ATP-sensitive potassium channel opener iptakalim protects against MPP-induced astrocytic apoptosis via mitochondria and mitogen-activated protein kinase signal pathways

Inhibition of astrocytic apoptosis has been regarded as a novel prospective strategy for treating neurodegenerative disorders such as Parkinson's disease. In the present study, we demonstrated that iptakalim (IPT), an ATP-sensitive potassium channel (K(ATP) channel) opener, exerted protective effect on MPP(+)-induced astrocytic apoptosis, which was reversed by selective mitochondrial K(ATP) channel blocker 5-hydroxydecanoate. Further study revealed that IPT inhibited glutathione (GSH) depletion, mitochondrial membrane potential loss and subsequent release of pro-apoptotic factors (cytochrome c and apoptosis-inducing factor (AIF), and c-Jun NH(2)-terminal kinase/mitogen-activated protein kinases (MAPK) phosphorylation induced by MPP(+). Meanwhile, extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 inhibited the protective effect of IPT on MPP(+)-induced astrocytic apoptosis. Furthermore, IPT could also activate ERK/MAPK and maintain increased phospho-ERK1/2 level after MPP(+) exposure. Taken together, these findings reveal for the first time that IPT protects against MPP(+)-induced astrocytic apoptosis via inhibition of mitochondria apoptotic pathway and regulating the MAPK signal transduction pathways by opening mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels in astrocytes. And targeting K(ATP) channels expressed in astrocytes may provide a novel therapeutic strategy for neurodegenerative disorders.     

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.     

The parkinsonism-inducing drug 1-methyl-4-phenylpyridinium triggers intracellular dopamine oxidation. A novel mechanism of toxicity

Uptake of the Parkinsonism-inducing toxin, 1-methyl-4-phenylpyridinium (MPP(+)), into dopaminergic terminals is thought to block Complex I activity leading to ATP loss and overproduction of reactive oxygen species (ROS). The present study indicates that MPP(+)-induced ROS formation is not mitochondrial in origin but results from intracellular dopamine (DA) oxidation. Although a mean lethal dose of MPP(+) led to ROS production in identified dopaminergic neurons, toxic doses of the Complex I inhibitor rotenone did not. Concurrent with ROS formation, MPP(+) redistributed vesicular DA to the cytoplasm prior to its extrusion from the cell by reverse transport via the DA transporter. MPP(+)-induced DA redistribution was also associated with cell death. Depleting cells of newly synthesized and/or stored DA significantly attenuated both superoxide production and cell death, whereas enhancing intracellular DA content exacerbated dopaminergic sensitivity to MPP(+). Lastly, depleting cells of DA in the presence of succinate completely abolished MPP(+)-induced cell death. Thus, MPP(+) neurotoxicity is a multi-component process involving both mitochondrial dysfunction and ROS generated by vesicular DA displacement. These results suggest that in the presence of a Complex I defect, misregulation of DA storage could lead to the loss of nigrostriatal neurons in Parkinson's disease.     

1-Methyl-4-phenylpyridinium-induced apoptosis in cerebellar granule neurons is mediated by transferrin receptor iron-dependent depletion of tetrahydrobiopterin and neuronal nitric-oxide synthase-derived superoxide

In this study, we investigated the molecular mechanisms of toxicity of 1-methyl-4-phenylpyridinium (MPP(+)), an ultimate toxic metabolite of a mitochondrial neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, that causes Parkinson-like symptoms in experimental animals and humans. We used rat cerebellar granule neurons as a model cell system for investigating MPP(+) toxicity. Results show that MPP(+) treatment resulted in the generation of reactive oxygen species from inhibition of complex I of the mitochondrial respiratory chain, and inactivation of aconitase. This, in turn, stimulated transferrin receptor (TfR)-dependent iron signaling via activation of the iron-regulatory protein/iron-responsive element interaction. MPP(+) caused a time-dependent depletion of tetrahydrobiopterin (BH(4)) that was mediated by H(2)O(2) and transferrin iron. Depletion of BH(4) decreased the active, dimeric form of neuronal nitric-oxide synthase (nNOS). MPP(+)-mediated "uncoupling" of nNOS decreased *NO and increased superoxide formation. Pretreatment of cells with sepiapterin to promote BH(4) biosynthesis or cell-permeable iron chelator and TfR antibody to prevent iron-catalyzed BH(4) decomposition inhibited MPP(+) cytotoxicity. Preincubation of cerebellar granule neurons with nNOS inhibitor exacerbated MPP(+)-induced iron uptake, BH(4) depletion, proteasomal inactivation, and apoptosis. We conclude that MPP(+)-dependent aconitase inactivation, Tf-iron uptake, and oxidant generation result in the depletion of intracellular BH(4), leading to the uncoupling of nNOS activity. This further exacerbates reactive oxygen species-mediated oxidative damage and apoptosis. Implications of these results in unraveling the molecular mechanisms of neurodegenerative diseases (Parkinson's and Alzheimer's disease) are discussed.     

