Mitochondrial dysfunction is involved in P2X7 receptor-mediated neuronal cell death

J. Neurochem. (2012) 122, 1118-1128. ABSTRACT: P2X7 receptor (P2X7R) is known to be a 'death receptor' in immune cells, but its functional expression in non-immune cells such as neurons is controversial. Here, we examined the involvement of P2X7R activation and mitochondrial dysfunction in ATP-induced neuronal death in cultured cortical neurons. In P2X7R- and pannexin-1-expressing neuron cultures, 5 or more mM ATP or 0.1 or more mM BzATP induced neuronal death including apoptosis, and cell death was prevented by oxATP, P2X7R-selective antagonists. ATP-treated neurons exhibited Ca(2+) entry and YO-PRO-1 uptake, the former being inhibited by oxATP and A438079, and the latter by oxATP and carbenoxolone, while P2X7R antagonism with oxATP, but not pannexin-1 blocking with carbenoxolone, prevented the ATP-induced neuronal death. The ATP treatment induced reactive oxygen species generation through activation of NADPH oxidase and activated poly(ADP-ribose) polymerase, but both of them made no or negligible contribution to the neuronal death. Rhodamine123 efflux from neuronal mitochondria was increased by the ATP-treatment and was inhibited by oxATP, and a mitochondrial permeability transition pore inhibitor, cyclosporine A, significantly decreased the ATP-induced neuronal death. In ATP-treated neurons, the cleavage of pro-caspase-3 was increased, and caspase inhibitors, Q-VD-OPh and Z-DEVD-FMK, inhibited the neuronal death. The cleavage of apoptosis-inducing factor was increased, and calpain inhibitors, MDL28170 and PD151746, inhibited the neuronal death. These findings suggested that P2X7R was functionally expressed by cortical neuron cultures, and its activation-triggered Ca(2+) entry and mitochondrial dysfunction played important roles in the ATP-induced neuronal death.     

The role of NADPH oxidase,neuronal nitric oxide synthase and poly(ADP ribose) polymerase in oxidative neuronal death induced in cortical cultures by brain-derived neurotrophic factor and neurotrophin-4/5

Certain neurotrophins promote or induce oxidative neuronal death in cortical cultures. However, the effector mechanisms mediating this phenomenon have not been delineated. In this study, we investigated the possibility that NADPH oxidase and nitric oxide synthase (NOS) function as such effectors. Western blot analysis showed that treatment with brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)-4/5 increased the levels of NADPH oxidase subunits. Moreover, neurotrophin treatment resulted in membrane translocation of p67phox, a characteristic feature of NADPH oxidase activation. Administration of the specific NADPH oxidase inhibitor, 4-(2-aminoethyl)benzenesulfonylfluoride (AEBSF), attenuated increases in oxygen free radicals thereby suggesting that NADPH oxidase contributes to the oxidative stress induced by neurotrophins. Furthermore, neuronal death induced by BDNF or NT-4/5 was significantly attenuated by AEBSF. Treatment with BDNF has previously been shown to induce neuronal NOS (nNOS). Our data indicated that inhibitors of nNOS attenuated neuronal death induced by BDNF or NT-4/5, consistent with an active role of nNOS in the mediation of neurotrophin neurotoxicity. As in other models of oxidative cell death, BDNF-induced neuronal death was accompanied by poly(ADP ribose) polymerase (PARP) activation. AEBSF or N-nitro-l-arginine (NNA) reduced BDNF-mediated PARP activation. PARP and poly(ADP ribose) glycohydrolase (PARG) are actively involved in mediating neurotrophin neurotoxicity since inhibitors of PARP and PARG significantly reduced levels of cell death. These results suggest that NADPH oxidase and nNOS contribute to increased oxidative stress, subsequent activation of PARP/PARG, and neuronal death induced by prolonged neurotrophin exposure.     

Dietary folate deficiency and elevated homocysteine levels endanger dopaminergic neurons in models of Parkinson's disease.

