Mitochondrial dysfunction mediated by quinone oxidation products of dopamine: Implications in dopamine cytotoxicity and pathogenesis of Parkinson's disease

The study has demonstrated that dopamine induces membrane depolarization and a loss of phosphorylation capacity in dose-dependent manner in isolated rat brain mitochondria during extended in vitro incubation and the phenomena are not prevented by oxyradical scavengers or metal chelators. Dopamine effects on brain mitochondria are, however, markedly prevented by reduced glutathione and N-acetyl cysteine and promoted by tyrosinase present in the incubation medium. The results imply that quinone oxidation products of dopamine are involved in mitochondrial damage under this condition. When PC12 cells are exposed to dopamine in varying concentrations (100-400 μM) for up to 24 h, a pronounced impairment of mitochondrial bio-energetic functions at several levels is observed along with a significant (nearly 40%) loss of cell viability with features of apoptotic nuclear changes and increased activities of caspase 3 and caspase 9 and all these effects of dopamine are remarkably prevented by N-acetyl cysteine. N-acetyl cysteine also blocks nearly completely the dopamine induced increase in reactive oxygen species production and the formation of quinoprotein adducts in mitochondrial fraction within PC12 cells and also the accumulation of quinone products in the culture medium. Clorgyline, an inhibitor of MAO-A, markedly decreases the formation of reactive oxygen species in PC12 cells upon dopamine exposure but has only mild protective actions against quinoprotein adduct formation, mitochondrial dysfunctions, cell death and caspase activation induced by dopamine. The results have indicated that quinone oxidation products and not reactive oxygen species are primarily involved in cytotoxic effects of dopamine and the mitochondrial impairment plays a central role in the latter process. The data have clear implications in the pathogenesis of Parkinson's disease.     

Inhibition of rat brain mitochondrial electron transport chain activity by dopamine oxidation products during extended in vitro incubation: implications for Parkinson's disease

Several studies on mitochondrial functions following brief exposure (5-15 min) to dopamine (DA) in vitro have produced extremely variable results. In contrast, this study demonstrates that a prolonged exposure (up to 2 h) of disrupted or lysed mitochondria to DA (0.1-0.4 mM) causes a remarkable and dose-dependent inhibition of complex I and complex IV activities. The inhibition of complex I and complex IV activities is not prevented by the antioxidant enzyme catalase (0.05 mg/ml) or the metal-chelator diethylenetriaminepentaacetic acid (0.1 mM) or the hydroxyl radical scavengers like mannitol (20 mM) and dimethyl sulphoxide (20 mM) indicating the non-involvement of *OH radicals and Fenton's chemistry in this process. However, reduced glutathione (5 mM), a quinone scavenger, almost completely abolishes the DA effect on mitochondrial complex I and complex IV activities, while tyrosinase (250 units/ml) which catalyses the conversion of DA to quinone products dramatically enhances the former effect. The results suggest the predominant involvement of quinone products instead of reactive oxygen radicals in long-term DA-mediated inactivation of complex I and complex IV. This is further indicated from the fact that significant amount of quinones and quinoprotein adducts (covalent adducts of reactive quinones with protein thiols) are formed during incubation of mitochondria with DA. Monoamine oxidase A (MAO-A) inhibitor clorgyline also provides variable but significant protection against DA induced inactivation of complex I and complex IV activities, presumably again through inhibition of quinoprotein formation. Mitochondrial ability to reduce tetrazolium dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) in presence of a respiratory substrate like succinate (10 mM) is also reduced by nearly 85% following 2 h incubation with 0.4 mM DA. This effect of DA on mitochondrial function is also dose-dependent and presumably mediated by quinone products of DA oxidation. The mitochondrial dysfunction induced by dopamine during extended periods of incubation as reported here have important implications in the context of dopaminergic neuronal death in Parkinson's disease (PD).     