Protection against MPP+ neurotoxicity in cerebellar granule cells by antioxidants

The neuropathology associated with Parkinson's disease (PD) is thought to involve excessive production of free radicals, dopamine autoxidation, defects in glutathione peroxidase expression, attenuated levels of reduced glutathione, altered calcium homeostasis, excitotoxicity and genetic defects in mitochondrial complex I activity. While the neurotoxic mechanisms are vastly different for excitotoxins and 1-methyl-4-phenylpyridinium ion (MPP(+)), both are thought to involve free radical production, compromised mitochondrial activity and excessive lipid peroxidation. We show here that the levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) increased significantly after treatment of cultured cerebellar granule cells (CGCs) with 50 microM MPP(+). Co-treatment with antioxidants such as ascorbate (ASC), catalase, alpha-tocopherol (alpha-TOH), coenzyme Q(10) (CoQ(10)) or superoxide dismutase (SOD) rescued the cells from MPP(+)-induced death. MPP(+)-induced cell death was also abolished by co-treatment with nitric oxide synthase (NOS) inhibitors such as 7-nitroindazole (7-NI), 2-ethyl-2-thiopseudourea hydrobromide (EPTU) or S-methylisothiourea sulphate (MPTU). We also tested the protective effects of an iron chelator (deferoxamine mesylate, DFx) and a peroxynitrite scavenger (FeTTPS) and the results lend further support to the view that the free radical cytotoxicity plays an essential role in MPP(+)-induced death in primary cultures of CGC.     

TRPC1-mediated inhibition of 1-methyl-4-phenylpyridinium ion neurotoxicity in human SH-SY5Y neuroblastoma cells

Mammalian homologues of the Drosophila canonical transient receptor potential (TRP) proteins have been implicated to function as plasma membrane Ca(2+) channels. This study examined the role of TRPC1 in human neuroblastoma (SH-SY5Y) cells. SH-SY5Y cells treated with an exogenous neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP(+)) significantly decreased TRPC1 protein levels. Confocal microscopy on SH-SY5Y cells treatment with MPP(+) showed decreased plasma membrane staining of TRPC1. Importantly, overexpression of TRPC1 reduced neurotoxicity induced by MPP(+). MPP(+)-induced alpha-synuclein expression was also suppressed by TRPC1 overexpression. Protection of SH-SY5Y cells against MPP(+) was significantly decreased upon the overexpression of antisense TRPC1 cDNA construct or the addition of a nonspecific transient receptor potential channel blocker lanthanum. Activation of TRPC1 by thapsigargin or carbachol decreased MPP(+) neurotoxicity, which was partially dependent on external Ca(2+). Staining of SH-SY5Y cells with an apoptotic marker (YO-PRO-1) showed that TRPC1 protects SH-SY5Y neuronal cells against apoptosis. Further, TRPC1 overexpression inhibited cytochrome c release and decreased Bax and Apaf-1 protein levels. Interpretation of the above data suggests that reduction in the cell surface expression of TRPC1 following MPP(+) treatment may be involved in dopaminergic neurodegeneration. Furthermore, TRPC1 may inhibit degenerative apoptotic signaling to provide neuroprotection against Parkinson's disease-inducing agents.     

PEP-1-heat shock protein 27 protects from neuronal damage in cells and in a Parkinson's disease mouse model

Heat shock proteins (HSPs) are a highly conserved family of proteins that are induced in response to various environmental stressors including reactive oxygen species. HSP27 is a chaperone protein with the ability to increase cell survival in response to oxidative stress. Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons. Although the mechanism of PD remains unclear, oxidative stress is known to be important in its pathogenesis. This study investigated the protective effects of PEP-1-HSP27 on neuronal damage induced by 1-methyl-4-phenyl pyridinium (MPP(+) ) in SH-SY5Y cells and in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. PEP-1-HSP27 rapidly entered the cells and protected them against MPP(+) -induced toxicity by inhibiting the reactive oxygen species levels and DNA fragmentation. Furthermore, transduced PEP-1-HSP27 prevented dopaminergic neuronal cell death in the substantia nigra of MPTP-induced PD mouse models. These results demonstrate that PEP-1-HSP27 provides a potential strategy for therapeutic delivery against various diseases and is a potential tool for the treatment of PD.     