Although the cause of Parkinson's disease (PD) is unknown, data suggest roles for environmental factors that may sensitize dopaminergic neurons to age-related dysfunction and death. Based upon epidemiological data suggesting roles for dietary factors in PD and other age-related neurodegenerative disorders, we tested the hypothesis that dietary folate can modify vulnerability of dopaminergic neurons to dysfunction and death in a mouse model of PD. We report that dietary folate deficiency sensitizes mice to MPTP-induced PD-like pathology and motor dysfunction. Mice on a folate-deficient diet exhibit elevated levels of plasma homocysteine. When infused directly into either the substantia nigra or striatum, homocysteine exacerbates MPTP-induced dopamine depletion, neuronal degeneration and motor dysfunction. Homocysteine exacerbates oxidative stress, mitochondrial dysfunction and apoptosis in human dopaminergic cells exposed to the pesticide rotenone or the pro-oxidant Fe(2+). The adverse effects of homocysteine on dopaminergic cells is ameliorated by administration of the antioxidant uric acid and by an inhibitor of poly (ADP-ribose) polymerase. The ability of folate deficiency and elevated homocysteine levels to sensitize dopaminergic neurons to environmental toxins suggests a mechanism whereby dietary folate may influence risk for PD.     

Involvement of the proteasome in the programmed cell death of NGF-deprived sympathetic neurons.

Sympathetic neurons undergo programmed cell death (PCD) upon deprivation of nerve growth factor (NGF). PCD of neurons is blocked by inhibitors of the interleukin-1beta converting enzyme (ICE)/Ced-3-like cysteine protease, indicating involvement of this class of proteases in the cell death programme. Here we demonstrate that the proteolytic activities of the proteasome are also essential in PCD of neurons. Nanomolar concentrations of several proteasome inhibitors, including the highly selective inhibitor lactacystin, not only prolonged survival of NGF-deprived neurons but also prevented processing of poly(ADP-ribose) polymerase which is known to be cleaved by an ICE/Ced-3 family member during PCD. These results demonstrate that the proteasome is a key regulator of neuronal PCD and that, within this process, it is involved upstream of proteases of the ICE/Ced-3 family. This order of events was confirmed in macrophages where lactacystin inhibited the proteolytic activation of precursor ICE and the subsequent generation of active interleukin-1beta.     

Kynurenine 3-mono-oxygenase inhibitors attenuate post-ischemic neuronal death in organotypic hippocampal slice cultures

Kynurenine 3-mono-oxygenase (KMO) inhibitors reduce 3-hydroxykynurenine (3-HK) and quinolinic acid (QUIN) neosynthesis and facilitate kynurenine metabolism towards kynurenic acid (KYNA) formation. They also reduce tissue damage in models of focal or transient global cerebral ischemia in vivo. We used organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation (OGD) to investigate KMO mechanism(s) of neuroprotective activity. Exposure of the slices to 30 min of OGD caused CA1 pyramidal cell death and significantly decreased the amount of KYNA released in the incubation medium. The KMO inhibitors (m-nitrobenzoyl)-alanine (30-100 micro m) or 3,4-dimethoxy-[-N-4-(nitrophenyl)thiazol-2yl]-benzenesulfonamide (1-10 micro m) reduced post-ischemic neuronal death and increased KYNA concentrations in slice incubation media. The maximal concentration of KYNA detected in the incubation media of slices treated with KMO inhibitors was approximately 50 nm and was too low to efficiently interact with alpha7 nicotinic acetylcholine receptors or with the glycineb site of N-methyl-d-aspartate (NMDA) receptors. On the other hand, the addition of either 3-HK or QUIN (1-10 micro m) to OGD-exposed hippocampal slices prevented the neuroprotective activity of KMO inhibitors. Our results suggest that KMO inhibitors reduce the neuronal death found in the CA1 region of organotypic hippocampal slices exposed to 30 min of OGD by decreasing the local synthesis of 3-HK and QUIN.     

