attain a size of 200-300 residues but are rapidly degraded after synthesis. The function of poly(ADP-ribose) synthesis is not clear, although it seems to be required for DNA repair. Here we describe a human cell-free system that enables the role of poly(ADP-ribose) synthesis in DNA repair to be characterized. The results indicate that unmodified polymerase molecules bind tightly to DNA strand breaks; auto-poly(ADP-ribosyl)ation of the protein then effects its release and allows access to lesions for DNA repair enzymes.
The vascular endothelium is a significant site for tissue injury following exposure to reactive oxygen species derived from a number of sources. In order to develop a better understanding of the mechanism(s) of oxidative damage, monolayer cultures of endothelial cells obtained from bovine pulmonary arteries were exposed to reactive oxygen species generated from the oxidation of dihydroxyfumarate (DHF) to diketosuccinate. Exposure to oxidizing DHF caused a loss of cell membrane integrity that was delayed in onset; that is, it did not begin until 2 h after the addition of DHF although reactive oxygen species are produced immediately by DHF in solution. Endothelial cell lysis by DHF was prevented by the simultaneous addition of superoxide dismutase (SOD), catalase (CAT), or deferoximine (DFX). This oxidant-induced lysis was unaffected by N,N,-diphenyl-p-phenylenediamine (DPPD), a potent inhibitor of lipid peroxidation. However, simultaneous addition of 3-aminobenzamide (3AB) and nicotinamide (NA), inhibitors of poly(ADP-ribose) polymerase, prevented cell lysis. Oxidant-induced loss of membrane integrity was preceded by the early appearance of DNA strand breaks, by increased levels of poly(ADP-ribose), the product of polymerase activity, and by depletion of NAD+ and ATP, followed by a decline in the energy charge ratio of the cells. None of these intracellular changes occurred when either SOD, CAT, or DFX were added at the same time as DHF, suggesting that O2-., H2O2, and HO. mediated these changes. The O2-. appears to be important in the autoxidation reaction of DHF. The latter two reactive oxygen species may be part of cellular-catalyzed Fenton chemistry. The increase in poly(ADP-ribose), depletion of NAD+, and the decline in ATP were also prevented by the addition of 3AB. The oxidant-induced DNA strand breakage was, however, unaffected by either 3AB or NA. Addition of 3AB immediately prior to the onset of cell lysis (2 h after the addition of DHF), prevented cell lysis, i.e., "rescued" the cells when neither SOD, CAT, nor DFX addition were effective. Concurrent with the "rescue" from lysis by 3AB, there was an increase in NAD+ content and a return of the energy charge ratio to control levels. The data presented in this study suggests that in endothelial cells, DNA is a very sensitive target for reactive oxygen species and HO. is the likely proximal damaging species.(ABSTRACT TRUNCATED AT 400 WORDS)
Although hyperglycemia has been shown to consistently exacerbate ischemia brain injury following global or diffuse cerebral ischemia, the effect of hyperglycemia in unilateral focal cerebral ischemia remains controversial. Recent advances in thrombolytic therapy have enhanced the clinical significance of postischemic reperfusion. We studied the effect of plasma glucose on ischemic brain injury in a newly developed focal cerebral ischemia-reperfusion model. Rats allowed free access to food until ischemic insult developed intra- and postischemic hyperglycemia and cortical infarction. Rats fasted for 24 hours had blunted hyperglycemic responses. Infarct volumes were correspondingly smaller. The protective effect of fasting was partially abolished by glucose loading during ischemia to induce intra-ischemic hyperglycemia. Glucose loading immediately or 3 hours after focal cerebral ischemia did not significantly alter the protective effect of fasting. Insulin treatment in fed rats before ischemia also reduced hyperglycemic responses and infarct volume. Timing of insulin treatment was also critical in the reduction of ischemic injury. These findings indicate that plasma glucose during the period of ischemia is an important determinant of brain injury in focal cerebral ischemia-reperfusion and there is a therapeutic window for normalization of plasma glucose to be efficacious.
The expression level of poly(ADP-ribose) polymerase mRNA as well as the level of enzymatic activity were examined in various mouse organs by northern blot and activity gel analyses. High levels of the mRNA expression and enzymatic activity were observed in testis, thymus, spleen, and brain. On the other hand, low levels of the mRNA expression and enzymatic activity were observed in liver and kidney. These findings suggest that the expression of the poly(ADP-ribose) polymerase is mainly regulated by transcription. Striking similarity was observed between the patterns of organ distribution of enzymatic activities of poly(ADP-ribose) polymerase and DNA polymerase beta in various mouse organs.