银杏叶提取物对过氧化氢诱导的星形胶质细胞氧化损伤的保护作用

目的 研究银杏叶提取物（EGb761）对H2O2所致星形胶质细胞氧化损伤的保护作用。方法 用不同浓度的EGb761预处理细胞,再加入H2O2,通过噻唑蓝（MTT）实验、线粒体跨膜电位（△ψm）及细胞色素C释放实验、DNA损伤实验及半胱氨酰天冬氨酸特异性蛋白酶-3（Caspase-3）活性测定,观察EGb761对细胞存活率、线粒体膜通透性、DNA氧化损伤及Caspase-3活性的影响。结果 EGb761能明显降低Hz02对星形胶质细胞的氧化损伤,提高细胞的存活率;维持线粒体膜的完整性,抑制跨膜电位的耗散和细胞色素C的释放;抑制Caspase-3的活化和DNA的降解。结论 EGb761具有清除活性氧,减轻H2O2所致星形胶质细胞的氧化损伤,对星形胶质细胞有保护作用。     

Nitric oxide enhances MPP(+) inhibition of complex I

There is evidence that 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) toxicity is mediated through both inhibition of mitochondrial complex I and free radical generation. 7-Nitroindazole protects against MPTP toxicity in vitro and in vivo, and this appears to be related to its inhibition of nitric oxide (NO(*-)) synthase. We now show that the NO(*-) generator, glutathione-N-oxide, enhances the inhibitory action of 1-methyl-4-phenylpyridinium (MPP(+)) on complex I activity in brain submitochondrial particles. We propose that the NO(*-)-induced reversible inhibition of complex IV (cytochrome oxidase) potentiates the MPP(+)-induced irreversible free radical-mediated inhibition of complex I. Thus, NO(*-) may 'prime' the respiratory chain to the effects of MPP(+). These data provide evidence for an interaction between NO(*-) and MPP(+) at the level of the respiratory chain.     

Oxidative stress in Ca2+‐induced membrane permeability transition in brain mitochondria

Mitochondrial permeability transition (PT) is a non-selective inner membrane permeabilization, typically promoted by the accumulation of excessive quantities of Ca(2+) ions in the mitochondrial matrix. This phenomenon may contribute to neuronal cell death under some circumstances, such as following brain trauma and hypoglycemia. In this report, we show that Ca(2+)-induced brain mitochondrial PT was stimulated by Na(+) (10 mM) and totally prevented by the combination of ADP and cyclosporin A. Removal of Ca(2+) from the mitochondrial suspension by EGTA or inhibition of Ca(2+) uptake by ruthenium red partially reverted the dissipation of the membrane potential associated with PT. Ca(2+)-induced brain mitochondrial PT was significantly inhibited by the antioxidant catalase, indicating the participation of reactive oxygen species in this process. An increased detection of reactive oxygen species, measured through dichlorodihydrofluorescein oxidation, was observed after mitochondrial Ca(2+) uptake. Ca(2+)-induced dichlorodihydrofluorescein oxidation was enhanced by Na(+) and prevented by ADP and cyclosporin A, indicating that PT enhances mitochondrial oxidative stress. This could be at least in part a consequence of the extensive depletion in NAD(P)H that accompanied this Ca(2+)-induced mitochondrial PT. NADPH is known to maintain the antioxidant function of the glutathione reductase/peroxidase and thioredoxin reductase/peroxidase systems. In addition, the occurrence of mitochondrial PT was associated with membrane lipid peroxidation. We conclude that PT further increases Ca(2+)-induced oxidative stress in brain mitochondria leading to secondary damage such as lipid peroxidation.     