Glia Modulate NMDA-Mediated Signaling in Primary Cultures of Cerebellar Granule Cells

Abstract: Excessive activation of N-methyl-d -aspartate (NMDA) receptor channels (NRs) is a major cause of neuronal death associated with stroke and ischemia. Cerebellar granule neurons in vivo, but not in culture, are relatively resistant to toxicity, possibly owing to protective effects of glia. To evaluate whether NR-mediated signaling is modulated when developing neurons are cocultured with glia, the neurotoxic responses of rat cerebellar granule cells to applied NMDA or glutamate were compared in astrocyte-rich and astrocyte-poor cultures. In astrocyte-poor cultures, significant neurotoxicity was observed in response to NMDA or glutamate and was inhibited by an NR antagonist. Astrocyte-rich neuronal cultures demonstrated three significant differences, compared with astrocyte-poor cultures: (a) Neuronal viability was increased; (b) glutamate-mediated neurotoxicity was decreased, consistent with the presence of a sodium-coupled glutamate transport system in astrocytes; and (c) NMDA- but not kainate-mediated neurotoxicity was decreased, in a manner that depended on the relative abundance of glia in the culture. Because glia do not express NRs or an NMDA transport system, the mechanism of protection is distinct from that observed in response to glutamate. No differences in NR subunit composition (evaluated using RT-PCR assays for NR1 and NR2 subunit mRNAs), NR sensitivity (evaluated by measuring NR-mediated changes in intracellular Ca²⁺ levels), or glycine availability as a coagonist (evaluated in the presence and absence of exogenous glycine) were observed between astrocyte-rich and astrocyte-poor cultures, suggesting that glia do not directly modulate NR composition or function. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, blocked NMDA-mediated toxicity in astrocyte-poor cultures, raising the possibility that glia effectively reduce the accumulation of highly diffusible and toxic arachidonic acid metabolites in neurons. Alternatively, glia may alter neuronal development/phenotype in a manner that selectively reduces susceptibility to NR-mediated toxicity.     

Cerebellar Granule Neurons are More Vulnerable to Transient Transport-Mediated Glutamate Release than to Glutamate Uptake Blockade. Correlation with Excitatory Amino Acids Levels

The extracellular concentration of glutamate is highly regulated due to its excitotoxic nature. Failure of glutamate uptake or reversed activation of its transporters contributes to neurodegeneration related to some pathological conditions. We have compared the neurotoxicity of the substrate glutamate uptake inhibitor, l-trans-pyrrolidine-2,4-dicarboxylate (PDC), which promotes glutamate release by heteroexchange, with that of DL-threo-beta-benzyloxyaspartate (DL-TBOA), a non-substrate inhibitor, in cerebellar granule cell cultures. PDC substantially increases the extracellular concentration of glutamate during 30 min exposure and causes neuronal death at high concentrations, while DL-TBOA neurotoxicity is only observed after long-term exposure (8–24 h). During mitochondrial inhibition by 3-nitropropionic acid (3-NP), PDC-induced neuronal death is facilitated, but not that of DL-TBOA. In cultures containing a higher population of astrocytes DL-TBOA-induced increase in glutamate levels is more pronounced, but neuronal death is only triggered in the presence of 3-NP. Results suggest that cerebellar granule neurons are more vulnerable to acute transport-mediated glutamate release than to uptake blockade, which correlates with the extracellular excitatory amino acids levels.     

Glucose deprivation and chemical hypoxia: neuroprotection by P2 receptor antagonists

In this work we investigate cell survival after glucose deprivation and/or chemical hypoxia and we analyse the neuroprotective properties of selected antagonists of P2 ATP receptors. We find that in rat cerebellar granule neurones, the antagonist basilen blue prevents neuronal death under hypoglycaemia. Basilen blue acts through a wide temporal range and it retains its efficacy under chemically induced hypoxic conditions, in the presence of the respiratory inhibitors of mitochondria electron transport chain complexes II (3-nitropropionic acid) and III (antimycin A). In spite of the presence of these compounds, basilen blue maintains normal intracellular ATP levels. It furthermore prevents neuronal death caused by agents blocking the mitochondrial calcium uptake (ruthenium red) or discharging the mitochondrial membrane potential (carbonyl cyanide m-chlorophenylhydrazone). Inhibition of poly (ADP-ribose) polymerase, modulation of the enzyme GAPDH and mitochondrial transport of mono-carboxylic acids are not conceivable targets for the action of basilen blue. Survival is sustained by basilen blue also in CNS primary cultures from hippocampus and in PNS sympathetic-like neurones. Partial neuroprotection is furthermore provided by three additional P2 receptor antagonists: suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium and 4,4'-diisothiocyanatostilbene-2,2'disulphonic acid. Our data suggest the exploitation of selected P2 receptor antagonists as potential neuroprotective agents.     