The objective of this study was to determine whether nitric oxide (NO) is responsible for the vascular smooth muscle relaxation elicited by endothelium-derived relaxing factor (EDRF). EDRF is an unstable humoral substance released from artery and vein that mediates the action of endothelium-dependent vasodilators. NO is an unstable endothelium-independent vasodilator that is released from vasodilator drugs such as nitroprusside and glyceryl trinitrate. We have repeatedly observed that the actions of NO on vascular smooth muscle closely resemble those of EDRF. In the present study the vascular effects of EDRF released from perfused bovine intrapulmonary artery and vein were compared with the effects of NO delivered by superfusion over endothelium-denuded arterial and venous strips arranged in a cascade. EDRF was indistinguishable from NO in that both were labile (t1/2 = 3-5 sec), inactivated by pyrogallol or superoxide anion, stabilized by superoxide dismutase, and inhibited by oxyhemoglobin or potassium. Both EDRF and NO produced comparable increases in cyclic GMP accumulation in artery and vein, and this cyclic GMP accumulation was inhibited by pyrogallol, oxyhemoglobin, potassium, and methylene blue. EDRF was identified chemically as NO, or a labile nitroso species, by two procedures. First, like NO, EDRF released from freshly isolated aortic endothelial cells reacted with hemoglobin to yield nitrosylhemoglobin. Second, EDRF and NO each similarly promoted the diazotization of sulfanilic acid and yielded the same reaction product after coupling with N-(1-naphthyl)-ethylenediamine. Thus, EDRF released from artery and vein possesses identical biological and chemical properties as NO.
In the vascular system, endothelium-derived relaxing factor (EDRF) is the name of the local hormone released from endothelial cells in response to vasodilators such as acetylcholine, bradykinin and histamine. It diffuses into underlying smooth muscle where it causes relaxation by activating guanylate cyclase, so producing a rise in cyclic GMP levels. It has been known for many years that in the central nervous system (CNS) the excitatory neurotransmitter glutamate can elicit large increases in cGMP levels, particularly in the cerebellum where the turnover rate of cGMP is low. Recent evidence indicates that cell-cell interactions are involved in this response. We report here that by acting on NMDA (N-methyl-D-aspartate) receptors on cerebellar cells, glutamate induces the release of a diffusible messenger with strikingly similar properties to EDRF. This messenger is released in a Ca2+-dependent manner and its activity accounts for the cGMP responses that take place following NMDA receptor activation. In the CNS, EDRF may link activation of postsynaptic NMDA receptors to functional modifications in neighbouring presynaptic terminals and glial cells.
In the search for a more reproducible focal ischemic stroke model in the rat, we systematically interrupted blood flow to the right middle cerebral artery territory by ligating the right middle cerebral artery, and the right and left common carotid arteries in succession. Using a laser-Doppler flowmeter, we found that the relative surface blood flow in cerebral cortex supplied by the right middle cerebral artery decreased to 62, 48, and 18% of baseline respectively after successive ligation of the right middle cerebral artery, and the right and left common carotid arteries. A focal infarct in the cerebral cortex supplied by the right middle cerebral artery was consistently noted after ligation of the right middle cerebral and the right common carotid arteries and temporary clip occlusion of the left common carotid artery for 60 min. The surface areas of infarction measured 100 +/- 6 mm2 and the maximal cross-sectional area of infarction was 10.4 +/- 1.1 mm2 (N = 10). The mortality rate was 7% (N = 70). The characteristic changes of ischemic necrosis were limited to the cortex with sparing of subcortical structures. No motor deficits occurred. Occlusion of the right middle cerebral artery alone or together with the right common carotid artery did not consistently cause gross infarction and the maximal cross-sectional area of infarction was smaller (the right middle cerebral artery, 1.7 +/- 0.8 mm2, N = 10; the right middle cerebral artery plus the right common carotid artery, 4.8 +/- 1.9 mm2, N = 10). Permanent ligation of the right middle cerebral artery and both common carotid arteries had a high mortality (60% in 3 days, N = 10).(ABSTRACT TRUNCATED AT 250 WORDS)
The poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide had dramatically different effects on X-ray-induced cytogenetic damage in human lymphocytes depending on the stage of the cell cycle in which cells were irradiated. 3-Aminobenzamide (0.08-3.00 mM) potentiated the frequency of chromosomal aberrations when lymphocytes were irradiated in G1, S, or late G2. No effect was observed, however, when lymphocytes were irradiated in G0 or at the S/G2 boundary 6 h before termination of culture. These results indicate that poly(ADP-ribose) polymerase may be involved in chromosomal repair of radiation damage only during specific stages of the cell cycle.