Combined effect of dopamine and MPP+ on membrane permeability in mitochondria and cell viability in PC12 cells

The present study examined the combined effect of dopamine and 1-methyl-4-phenylpyridinium (MPP(+)) on the membrane permeability in isolated brain mitochondria and on cell viability in PC12 cells. MPP(+) increased effect of dopamine against the swelling, membrane potential, and Ca(2+) transport in isolated mitochondria, which was not inhibited by the addition of antioxidant enzymes (SOD and catalase). Dopamine or MPP(+) caused the decrease in transmembrane potential, increase in reactive oxygen species, depletion of GSH, and cell death in PC12 cells. Antioxidant enzymes reduced each effect of dopamine and MPP(+) against PC12 cells. Co-addition of dopamine and MPP(+) caused the decrease in the transmembrane potential and increase in the formation of reactive oxygen species in PC12 cells, in which they showed an additive effect. Dopamine plus MPP(+)-induced the depletion of GSH and cell death in PC12 cells were not decreased by the addition of antioxidant enzymes, rutin, diethylstilbestrol, and ascorbate. Melanin caused a cell viability loss in PC12 cells. The N-acetylcysteine, N-phenylthiourea, and 5-hydroxyindole decreased the cell death and the formation of dopamine quinone and melanin induced by co-addition of dopamine and MPP(+), whereas deprenyl and chlorgyline did not show an inhibitory effect. The results suggest that co-addition of dopamine and MPP(+) shows an enhancing effect on the change in mitochondrial membrane permeability and cell death, which may be accomplished by toxic quinone and melanin derived from the MPP(+)-stimulated dopamine oxidation.     

Dopamine Oxidation Alters Mitochondrial Respiration and Induces Permeability Transition in Brain Mitochondria

Both reactive dopamine metabolites and mitochondrial dysfunction have been implicated in the neurodegeneration of Parkinson's disease. Dopamine metabolites, dopamine quinone and reactive oxygen species, can directly alter protein function by oxidative modifications, and several mitochondrial proteins may be targets of this oxidative damage. In this study, we examined, using isolated brain mitochondria, whether dopamine oxidation products alter mitochondrial function. We found that exposure to dopamine quinone caused a large increase in mitochondrial resting state 4 respiration. This effect was prevented by GSH but not superoxide dismutase and catalase. In contrast, exposure to dopamine and monoamine oxidase-generated hydrogen peroxide resulted in a decrease in active state 3 respiration. This inhibition was prevented by both pargyline and catalase. We also examined the effects of dopamine oxidation products on the opening of the mitochondrial permeability transition pore, which has been implicated in neuronal cell death. Dopamine oxidation to dopamine quinone caused a significant increase in swelling of brain and liver mitochondria. This was inhibited by both the pore inhibitor cyclosporin A and GSH, suggesting that swelling was due to pore opening and related to dopamine quinone formation. In contrast, dopamine and endogenous monoamine oxidase had no effect on mitochondrial swelling. These findings suggest that mitochondrial dysfunction induced by products of dopamine oxidation may be involved in neurodegenerative conditions such as Parkinson's disease and methamphetamine-induced neurotoxicity.     

Quinoprotein Adducts Accumulate in the Substantia Nigra of Aged Rats and Correlate with Dopamine-Induced Toxicity in SH-SY5Y Cells

Parkinson’s disease (PD) is an age-dependent neurodegenerative disorder characterized by dopaminergic neuron loss in substantia nigra. Previous studies have implicated a role of dopamine oxidation in PD. Dopamine oxidation leads to the formation of dopamine quinone, which generates reactive oxygen species and covalently modifies cysteinyl proteins to form quinoprotein adduct. We compared quinoprotein adduct formation and lipid peroxidation in different brain regions of young and old rats. We found a prominent age-dependent accumulation of quinoprotein adducts in the substantia nigra, while no significant change of lipid peroxidation was detected in any brain regions of 2- to 15-month old rats. To determine whether quinoprotein adduct formation correlates with dopamine-induced cytotoxicity, we analyzed dopamine treated SH-SY5Y cells and found a strong correlation between quinoprotein adduct formation and cytotoxicity. Together, our results indicate that quinoprotein adduct formation may play a role in the age-dependent selective vulnerability of dopaminergic neurons in PD.     