Glutamate Neurotoxicity Is Independent of Calpain I Inhibition in Primary Cultures of Cerebellar Granule Cells

Glutamate-induced neurotoxicity and calpain activity were studied in primary cultures of rat cerebellar granule neurons and glial cells. Calpain activation, as monitored by quantitative immunoblotting of spectrin, required micromolar concentrations of Ca2+ in neuronal homogenates (calpain I) and millimolar Ca2+ concentrations in glial homogenates (calpain II). Glutamate-induced toxicity and calpain activation were observed in neuronal, but not in glial, cultures. In neurons, calpain I activation by glutamate was dose-dependent and persisted after withdrawal of neurotoxic doses of glutamate. Natural (GM1) and semisynthetic (LIGA4) gangliosides or the glutamate receptor blocker MK-801 prevented calpain I activation and delayed neuronal death elicited by glutamate. GM1 and LIGA4 had no effect on calpain I activity in neuronal homogenates, however. Furthermore, two calpain I inhibitors (leupeptin and N-acetyl-Leu-Leu-norleucinal) prevented glutamate-induced spectrin degradation, but failed to affect glutamate neurotoxicity. These results thus suggest that glutamate-induced neurotoxicity is independent of calpain I activation.     

Poly(ADP-ribose) polymerase inhibitors attenuate necrotic but not apoptotic neuronal death in experimental models of cerebral ischemia

An excessive activation of poly(ADP-ribose) polymerase (PARP) has been proposed to play a key role in post-ischemic neuronal death. We examined the neuroprotective effects of the PARP inhibitors benzamide, 6(5H)-phenanthridinone, and 3,4-dihydro-5-[4-1(1-piperidinyl)buthoxy]-1(2H)-isoquinolinone in three rodent models of cerebral ischemia. Increasing concentrations of the three PARP inhibitors attenuated neuronal injury induced by 60 min oxygen-glucose deprivation (OGD) in mixed cortical cell cultures, but were unable to reduce CA1 pyramidal cell loss in organotypic hippocampal slices exposed to 30 min OGD or in gerbils following 5 min bilateral carotid occlusion. We then examined the necrotic and apoptotic features of OGD-induced neurodegeneration in cortical cells and hippocampal slices using biochemical and morphological approaches. Cortical cells exposed to OGD released lactate dehydrogenase into the medium and displayed ultrastructural features of necrotic cell death, whereas no caspase-3 activation nor morphological characteristics of apoptosis were observed at any time point after OGD. In contrast, a marked increase in caspase-3 activity was observed in organotypic hippocampal slices after OGD, together with fluorescence and electron microscope evidence of apoptotic neuronal death in the CA1 subregion. Moreover, the caspase inhibitor Z-VAD-FMK reduced OGD-induced CA1 pyramidal cell loss. These findings suggest that PARP overactivation may be an important mechanism leading to post-ischemic neurodegeneration of the necrotic but not of the apoptotic type.     