Cytoprotective activity of annphenone against oxidative stress-induced apoptosis in V79-4 lung fibroblast cells

We have elucidated the cytoprotective effect of annphenone (2,4-dihyroxy-6-methoxy-acetophenone 4-O-beta-d-glucopyranoside) against oxidative stress-induced apoptosis. Annphenone scavenged intracellular reactive oxygen species (ROS) and increased antioxidant enzyme activities. It thereby prevented lipid peroxidation and DNA damage, which was demonstrated by the inhibition of the formation of thiobarbituric acid reactive substance (TBARS), inhibition of the comet tail and decreased phospho-H2A.X expression. Annphenone protected Chinese hamster lung fibroblast (V79-4) cells from cell death via the inhibition of apoptosis induced by hydrogen peroxide (H(2)O(2)), as shown by decreased apoptotic nuclear fragmentation, decreased sub-G(1) cell population and inhibited mitochondrial membrane potential (Deltapsi) loss. Taken together, these findings suggest that annphenone exhibits antioxidant properties by inhibiting ROS generation and thus protecting cells from H(2)O(2)-induced cell damage.     

The endogenous amine 1-methyl-1,2,3,4- tetrahydroisoquinoline prevents the inhibition of complex I of the respiratory chain produced by MPP(+)

The endogenous monoamine 1-methyl-1,2,3,4-tetrahydroisoquinoline has been shown to prevent the neurotoxic effect of MPP(+) and other endogenous neurotoxins, which produce a parkinsonian-like syndrome in humans. We have tested its potential protective effect in vivo by measuring the protection of 1-methyl-1,2,3,4-tetrahydroisoquinoline in the neurotoxicity elicited by MPP(+) in rat striatum by tyrosine hydroxylase immunocytochemistry. Because we know that cellular damage caused by MPP(+) is primarily the result of mitochondrial respiratory inhibition at the complex I level, we have extended the study further to understand this protective mechanism. We found that the inhibitory effect on the mitochondrial respiration rate induced by MPP(+) in isolated rat liver mitochondria and striatal synaptosomes was prevented by addition of 1-methyl-1,2,3,4-tetrahydroisoquinoline. This compound has no antioxidant capacity; therefore, this property is not involved in its protective effect. Thus, we postulate that the preventive effect that 1-methyl-1,2,3,4-tetrahydroisoquinoline has on mitochondrial inhibition for MPP(+) could be due to a "shielding effect," protecting the energetic machinery, thus preventing energetic failure. These results suggest that this endogenous amine may protect against the effect of several parkinsonism-inducing compounds that are associated with progressive impairment of the mitochondrial function.     

Short-term and long-term effects of fatty acids in rat hepatoma AS-30D cells: the way to apoptosis

Arachidonic acid and, to a smaller extent, oleic acid at micromolar concentrations decreased the mitochondrial membrane potential within AS-30D rat hepatoma cells cultivated in vitro and increased cell respiration. The uncoupling effect of both fatty acids on cell respiration was partly prevented by cyclosporin A, blocker of the mitochondrial permeability transition pore. Arachidonic acid increased the rate of reactive oxygen species (ROS) production, while oleic acid decreased it. Both fatty acids induced apoptotic cell death of AS-30D cells, accompanied by the release of cytochrome c from mitochondria to the cytosol, activation of caspase-3 and association of proapoptotic Bax protein with mitochondria; arachidonic acid being a more potent inducer than oleic acid. Trolox, a potent antioxidant, prevented ROS increase induced by arachidonic acid and protected the cells against apoptosis produced by this fatty acid. It is concluded that arachidonic and oleic acids induce apoptosis of AS-30D hepatoma cells by the mitochondrial pathway but differ in the mechanism of their action: Arachidonic acid induces apoptosis mainly by stimulating ROS production, whereas oleic acid may contribute to programmed cell death by activation of the mitochondrial permeability transition pore.     

Effects of 1,4-dihydropyridine derivatives (cerebrocrast, gammapyrone, glutapyrone, and diethone) on mitochondrial bioenergetics and oxidative stress: a comparative study