Poly(ADP-ribose) polymerase is a chromatin-bound enzyme which, on activation by DNA strand breaks, catalyzes the successive transfer of ADP-ribose units from NAD to nuclear proteins. Poly(ADP-ribose) synthesis is stimulated by DNA strand breaks, and the polymer may alter the structure and/or function of chromosomal proteins to facilitate the DNA repair process. Electronmicroscopic studies show that poly(ADP-ribose) unwinds the tightly packed nucleosomal structure of isolated chromatin. Recent studies also show that the presence of poly(ADP-ribose) enhances the activity of DNA ligase. This may increase the capacity of the cell to complete DNA repair. Inhibitors of poly(ADP-ribose) polymerase or deficiencies of the substrate, NAD, lead to retardation of the DNA repair process. When DNA strand breaks are extensive or when breaks fail to be repaired, the stimulus for activation of poly(ADP-ribose) persists and the activated enzyme is capable of totally consuming cellular pools of NAD. Depletion of NAD and consequent lowering of cellular ATP pools, due to activation of poly(ADP-ribose) polymerase, may account for rapid cell death before DNA repair takes place and before the genetic effects of DNA damage become manifest.
The activity of poly(ADP-ribose) polymerase is stimulated by DNA damage resulting from treatment of cells with ionizing radiation, as well as with DNA-damaging chemicals. The elevated polymerase activity can be observed at doses lower than those necessary for measurable reduction in cellular NAD concentration (less than 20 Gy). Several nuclear proteins, including the polymerase itself, are poly(ADP-ribosylated) at elevated levels in irradiated Chinese hamster cells. The addition of inhibitors of poly(ADP-ribose) polymerase to irradiated cells has been found to sensitize the cells to the lethal effects of the radiation, to inhibit the repair of potentially lethal damage, and to delay DNA strand break rejoining. Because of the nonspecificity of the inhibitors, however, it is as yet unknown whether their effects are directly related to the inhibition of poly(ADP-ribose) polymerase, to interference with the poly(ADP-ribosylation) of one or more chromosomal proteins, or to effects unrelated to the poly(ADP-ribosylation) process. The data are consistent with the involvement of poly(ADP-ribose) in the repair of radiation damage, but the nature of this involvement remains to be elucidated.
The inter- and intracellular localization of poly(adenosine diphosphate-ribose)(poly(ADP-ribose] synthetase was investigated using an indirect immunofluorescence technique and a specific antibody against the enzyme purified from calf thymus. In various bovine tissues, including liver, heart, pancrease, thyroid, spleen, adrenal, and skeletal muscle, the specific immunofluorescence of poly(ADP-ribose) synthetase was localized exclusively in the nucleus. Immunostaining was inhibited by preabsorption of the antibody with purified calf thymus poly(ADP-ribose) synthetase. Nuclear immunofluorescence appeared to be more prominent in the marginal area than in the central region in most nuclei. This staining pattern is similar to that of naturally occurring poly(ADP-ribose). In bovine peripheral blood the immunofluorescence of poly(ADP-ribose) synthetase was detected in nuclei of lymphocytes, but not in granulocytes, in agreement with the finding that the enzymatic activity of poly(ADP-ribose) synthetase was barely detectable in nuclei isolated from granulocytes.
Natural distribution of poly(adenosine diphosphate-ribose), a novel macromolecule in eukaryotes, was investigated using an indirect immunofluorescence technique. The antibody, produced in a rabbit toward poly(ADP-ribose), was most reactive with polymers having the chain length of about 25 ADP-ribose units and weakly reactive with short oligomers; it was totally inert with monomers. Immunostaining with this antibody revealed the existence of the polymer in various rat tissues. The immunostaining seems to be specific for poly(ADP-ribose), as judged by its disappearance by preabsorption of the antiserum with purified poly(ADP-ribose) or pretreatment of tissue sections with poly(ADP-ribose)-degrading enzymes. Intensification of the fluorescence by preincubation with nicotinamide adenine dinucleotide (NAD), a substrate for poly(ADP-ribose) synthesis, also supported this view. The immunofluorescence of poly(ADP-ribose) was found exclusively in the nucleus of almost all tissues tested, including liver (adult, newborn, regenerating, and hepatoma), brain, heart, intestine, pancreas, kidney, spleen, testis, thyroid gland, and skeletal muscle. Exceptions were blood cells; little fluorescence was detectable in nuclei of peripheral leukocytes. Only after preincubation with NAD, did lymphocytes and monocytes exhibit fluorescence, however, granulocytes never did exhibit fluorescence. The cells appeared to represent the first instance where poly(ADP-ribose) synthesis activity among eukaryotic cells was missing.