Mechanism of LIGHT/interferon-gamma-induced cell death in HT-29 cells

LIGHT is a member of tumor necrosis factor (TNF) superfamily, and previous studies have indicated that in the presence of interferon-gamma (IFN-gamma), LIGHT through LTbetaR signaling can induce cell death with features unlike classic apoptosis. In present study, we investigated the mechanism of LIGHT/IFN-gamma-induced cell death in HT-29 cells, where the cell death was profoundly induced when sub-toxic concentrations of LIGHT and IFN-gamma were co-treated. LIGHT/IFN-gamma-induced cell death was accompanied by DNA fragmentation and slight LDH release. This effect was not affected by caspase, JNK nor cathepsin B inhibitors, but was partially prevented by p38 mitogen-activated protein kinase (MAPK) and poly (ADP-ribose) polymerase (PARP) inhibitors, and abolished by aurintricarboxylic acid (ATA), which is an inhibitor of endonuclease and STATs signaling of IFN-gamma. Immunobloting reveals that LIGHT/IFN-gamma could induce p38 MAPK activity, Bak and Fas expression, but down-regulate Mcl-1. Besides, LIGHT/IFN-gamma could not activate caspase-3 and -9, but decreased mitochondrial membrane potential. Although LIGHT could not affect IFN-gamma-induced STAT1 phosphorylation and transactivation activity, which was required for the sensitization of cell death, survival NF-kappaB signaling of LIGHT was inhibited by IFN-gamma. These data suggest that co-presence of LIGHT and IFN-gamma can induce an integrated interaction in signaling pathways, which lead to mitochondrial dysfunction and mix-type cell death, not involving caspase activation.     

HIV-1 Tat through phosphorylation of NMDA receptors potentiates glutamate excitotoxicity

Toxic effects of HIV-1 proteins contribute to altered function and decreased survival of select populations of neurons in HIV-1-infected brain. One such HIV-1 protein, Tat, can activate calcium release from IP3-sensitive intracellular pools, induce calcium influx in neural cells, and, as a result, can increase neuronal cell death. Here, we provide evidence that Tat potentiates excitatory amino acid (glutamate and NMDA) triggered calcium flux, as well as glutamate- and staurosporine-mediated neurotoxicity. Calcium flux in cultured rat hippocampal neurons triggered by the transient application of glutamate or NMDA was facilitated by pre-exposure to Tat. Facilitation of glutamate-triggered calcium flux by Tat was prevented by inhibitors of ADP-ribosylation of G(i)/G(o) proteins (pertussis toxin), protein kinase C (H7 and bisindolymide), and IP3-mediated calcium release (xestospongin C), but was not prevented by an activator of G(s) (cholera toxin) or an inhibitor of protein kinase A (H89). Facilitation of NMDA-triggered calcium flux by Tat was reversed by inhibitors of tyrosine kinase (genestein and herbimycin A) and by an inhibitor of NMDA receptor function (zinc). Tat increased 32P incorporation into NMDA receptor subunits NR2A and NR2B and this effect was blocked by genestein. Subtoxic concentrations of Tat combined with subtoxic concentrations of glutamate or staurosporine increased neuronal cell death significantly. Together, these findings suggest that NMDA receptors play an important role in Tat neurotoxicity and the mechanisms identified may provide additional therapeutic targets for the treatment of HIV-1 associated dementia.     

Possible role of nicaraven in neuroprotective effect on hippocampal slice culture

Nicaraven is an agent that is especially beneficial in vasospasm or brain damage caused by subarachnoid hemorrhage. It ameliorates neurological deficits of patients and protects the central nervous system from ischemia. We investigated the neuroprotective effect of nicaraven against oxygen-glucose deprivation (OGD) induced or N-methyl-D-aspartic acid (NMDA) induced hippocampal neuronal cell death in organotypic brain slice cultures. The effect of nicaraven on hippocampal neuronal injury was evaluated by inhibition of uptake of propidium iodide (PI) into dead cells. The results demonstrated that nicaraven protected neuronal cells from both OGD- and NMDA-induced cell death. While nicaraven has a strong hydroxyl radical scavenging effect, another radical scavenger, N-acetyl-L-cysteine (NAC), inhibited cell death only caused by OGD. In contrast, the poly(ADP-ribose) synthetase (PARS) inhibitors 3-aminobenzamide (3-AB) and theophylline protected cells from both OGD- and NMDA-induced cell death. Since nicaraven has an inhibitory effect in PARS, as well as a radical scavenging effect, these results suggest that inhibition of hippocampal cell death caused by NMDA may be attributable to PARS inhibition by nicaraven.     