Mitochondrial permeability transition in neuronal damage promoted by Ca2+ and respiratory chain complex II inhibition

Changes in mitochondrial integrity, reactive oxygen species release and Ca2+ handling are proposed to be involved in the pathogenesis of many neurological disorders including methylmalonic acidaemia and Huntington's disease, which exhibit partial mitochondrial respiratory inhibition. In this report, we studied the mechanisms by which the respiratory chain complex II inhibitors malonate, methylmalonate and 3-nitropropionate affect rat brain mitochondrial function and neuronal survival. All three compounds, at concentrations which inhibit respiration by 50%, induced mitochondrial inner membrane permeabilization when in the presence of micromolar Ca2+ concentrations. ADP, cyclosporin A and catalase prevented or delayed this effect, indicating it is mediated by reactive oxygen species and mitochondrial permeability transition (PT). PT induced by malonate was also present in mitochondria isolated from liver and kidney, but required more significant respiratory inhibition. In brain, PT promoted by complex II inhibition was stimulated by increasing Ca2+ cycling and absent when mitochondria were pre-loaded with Ca2+ or when Ca2+ uptake was prevented. In addition to isolated mitochondria, we determined the effect of methylmalonate on cultured PC12 cells and freshly prepared rat brain slices. Methylmalonate promoted cell death in striatal slices and PC12 cells, in a manner attenuated by cyclosporin A and bongkrekate, and unrelated to impairment of energy metabolism. We propose that under conditions in which mitochondrial complex II is partially inhibited in the CNS, neuronal cell death involves the induction of PT.     

Inhibition of SIN-1–Induced Change in Mitochondrial Membrane Permeability in PC12 Cells by Dopamine

Dopamine (50 or 100 microM) attenuated the nuclear damage and cell death due to 500 microM SIN-1, a donor of superoxide and nitric oxide, in differentiated PC12 cells whereas 200 microM dopamine did not depress cell death. Dopamine at 50-100 microM for a 4-h treatment did not show a significant cytotoxic effect on PC12 cells. Dopamine (100 microM) inhibited the decrease in mitochondrial transmembrane potential, cytochrome c release, activation of caspase-3, formation of reactive oxygen species, and depletion of glutathione (GSH) due to 500 microM SIN-1 in PC12 cells. The reaction of dopamine with peroxynitrite reduced an amount of peroxynitrite. The results suggest that dopamine exhibits a biphasic effect against the cytotoxicity of SIN-1 depending on concentrations. Dopamine at 50-100 microM may attenuate the reactive nitrogen species-induced viability loss in PC12 cells by suppressing the mitochondrial membrane permeability change through inhibition of the formation of reactive species, including peroxynitrite.     

Dopamine as a Potent Inducer of Cellular Glutathione and NAD(P)H:Quinone Oxidoreductase 1 in PC12 Neuronal Cells: A Potential Adaptive Mechanism for Dopaminergic Neuroprotection

Dopamine auto-oxidation and the consequent formation of reactive oxygen species and electrophilic quinone molecules have been implicated in dopaminergic neuronal cell death in Parkinson’s disease. We reported here that in PC12 dopaminergic neuronal cells dopamine at noncytotoxic concentrations (50–150 μM) potently induced cellular glutathione (GSH) and the phase 2 enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1), two critical cellular defenses in detoxification of ROS and electrophilic quinone molecules. Incubation of PC12 cells with dopamine also led to a marked increase in the mRNA levels for γ-glutamylcysteine ligase catalytic subunit (GCLC) and NQO1. In addition, treatment of PC12 cells with dopamine resulted in a significant elevation of GSH content in the mitochondrial compartment. To determine whether treatment with dopamine at noncytotoxic concentrations, which upregulated the cellular defenses could protect the neuronal cells against subsequent lethal oxidative and electrophilic injury, PC12 cells were pretreated with dopamine (150 μM) for 24 h and then exposed to various cytotoxic concentrations of dopamine or 6-hydroxydopamine (6-OHDA). We found that pretreatment of PC12 cells with dopamine at a noncytotoxic concentration led to a remarkable protection against cytotoxicity caused by dopamine or 6-OHDA at lethal concentrations, as detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium reduction assay. In view of the critical roles of GSH and NQO1 in protecting against dopaminergic neuron degeneration, the above findings implicate that upregulation of both GSH and NQO1 by dopamine at noncytotoxic concentrations may serve as an important adaptive mechanism for dopaminergic neuroprotection.     