DNA damage activates a nuclear enzyme poly(ADP-ribose) synthetase (PARS) that facilitates DNA repair by adding multiple ADP-ribose groups to nuclear proteins such as histones and PARS itself. N-Methyl-D-aspartate neurotoxicity may involve DNA damage excessively activating PARS to deplete its substrate NAD, as PARS inhibitors prevent this toxicity. We now show that PARS is rapidly and markedly activated in PC12 cells following treatment with neurotoxic agents, including the amyloid beta-protein, hydrogen peroxide, N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and its active metabolite N-methyl-4-phenylpyridine (MPP+). With MPP+, PARS activity is increased fivefold in 1 h and 20-fold by 3 h. By contrast, direct measurement of DNA damage by the terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay shows no significant increase by 3 h and less than fourfold by 24 h. These findings indicate that PARS activity can provide a simple, sensitive, and early index of DNA damage following neurotoxic insults.
Poly(ADP-ribosyl)ation is catalyzed by NAD+: protein(ADP-ribosyl) transferase (ADPRT), a chromatin-associated enzyme which, in the presence of DNA breaks, transfers ADP-ribose from NAD+ to nuclear proteins. This post-translational modification has been implicated in many fundamental processes, like DNA repair, chromatin stability, cell proliferation, and cell death. To elucidate the biological function of ADPRT and poly(ADP-ribosyl)ation in vivo the gene was inactivated in the mouse germ line. Mice homozygous for the ADPRT mutation are healthy and fertile. Analysis of mutant tissues and fibroblasts isolated from mutant fetuses revealed the absence of ADPRT enzymatic activity and poly(ADP-ribose), implying that no poly(ADP-ribosyl)ated proteins are present. Mutant embryonic fibroblasts were able to efficiently repair DNA damaged by UV and alkylating agents. However, proliferation of mutant primary fibroblasts as well as thymocytes following gamma-radiation in vivo was impaired. Moreover, mutant mice are susceptible to the spontaneous development of skin disease as approximately 30% of older mice develop epidermal hyperplasia. The generation of viable ADPRT-/-mice negates an essential role for this enzyme in normal chromatin function, but the impaired proliferation and the onset of skin lesions in older mice suggest a function for ADPRT in response to environmental stress.
Nitric oxide (NO) has been implicated as an immunological effector molecule that mediates beta-cell dysfunction associated with Type 1 diabetes. To assess whether NO induces poly(ADP-ribose) synthesis in islet cells, we examined the effect of nitroprusside on islet cells. The exposure of mouse islet cells and a beta-cell line (beta TC1) to 0.05-0.2 mM nitroprusside resulted in the reduction of intracellular nicotinamide adenine dinucleotide (NAD) levels. Nitroprusside stimulated poly(ADP-ribose) synthetase activity in beta TC1 cells. An inhibitor of poly(ADP-ribose) synthetase, 3-aminobenzamide, prevented both NAD decrease and poly ADP-ribosylation. These observations suggest that NO-induced pancreatic beta-cell damage may be ascribable to the activation of poly(ADP-ribose) synthetase that results in the decrease of NAD content.
Glutamate neurotoxicity is correlated with an increase of cytosolic free Ca2+. In some cell systems, activation of Ca2+ dependent endonucleases or formation of free radicals can damage DNA and activate the chromatin bound enzyme poly(ADP-ribose) polymerase (pADPRP). We have investigated whether pADPRP may be involved in glutamate neurotoxicity in vitro. Cerebellar granule cells at 12 days in culture when treated with a toxic dose of glutamate (100 microM) showed a rapid and transient increase of polyADP-ribose immunoreactivity. Cellular immunostaining was heterogeneous and returned to control levels after washout of glutamate. In the same cell preparations glutamate elicited a marked increase in enzyme protein immunoreactivity which persisted at later times. Non-toxic doses of glutamate did not affect immunostaining. In another set of experiments, pADPRP mRNA was increased 30 min after glutamate. In order to investigate the role of pADPRP in glutamate-mediated neurotoxicity, structurally different inhibitors of pADPRP (3-aminobenzamide, benzamide,3-aminophthalhydrazide) and their inactive analogues (benzoic acid and phthalimide) were tested in this model. Addition of the inhibitors to cultures 60 min before and during the 30 min of glutamate treatment prevented neuronal death by 60-100%, assessed 24 hr later. Glutamate-induced Ca2+ influx was not affected. Inactive analogues failed to afford neuroprotection. These data indicate that not only is pADPRP activated by the early, possibly Ca(2+)-mediated mechanisms initiated by glutamate, but that it might also actively contribute to the subsequent neuronal death.