Toxicity of Dopamine to Striatal Neurons In Vitro and Potentiation of Cell Death by a Mitochondrial Inhibitor

Abstract: Intrastriatal injections of the mitochondrial toxins malonate and 3-nitropropionic acid produce selective cell death similar to that seen in transient ischemia and Huntington's disease. The extent of cell death can be attenuated by pharmacological or surgical blockade of cortical glutamatergic input. It is not known, however, if dopamine contributes to toxicity caused by inhibition of mitochondrial function. Exposure of primary striatal cultures to dopamine resulted in dose-dependent death of neurons. Addition of medium supplement containing free radical scavengers and antioxidants decreased neuronal loss. At high concentrations of the amine, cell death was predominantly apoptotic. Methyl malonate was used to inhibit activity of the mitochondrial respiratory chain. Neither methyl malonate (50 µ M ) nor dopamine (2.5 µ M ) caused significant toxicity when added individually to cultures, whereas simultaneous addition of both compounds killed 60% of neurons. Addition of antioxidants and free radical scavengers to the incubation medium prevented this cell death. Dopamine (up to 250 µ M ) did not alter the ATP/ADP ratio after a 6-h incubation. Methyl malonate, at 500 µ M , reduced the ATP/ADP ratio by ∼30% after 6 h; this decrease was not augmented by coincubation with 25 µ M dopamine. Our results suggest that dopamine causes primarily apoptotic death of striatal neurons in culture without damaging cells by an early adverse action on oxidative phosphorylation. However, when combined with minimal inhibition of mitochondrial function, dopamine neurotoxicity is markedly enhanced.     

The Effect of Magnesium on Oxidative Neuronal Injury In Vitro

Abstract: The effect of magnesium on the oxidative neuronal injury induced by hemoglobin was assessed in murine cortical cell cultures. Exposure to 5 µ M hemoglobin in physiologic (1 m M ) magnesium for 26 h resulted in the death of about one-half the neurons and a sixfold increase in malondialdehyde production; glia were not injured. Increasing medium magnesium to 3 m M reduced neuronal death by about one-half and malondialdehyde production by about two-thirds; neuronal death and lipid peroxidation were approximately doubled in 0.3 m M magnesium. Comparable results were observed in spinal cord cultures. The NMDA antagonist MK-801 weakly attenuated hemoglobin neurotoxicity in low-magnesium medium, but tended to potentiate injury in physiologic magnesium. Incubation in low-magnesium medium alone for 24 h reduced cellular glutathione by ∼50% in mixed neuronal and glial cultures but by only 10% in pure glial cultures. The iron-dependent oxidation of phosphatidylethanolamine liposomes was attenuated in a concentration-dependent fashion by 2.5–10 m M magnesium; a similar effect was provided by 0.01–0.1 m M cobalt. However, oxidation was weakly enhanced by 0.5–1 m M magnesium. These results suggest that the vulnerability of neurons to iron-dependent oxidative injury is an inverse function of the extracellular magnesium concentration. At high concentrations, magnesium inhibits lipid peroxidation directly, perhaps by competing with iron for phospholipid binding sites. At low concentrations, enhancement of cell death may be due to the combined effect of increased NMDA receptor activity, glutathione depletion, and direct potentiation of lipid peroxidation.     

A novel in vivo post-translational modification of p53 by PARP-1 in MPTP-induced parkinsonism

Sporadic Parkinson's disease (PD) affects primarily dopaminergic neurons of the substantia nigra pars compacta. There is evidence of necrotic and apoptotic neuronal death in PD, but the mechanisms behind selected dopaminergic neuronal death remain unknown. The tumor suppressor protein p53 functions to selectively destroy stressed or abnormal cells during life and development by means of necrosis and apoptosis. Activation of p53 leads to death in a variety of cells including neurons. p53 is a target of the nuclear enzyme Poly(ADP-ribose)polymerase (PARP), and PARP is activated following DNA damage that occurs following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity. MPTP is the favored in vivo model of PD, and reproduces the pathophysiology, anatomy and biochemistry of PD. p53 protein normally exhibits a fleeting half-life, and regulation of p53 stability and activation is achieved mainly by post-translational modification. We find that p53 is heavily poly(ADP-ribosyl)ated by PARP-1 following MPTP intoxication. This post-translational modification serves to stabilize p53 and alters its transactivation of downstream genes. These influences of PARP-1 on p53 may underlie the mechanisms of MPTP-induced parkinsonism and other models of neuronal death.     