Protective Effect of 1-Methylated β-Carbolines Against 3-Morpholinosydnonimine-Induced Mitochondrial Damage and Cell Viability Loss in PC12 Cells

The present study investigated the effect of 1-methylated beta-carbolines (harmaline, harmalol and harmine) on change in the mitochondrial membrane permeability and cell death due to reactive nitrogen species in differentiated PC12 cells. beta-Carbolines, caspase inhibitors (z-LEHD.fmk and z-DQMD.fmk) and antioxidants (N-acetylcysteine, dithiothreitol, melatonin, carboxy-PTIO and uric acid) depressed cell viability loss due to 3-morpholinosydnonimine (SIN-1) in PC12 cells. beta-Carbolines inhibited the nuclear damage, the decrease in mitochondrial transmembrane potential, the cytochrome c release, the formation of reactive oxygen species and the depletion of GSH caused by SIN-1 in PC12 cells. beta-Carbolines decreased the SIN-1-induced formations of 3-nitrotyrosine, malondialdehyde and carbonyls in PC12 cells. The results show that 1-methylated beta-carbolines attenuate SIN-1-induced mitochondrial damage. This results in the inhibition of caspase-9 and -3 and apoptotic cell death in PC12 cells by suppressing the toxic actions of reactive oxygen and nitrogen species, including the GSH depletion.     

Altered dopamine homeostasis differentially affects mitochondrial voltage-dependent anion channels turnover

Altered dopamine homeostasis plays a key role in the pathogenesis of Parkinson's disease. The generation of reactive oxygen species by spontaneous dopamine oxidation impairs mitochondrial function, causing in turn an enhancement of oxidative stress. Recent findings have highlighted the role of mitochondrial outer membrane proteins in the regulation of the correct disposal of damaged mitochondria. Here, we report the effect of altered dopamine homeostasis on the mitochondrial functionality in human neuroblastoma SH-SY5Y cells, a cellular model widely used to reproduce impaired dopamine homeostasis. We observed that dopamine significantly and relevantly reduces VDAC1 and VDAC2 levels without any change in the mRNA levels. Although mitochondria are depolarized by dopamine and mitochondrial calcium influx is reduced, dysfunctional mitochondria are not removed by mitophagy as it would be expected. Thus, alteration of dopamine homeostasis induces a mitochondrial depolarization not counteracted by the mitophagy quality control. As a consequence, the elimination of VDACs may contribute to the altered mitochondrial disposal in PD pathogenesis, thus enhancing the role of oxidative stress.     

Mitochondrial damage by nitric oxide is potentiated by dopamine in PC12 cells

Mitochondrial damage in PC12 cells, a model for dopaminergic cells, was examined in terms of the contribution of oxidative stress, nitric oxide (*NO), and dopamine to impairment of mitochondrial respiratory control (RC). A kinetic analysis suggested that the oxidative deamination of dopamine catalyzed by monoamine oxidase (MAO) was not a significant source of hydrogen peroxide, because of constrains imposed by the low cytosolic level of dopamine. *NO induced irreversible damage of mitochondrial complex I in PC12 cells: this damage followed a sigmoid response on *NO concentration with a well-defined threshold level. Dopamine did not elicit damage of mitochondria in PC12 cells; however, the amine potentiated the effects of *NO at or near the threshold level, thus leading to irreversible impairment of mitochondrial respiration. This synergism between *NO and dopamine was not observed at *NO concentrations below the threshold level. Depletion of dopamine from the storage vesicles by reserpine protected mitochondria from *NO damage. Dopamine oxidation by *NO increased with pH, and occurred at modest levels at pH 5.5. In spite of this, calculations showed that the oxidation of dopamine in the storage vesicles (pH 5.5) was higher than that in the cytosol (pH 7.4), due to the higher dopamine concentration in the storage vesicles (millimolar range) compared to that in the cytosol (micromolar range). It is suggested that storage vesicles may be the cellular sites where the potential for dopamine oxidation by *NO is higher.These data provide further support to the hypothesis that dopamine renders dopaminergic cells more susceptible to the mitochondrial damaging effects of *NO. In the early stages of Parkinson's disease, *NO production increases until reaching a point near the threshold level that induces neuronal damage. Dopamine stored in dopaminergic cells may cause these cells to be more susceptible to the deleterious effects of *NO, which involve irreversible impairment of mitochondrial respiration.     