Poly(adenosine 5'-diphosphoribose) synthetase (PARS) is a nuclear enzyme which, when activated by DNA strand breaks, adds up to 100 adenosine 5'-diphosphoribose (ADP-ribose) units to nuclear proteins such as histones and PARS itself. This activation can lead to cell death through depletion of beta-nicotinamide adenine dinucleotide (the source of ADP-ribose) and adenosine triphosphate. Nitric oxide (NO) stimulated ADP-ribosylation of PARS in rat brain. Benzamide and other derivatives, which inhibit PARS, blocked N-methyl-D-aspartate- and NO-mediated neurotoxicity with relative potencies paralleling their ability to inhibit PARS. Thus, NO appeared to elicit neurotoxicity by activating PARS.
Previous studies have shown that DNA strand breaks are an early consequence of nitric oxide toxicity in pancreatic islet cells. We show here that exposure of islet cells to chemical NO donors causes the formation of ADP-ribose polymers in cell nuclei, with concomitant depletion of intracellular NAD+. Islet cell lysis was largely prevented by the ADP-ribosylation inhibitors nicotinamide, 3-aminobenzamide, and 4-amino-1,8-naphthalimide, the latter being a potent new-generation compound with high selectivity for poly(ADP-ribosyl)-ation. These findings indicate a key role of poly(ADP-ribose) polymerase activation in NO toxicity in islet cells.
Reactive oxygen metabolites have an important role in ischemia-reperfusion injury. One of the sources of reactive oxygen metabolites is xanthine oxidase, which is present in several tissues but is also released into the circulation after ischemia. We studied the effect of several potentially protective compounds on adenine nucleotide depletion induced by extracellular xanthine oxidase and hypoxanthine, in concentrations relevant to human pathophysiology. In umbilical vein endothelial cells prelabeled with 14C-adenine, cellular adenine nucleotides retained 64 +/- 9% of the initial radioactivity over a 4-h incubation with culture medium (controls), whereas in the presence of xanthine oxidase (80 mU/mL) and hypoxanthine (100 microM), only 3 +/- 4% of radioactivity remained in cellular nucleotides, the rest appearing in catabolic products in the medium. Glutathione and 3-aminobenzamide, an inhibitor of poly-ADP-ribose polymerase, partly prevented the nucleotide depletion (adenine nucleotide radioactivity 15 +/- 6% to 33 +/- 13% of total), but scavengers of the hydroxyl radical, dimethylthiourea and DMSO, as well as vitamins E and C, were without effect. Superoxide dismutase prevented the leakage of nucleotides into the culture medium but not intracellular nucleotide catabolism, whereas the latter process was decreased by catalase, consistent with predominant effects of superoxide and hydrogen peroxide at the cell membrane and interior, respectively.
We investigated the effect of nitric oxide (NO) upon CA1 neurons of the hippocampal slice. NO was given via perfusate without oxygen and with glucose concentration increased to 10 mM to prevent hypoxic injury. Exposure to NO for 10 min produced severe neuronal injury, with CA1 orthodromic and antidromic population spike regaining only 3 +/- 3% and 9 +/- 3% of initial amplitude after 1 h recovery. Hypoxic controls in contrast, showed orthodromic and antidromic recovery of 98 +/- 5% and 93 +/- 7%. Good protection from NO-induced injury was seen with 10 mM nicotinamide, an inhibitor of poly-ADP-ribosylation, with CA1 PS recovering to 116 +/- 10% orthodromically, and 96 +/- 4% antidromically. Protection was also seen with 3'-aminobenzamide, another poly-ADP-ribosylation inhibitor, suggesting that poly-ADP-ribosylation may play an important role in NO-mediated neuronal injury.
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