The potential protective action of 1,4-dihydropyridine derivatives (cerebrocrast, gammapyrone, glutapyrone, and diethone) against oxidative stress was assessed on mitochondrial bioenergetics, inner membrane anion channel (IMAC), Ca2+-induced opening of the permeability transition pore (PTP), and oxidative damage induced by the oxidant pair adenosine diphosphate (ADP)/Fe2+ (lipid peroxidation) of mitochondria isolated from rat liver. By using succinate as the respiratory substrate, respiratory control ratio (RCR), ADP to oxygen ratio (ADP/O), state 3, state 4, and uncoupled respiration rates were not significantly affected by gammapyrone, glutapyrone, and diethone concentrations up to 100 microM. Cerebrocrast at concentrations higher than 25 microM depressed RCR, ADP/O, state 3, and uncoupled respiration rates, but increased three times state 4 respiration rate. The transmembrane potential (deltapsi) and the phosphate carrier rate were also decreased. At concentrations lower than 25 microM, cerebrocrast inhibited the mitochondrial IMAC and partially prevented Ca2+-induced opening of the mitochondrial PTP, whereas gammapyrone, glutapyrone, and diethone were without effect. Cerebrocrast, gammapyrone, and glutapyrone concentrations up to 100 microM did not affect ADP/Fe2+-induced lipid peroxidation of rat liver mitochondria, while very low diethone concentrations (up to 5 microM) inhibited it in a dose-dependent manner, as measured by oxygen consumption and thiobarbituric acid reactive substances formation. Diethone also prevented deltapsi dissipation due to lipid peroxidation initiated by ADP/Fe2+. It can be concluded that: none of the compounds interfere with mitochondrial bioenergetics at concentrations lower than 25 microM; cerebrocrast was the only compound that affected mitochondrial bioenergetics, but only for concentrations higher than 25 microM; at concentrations that did not affect mitochondrial bioenergetics (< or = 25 microM), only cerebrocrast inhibited the IMAC and partially prevented Ca2+-induced opening of the PTP; diethone was the only compound that expressed antioxidant activity at very low concentrations (< or = 5 microM). Cerebrocrast acting as an inhibitor of the IMAC and diethone acting as an antioxidant could provide effective protective roles in preventing mitochondria from oxidative damage, favoring their therapeutic interest in the treatment of several pathological situations known to be associated with cellular oxidative stress.     

Death-associated protein kinase as a sensor of mitochondrial membrane potential: role of lysosome in mitochondrial toxin-induced cell death

We have investigated here the mechanism of dephosphorylation and activation of death-associated protein kinase (DAPK) and the role of lysosome in neuroblastoma cells (SH-SY5Y) treated with mitochondrial toxins, such as MPP(+) and rotenone. Mitochondrial respiratory chain inhibitors and uncouplers decreased mitochondrial membrane potential leading to DAPK dephosphorylation and activation. The class III phosphoinositide 3-kinase inhibitors attenuated DAPK dephosphorylation induced by mitochondrial toxins. Complex I inhibition by mitochondrial toxins (e.g. MPP(+)) resulted in mitochondrial swelling and lysosome reduction. Inhibition of class III phosphoinositide 3-kinase attenuated MPP(+)-induced lysosome reduction and cell death. The role of DAPK as a sensor of mitochondrial membrane potential in mitochondrial diseases was addressed.     

Ischemic preconditioning attenuates ischemia-reperfusion-induced mucosal apoptosis by inhibiting the mitochondria-dependent pathway in rat small intestine

Ischemic preconditioning provides a way of protecting organs from damage inflicted with prolonged ischemia-reperfusion. In this study, we investigated the mechanism of ischemic preconditioning involved in inhibition of prolonged ischemia-reperfusion-induced mucosal apoptosis in rat small intestine. Ischemic preconditioning was triggered by a transient occlusion of the superior mesenteric artery followed by reperfusion. Ischemia-reperfusion was induced by 60-min occlusion of the superior mesenteric artery followed by 60-min reperfusion in the small intestine. Ischemia-reperfusion alone induced mucosal apoptosis and mitochondrial respiratory dysfunction via promoted reactive oxygen species generation, reduced mitochondrial glutathione oxidation, increased mitochondrial lipid peroxidation, reduced mitochondrial membrane potential, and enhanced release of cytochrome c from mitochondria to activate caspase-9 and caspase-6 in the small intestine. Pretreatment with 20-min ischemia followed by 5-min reperfusion significantly inhibited the prolonged ischemia-reperfusion-induced mucosal apoptosis by 30%. Ischemic preconditioning ameliorated mitochondrial respiratory dysfunction by 50%, reduced reactive oxygen species generation by 38%, and suppressed mitochondrial lipid peroxidation by 36%, resulting in improvement of the mitochondrial membrane potential and prevention of cytochrome c release as well as caspase-6 activation. Results suggest that ischemic preconditioning attenuated ischemia-reperfusion-induced mucosal apoptosis partly by inhibiting the reactive oxygen species-mediated mitochondria-dependent pathway in the rat small intestine.     

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.     