Age-Associated Changes of Rat Brain Neuronal and Astroglial Poly(ADP-Ribose) Polymerase Activity

Abstract: Nuclear poly(ADP-ribose) polymerase levels as well as the DNA strand break levels of whole-brain neuronal and astroglial cells were investigated. Three- and 30-month-old rats were used. Low-molecular-weight neurofilaments and glutamine synthetase served as neuronal and astroglial markers, respectively. A large increase in the poly(ADP-ribose) polymerase activity was observed in the neurons (threefold) and astrocytes (3.7-fold) derived from 30-month-old rats. Similarly, the amount of poly(ADP-ribose) polymerase, evaluated per milligram of DNA, increased ∼3.5-fold in neurons and 3.9-fold in astrocytes prepared from 30-month-old rats. Whether the increase in the poly(ADP-ribose) polymerase activity was due to an enhanced rate of DNA strand break was investigated by determining the rate of DNA unwinding. A significant increase in DNA unwinding rate was detected in the neurons (2.7-fold), although a lower increase was observed in the astroglia (1.3-fold) of aged animals.     

Group IB secretory phospholipase A2 induces neuronal cell death via apoptosis

Group IB secretory phospholipase A2 (sPLA2-IB) mediates cell proliferation, cell migration, hormone release and eicosanoid production via its receptor in peripheral tissues. In the CNS, high-affinity binding sites of sPLA2-IB have been documented. However, it remains obscure whether sPLA2-IB causes biologic or pathologic response in the CNS. To this end, we examined effects of sPLA2-IB on neuronal survival in primary cultures of rat cortical neurons. sPLA2-IB induced neuronal cell death in a concentration-dependent manner. This death was a delayed response requiring a latent time for 6 h; sPLA2-IB-induced neuronal cell death was accompanied with apoptotic blebbing, condensed chromatin, and fragmented DNA, exhibiting apoptotic features. Before cell death, sPLA2-IB liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2) from neurons. PGD2 and its metabolite, Delta12-PGJ2, exhibited neurotoxicity. Inhibitors of sPLA2 and cyclooxygenase-2 (COX-2) significantly suppressed not only AA release, but also PGD2 generation. These inhibitors significantly prevented neurons from sPLA2-IB-induced neuronal cell death. In conclusion, we demonstrate a novel biological response, apoptosis, of sPLA2-IB in the CNS. Furthermore, the present study suggests that PGD2 metabolites, especially Delta12-PGJ2, might mediate sPLA2-IB-induced apoptosis.     

Role of N-methyl-D-aspartate receptors in the neuroprotective activation of extracellular signal-regulated kinase 1/2 by cisplatin

Neurons are exposed to damaging stimuli that can trigger cell death and subsequently cause serious neurological disorders. Therefore, it is important to define defense mechanisms that can be activated in response to damage to reduce neuronal loss. Here we report that cisplatin (CPDD), a neurotoxic anticancer drug that damages DNA, triggered apoptosis and activated the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in cultured rat cortical neurons. Inhibition of ERK1/2 activation using either pharmacological inhibitors or a dominant-negative mutant of the ERK1/2 activator, mitogen-activated protein kinase kinase 1, increased the toxicity of CPDD. Interestingly, N-methyl-d-aspartate (NMDA) receptor (NMDAR) antagonists reduced the ERK1/2 activation and exacerbated apoptosis in CPDD-treated neurons. Pre-treatment with CPDD increased ERK1/2 activation triggered by exogenous NMDA, suggesting that CPDD augmented NMDAR responsiveness. CPDD-enhanced response of NMDAR and CPDD-mediated ERK1/2 activation were both decreased by inhibition of poly(ADP-ribose) polymerase (PARP). Interestingly, PARP activation did not produce ATP depletion, suggesting involvement of a non-energetic mechanism in NMDAR regulation by PARP. Finally, CPDD toxicity was reduced by brain-derived neurotrophic factor, and this protection required ERK1/2. In summary, our data identify a novel compensatory circuit in central nervous system neurons that couples the DNA injury, through PARP and NMDAR, to the defensive ERK1/2 activation.