Mitochondrial dysfunction in schizophrenia: a possible linkage to dopamine

Mitochondria are not only the principal source of high energy intermediates, but play an important role in intracellular calcium buffering, are main producers of reactive oxygen species, and are the source of pro- and antiapoptotic key factors. Moreover, the mitochondria are of a ubiquitous nature and the respiratory chain has a dual genetic basis, i.e. the mitochondrial and the nuclear DNAs. Thus mitochondrial impairment could provide an explanation for the tremendous heterogeneity of clinical and pathological manifestations in schizophrenia. This article reviews several independent lines of evidence that suggest an involvement of mitochondrial dysfunction in schizophrenia. Among them are altered cerebral energy metabolism, mitochondrial hypoplasia, dysfunction of the oxidative phosphorylation system and altered mitochondrial related gene expression. In addition, the interaction between dopamine, a predominant etiological factor in schizophrenia, and mitochondrial respiration is considered as a possible mechanism underlying the hyper- and hypo-activity cycling in schizophrenia. Understanding the role of mitochondria in schizophrenia may encourage novel treatment approaches, the identification of candidate genes and new insights into the pathophysiology and etiology of the disorder.     

Early redox imbalance mediates hydroperoxide-induced apoptosis in mitotic competent undifferentiated PC-12 cells

Our recent study has demonstrated that cellular redox imbalance can directly initiate apoptosis in a mitotic competent PC-12 cell line without the involvement of reactive oxygen species (ROS). However, whether cell apoptosis induced by ROS is, in fact, mediated by a loss of redox balance caused by the oxidant is unresolved. The linkage between oxidant-mediated apoptosis and the induction of cellular redox was examined in PC-12 cells using the oxidant, tert-butylhydroperoxide (TBH). TBH caused cell apoptosis in 24 h that was preceded by an early increase (30 min) in oxidized glutathione (GSSG). Pretreatment with N-acetyl cysteine prevented TBH-induced GSSG increases and cell apoptosis. Altered Bax/BcL-2 expression and release of mitochondrial cytochrome c occurred post-redox imbalance and was kinetically linked to caspase-3 activation and poly ADP-ribose polymerase cleavage. Moreover, cell apoptosis was attenuated by inhibition of caspase-9, but not caspase-8, and blockade of mitochondrial ROS generation and permeability transition pore attenuated caspase 3 activation and cell apoptosis. Collectively, these results show that TBH-induced GSSG elevation is associated with the disruption of mitochondrial integrity, activation of caspase-3 and cell apoptosis. This redox induction of the apoptotic cascade was dissociated from cellular GSH efflux.     

Mitochondrial Dysfunction Promotes Breast Cancer Cell Migration and Invasion through HIF1α Accumulation via Increased Production of Reactive Oxygen Species

Although mitochondrial dysfunction has been observed in various types of human cancer cells, the molecular mechanism underlying mitochondrial dysfunction mediated tumorigenesis remains largely elusive. To further explore the function of mitochondria and their involvement in the pathogenic mechanisms of cancer development, mitochondrial dysfunction clones of breast cancer cells were generated by rotenone treatment, a specific inhibitor of mitochondrial electron transport complex I. These clones were verified by mitochondrial respiratory defect measurement. Moreover, those clones exhibited increased reactive oxygen species (ROS), and showed higher migration and invasive behaviors compared with their parental cells. Furthermore, antioxidant N-acetyl cysteine, PEG-catalase, and mito-TEMPO effectively inhibited cell migration and invasion in these clones. Notably, ROS regulated malignant cellular behavior was in part mediated through upregulation of hypoxia-inducible factor-1 α and vascular endothelial growth factor. Our results suggest that mitochondrial dysfunction promotes cancer cell motility partly through HIF1α accumulation mediated via increased production of reactive oxygen species.     

Protective effect of serotonin on 6-hydroxydopamine- and dopamine-induced oxidative damage of brain mitochondria and synaptosomes and PC12 cells

The present study elucidated the effects of indoleamines (serotonin, melatonin, and tryptophan) on oxidative damage of brain mitochondria and synaptosomes induced either by 6-hydroxydopamine (6-OHDA) or by iron plus ascorbate and on viability loss in dopamine-treated PC12 cells. Serotonin (1-100 microM), melatonin (100 microM), and antioxidant enzymes attenuated the effects of 6-OHDA, iron plus ascorbate, or 1-methyl-4-phenylpyridinium on mitochondrial swelling and membrane potential formation. Serotonin and melatonin decreased the attenuation of synaptosomal Ca(2+) uptake induced by either 6-OHDA alone or iron plus ascorbate. Serotonin and melatonin inhibited the production of reactive oxygen species, formation of malondialdehyde and carbonyls, and thiol oxidation in mitochondria and synaptosomes and decreased degradation of 2-deoxy-D-ribose. Unlike serotonin, melatonin did not reduce the iron plus ascorbate-induced thiol oxidation. Tryptophan decreased thiol oxidation and 2-deoxy-D-ribose degradation but did not inhibit the production of reactive oxygen species and formation of oxidation products in the brain tissues. Serotonin and melatonin attenuated the dopamine-induced viability loss, including apoptosis, in PC12 cells. The results suggest that serotonin may attenuate the oxidative damage of mitochondria and synaptosomes and the dopamine-induced viability loss in PC12 cells by a decomposing action on reactive oxygen species and inhibition of thiol oxidation and shows the effect comparable to melatonin. Serotonin may show a prominent protective effect on the iron-mediated neuronal damage.     