Kynurenic acid attenuates MPP(+)-induced dopaminergic neuronal cell death via a Bax-mediated mitochondrial pathway

Kynurenic acid (KYNA), a tryptophan metabolite in the kynurenine pathway, is protective against various insults. However, the molecular mechanism of this protective effect has not been identified. In this study, we examined the protective effects of KYNA against 1-methyl-4-phenylpyridinium (MPP(+)), the best-characterized toxin inducing pathological changes resembling Parkinson's disease (PD), using SH-SY5Y and SK-N-SH human neuroblastoma cells. Pre-treatment of KYNA attenuated MPP(+)-induced neuronal cell death in SH-SY5Y and SK-N-SH cells. MPP(+)-induced cell death was preceded by increases in Bax expression and mitochondrial dysfunction, such as collapse of mitochondrial membrane potential (DeltaPsi(m)), release of cytochrome c from mitochondria into the cytoplasm, and increases in caspase-9/-3 activities. KYNA effectively inhibited all of these mitochondrial apoptotic processes. Our results indicate that KYNA plays a protective role by down-regulating Bax expression and maintaining mitochondrial function in MPP(+)-induced neuronal cell death, and suggest that KYNA may have therapeutic potential in PD.     

Reactive oxygen species‐induced cell death of rat primary astrocytes through mitochondria‐mediated mechanism

Astrocytes, the most abundant glial cell population in the central nervous system (CNS), play physiological roles in neuronal activities. Oxidative insult induced by the injury to the CNS causes neural cell death through extrinsic and intrinsic pathways. This study reports that reactive oxygen species (ROS) generated by exposure to the strong oxidizing agent, hexavalent chromium (Cr(VI)) as a chemical‐induced oxidative stress model, caused astrocytes to undergo an apoptosis‐like cell death through a caspase‐3‐independent mechanism. Although activating protein‐1 (AP‐1) and NF‐κB were activated in Cr(VI)‐primed astrocytes, the inhibition of their activity failed to increase astrocytic cell survival. The results further indicated that the reduction in mitochondrial membrane potential (MMP) was accompanied by an increase in the levels of ROS in Cr(VI)‐primed astrocytes. Moreover, pretreatment of astrocytes with N‐acetylcysteine (NAC), the potent ROS scavenger, attenuated ROS production and MMP loss in Cr(VI)‐primed astrocytes, and significantly increased the survival of astrocytes, implying that the elevated ROS disrupted the mitochondrial function to result in the reduction of astrocytic cell viability. In addition, the nuclear expression of apoptosis‐inducing factor (AIF) and endonuclease G (EndoG) was observed in Cr(VI)‐primed astrocytes. Taken together, evidence shows that astrocytic cell death occurs by ROS‐induced oxidative insult through a caspase‐3‐independent apoptotic mechanism involving the loss of MMP and an increase in the nuclear levels of mitochondrial pro‐apoptosis proteins (AIF/EndoG). This mitochondria‐mediated but caspase‐3‐independent apoptotic pathway may be involved in oxidative stress‐induced astrocytic cell death in the injured CNS. J. Cell. Biochem. 107: 933–943, 2009. © 2009 Wiley‐Liss, Inc.     

Relationships between the mitochondrial transmembrane potential, ATP concentration, and cytotoxicity in isolated rat hepatocytes

The relationships between mitochondrial transmembrane potential, ATP concentration, and cytotoxicity were evaluated after exposure of isolated rat hepatocytes to different mitochondrial poisons. Both the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its fully oxidized metabolite, the 1-methyl-4-phenylpyridinium (MPP+) ion, caused a concentration- and time-dependent depolarization of mitochondrial membranes which followed ATP depletion and preceded cytotoxicity. The effect of MPTP, but not that of MPP+, was prevented by deprenyl, an inhibitor of MPTP conversion to MPP+ via monoamine oxidase type B. Addition of fructose to the hepatocyte incubations treated with either MPTP or MPP+ counteracted the loss of mitochondrial transmembrane potential. Fructose was also effective in protecting against the mitochondrial membrane depolarization as well as ATP depletion and cytotoxicity induced by antimycin. A, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, and valinomycin. Data confirm the key role played by MPP(+)-induced mitochondrial damage in MPTP toxicity and indicate that (i) ATP produced via the glycolytic pathway can be utilized by hepatocytes to maintain mitochondrial electrochemical gradient, and (ii) a loss of mitochondrial membrane potential may occur only when supplies of ATP are depleted.