Cyclooxygenase-Independent Neuroprotective Effects of Aspirin Against Dopamine Quinone-Induced Neurotoxicity

Prostaglandin H synthase exerts not only cyclooxygenase activity but also peroxidase activity. The latter activity of the enzyme is thought to couple with oxidation of dopamine to dopamine quinone. Therefore, it has been proposed that cyclooxygenase inhibitors could suppress dopamine quinone formation. In the present study, we examined effects of various cyclooxygenase inhibitors against excess methyl L-3,4-dihydroxyphenylalanine (L-DOPA)-induced quinoprotein (protein-bound quinone) formation and neurotoxicity using dopaminergic CATH.a cells. The treatment with aspirin inhibited excess methyl L-DOPA-induced quinoprotein formation and cell death. However, acetaminophen did not show protective effects, and indomethacin and meloxicam rather aggravated these methyl L-DOPA-induced changes. Aspirin and indomethacin did not affect the level of glutathione that exerts quenching dopamine quinone in dopaminergic cells. In contrast with inhibiting effects of higher dose in the previous reports, relatively lower dose of aspirin that affected methyl L-DOPA-induced quinoprotein formation and cell death failed to prevent cyclooxygenase-induced dopamine chrome generation in cell-free system. Furthermore, aspirin but not acetaminophen or meloxicam showed direct dopamine quinone-scavenging effects in dopamine-semiquinone generating systems. The present results suggest that cyclooxygenase shows little contribution to dopamine oxidation in dopaminergic cells and that protective effects of aspirin against methyl L-DOPA-induced dopamine quinone neurotoxicity are based on its cyclooxygenase-independent property.     

Nitric-oxide-induced necrosis and apoptosis in PC12 cells mediated by mitochondria

Nitric oxide (NO) can trigger either necrotic or apoptotic cell death. We have used PC12 cells to investigate the extent to which NO-induced cell death is mediated by mitochondria. Addition of NO donors, 1 mM S-nitroso-N-acetyl-DL-penicillamine (SNAP) or 1 mM diethylenetriamine-NO adduct (NOC-18), to PC12 cells resulted in a steady-state level of 1-3 microM: NO, rapid and almost complete inhibition of cellular respiration (within 1 min), and a rapid decrease in mitochondrial membrane potential within the cells. A 24-h incubation of PC12 cells with NO donors (SNAP or NOC-18) or specific inhibitors of mitochondrial respiration (myxothiazol, rotenone, or azide), in the absence of glucose, caused total ATP depletion and resulted in 80-100% necrosis. The presence of glucose almost completely prevented the decrease in ATP level and the increase in necrosis induced by the NO donors or mitochondrial inhibitors, suggesting that the NO-induced necrosis in the absence of glucose was due to the inhibition of mitochondrial respiration and subsequent ATP depletion. However, in the presence of glucose, NO donors and mitochondrial inhibitors induced apoptosis of PC12 cells as determined by nuclear morphology. The presence of apoptotic cells was prevented completely by benzyloxycarbonyl-Val-Ala-fluoromethyl ketone (a nonspecific caspase inhibitor), indicating that apoptosis was mediated by caspase activation. Indeed, both NO donors and mitochondrial inhibitors in PC12 cells caused the activation of caspase-3- and caspase-3-processing-like proteases. Caspase-1 activity was not activated. Cyclosporin A (an inhibitor of the mitochondrial permeability transition pore) decreased the activity of caspase-3- and caspase-3-processing-like proteases after treatment with NO donors, but was not effective in the case of the mitochondrial inhibitors. The activation of caspases was accompanied by the release of cytochrome c from mitochondria into the cytosol, which was partially prevented by cyclosporin A in the case of NO donors. These results indicate that NO donors (SNAP or NOC-18) may trigger apoptosis in PC12 cells partially mediated by opening the mitochondrial permeability transition pores, release of cytochrome c, and subsequent caspase activation. NO-induced apoptosis is blocked completely in the absence of glucose, probably due to the lack of ATP. Our findings suggest that mitochondria may be involved in both types of cell death induced by NO donors: necrosis by respiratory inhibition and apoptosis by opening the permeability transition pore. Further, our results indicate that the mode of cell death (necrosis versus apoptosis) induced by either NO or mitochondrial inhibitors depends critically on the glycolytic capacity of the cell.     

Paclitaxel targets mitochondria upstream of caspase activation in intact human neuroblastoma cells

We previously reported that paclitaxel acted directly on mitochondria isolated from human neuroblastoma SK-N-SH cells. Here, we demonstrate that the direct mitochondrial effect of paclitaxel observed in vitro is relevant in intact SK-N-SH cells. After a 2 h incubation with 1 microM paclitaxel, the mitochondria were less condensed. Paclitaxel (1 microM, 1-4 h) also induced a 20% increase in respiration rate and a caspase-independent production of reactive oxygen species by mitochondria. The paclitaxel-induced release of cytochrome c was detected only after 24 h of incubation, was caspase-independent and permeability transition pore-dependent. Thus, paclitaxel targets mitochondria upstream of caspase activation, early during the apoptotic process in intact human neuroblastoma cells.     

Nerve growth factor prevents the apoptosis-associated increase in acetylcholinesterase activity after hydrogen peroxide treatment by activating Akt

Acetylcholinesterase (ACHE) is thought to play an important role during apoptosis.Our resultsshowed that H_2O_2 induced AChE activity,a functional marker in apoptosis,increases in neuronal-like PC 12cells.Glutathione, which is involved in cellular redox homeostasis,inhibited the increase of AChE activity,suggesting that reactive oxygen species (ROS) play a key role in this process.Further investigation showedthat the elevation of AChE was observed after the degradation of Akt, release of cytochrome c from mitochondriainto the cytosol,and activation of caspase family members.When nerve growth factor (NGF) was present,with the maintenance of Akt level,the elevation of AChE,the cytochrome c diffusion,as well as apoptosiswere markedly attenuated in H202-treated PC 12 cells. However,wortmannin,an inhibitor of the PI3K/Aktpathway,accelerated the apoptosis and increased the AChE activity.The overexpression of constitutivelyactivated Akt,which is a downstream signalling element of the NGF receptor TrkA,delayed mitochondrialcollapse and inhibited elevation of AChE activity.Thus, NGF prevented apoptosis and elevation of AChEactivity by activating the Akt pathway and stabilizing the function of mitochondria.     

1,2-bis(2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid induces caspase-mediated apoptosis and reactive oxygen species-mediated necrosis in cultured cortical neurons.

Sustained alteration in [Ca(2+)]i triggers neuronal death. We examined morphological and signaling events of Ca(2+)-deficiency-induced neuronal death. Cortical cell cultures exposed to 20 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), an intracellular calcium chelator, underwent neuronal apoptosis within 12 h that was evident by shriveled cell bodies, aggregated and condensed nuclear chromatin, and disrupted nuclear membrane. Thereafter, surviving neurons revealed typical necrosis, accompanied by swelling of cell body and mitochondria, over 24 h. Both apoptosis and necrosis were prevented by inclusion of 1 microg/mL cycloheximide, a protein synthesis inhibitor. Treatment with BAPTA-AM induced translocation of Bax into mitochondria within 4 h and release of cytochrome c from mitochondria over 4-12 h. An active fragment of caspase-3, a downstream mediator of cytochrome c, was observed within 8 h and cleaved PHF-1-positive tau. Administration of zVAD-fmk, a broad inhibitor of caspases, or DEVD-amc, a selective inhibitor of caspase-3, selectively prevented the apoptosis component of BAPTA-AM neurotoxicity. In contrast, BAPTA-AM-induced necrosis was propagated through sequential production of superoxide, mitochondrial and cytoplasmic reactive oxygen species. Combined treatment with caspase inhibitors and antioxidants blocked BAPTA-AM neurotoxicity. The present study suggests that neurons deficient in [Ca(2+)]i undergo caspase-3-mediated apoptosis and reactive oxygen species (ROS)-mediated necrosis.