Rapid reduction of ATP synthesis and lack of free radical formation by MPP+ in rat brain synaptosomes and mitochondria

MPTP is a neurotoxin thought to damage dopaminergic neurons through free radical formation. MPTP is metabolized in the brain to MPP(+), which is taken up into dopaminergic neurons via the dopamine transporter and assumed to impair mitochondrial function. We used striatal synaptosomes and telencephalic mitochondria to further investigate MPP(+) mechanism of action. For comparison, the respiratory toxins FCCP, a cyanide analog that uncouples mitochondrial ATP production, and rotenone, a NADH dehydrogenase inhibitor, were also tested. FCCP, MPP(+) and rotenone caused a rapid but stable decrease in [3H]dopamine (DA) uptake by striatal synaptosomes. Two free radical scavengers, the salen-manganese complex EUK-134, and the spin trap s-PBN, did not prevent MPP(+)-induced decrease in DA uptake. However, addition of ATP during synaptosome preparation resulted in partial recovery of MPP(+)-induced [3H]DA uptake decrease. Generation of oxygen free radicals by treatment of telencephalic mitochondria with MPP(+), FCCP, or rotenone, was evaluated by measuring DCF fluorescence, while light emission by the luciferin-luciferase complex was used to determine ATP levels. MPP(+), unlike rotenone, did not produce oxygen free radicals, but rather blocked ATP production in mitochondria, as did FCCP and rotenone. Taken together, these results suggest that MPP(+) toxicity, at least during its initial stages, is primarily due to a decrease in ATP synthesis by mitochondria and not to free radical formation.     

Heat shock protects PC12 cells against MPP+ toxicity

A mild heat shock preconditioning has been shown to induce thermotolerance and protection against a number of cytotoxic agents that may induce cell death by either apoptosis or necrosis. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin that selectively targets dopaminergic cells of the substantia nigra and, as such, it is often used to induce neuronal cell death in models of Parkinson's disease. PC12 cells were heat-shocked for 1 h at 41.5 °C. This led to a rapid induction of Hsp25 and Hsp70. Levels of these proteins remained elevated for at least 24 h post heat shock. Treatment of PC12 cells with 1-methyl-4-phenylpyridinium (MPP⁺), the active metabolite of MPTP, resulted in cell death. Morphological analysis and the lack of caspase activity suggested that cell death was by necrosis. Heat shocking the cells 6 h prior to addition of MPP⁺ significantly inhibited the induction of cell death by MPP⁺. These results indicated that heat shock is protective against MPP⁺ neurotoxicity in PC12 cells.     

Protective effect of BAG5 on MPP+-induced apoptosis in PC12 cells

Abstract

Objectives: Parkinson’s disease (PD) is the most common neurodegenerative disease in humans, and an abundance of evidence has implicated apoptosis signaling pathways in the neurodegeneration of PD. The purpose of this study was to assess the role of B-cell lymphoma 2 (Bcl-2)-associated athanogene 5 (BAG5) protein, which was previously confirmed to play an important role in the pathogenesis of PD, in the regulation of apoptosis induced by 1-methyl-4-phenyl-pyridinium (MPP⁺) in PC12 cells.

Methods: PC12 cells were treated with MPP⁺ for 48 hours to induce apoptosis, and activation of Bcl-2, Bcl-xl, and caspase 3 was measured by western blot.

Results: The upregulation of BAG5 in PC12 cells inhibited apoptosis and increased the expression of anti-apoptotic proteins, including Bcl-2 and Bcl-xl, after MPP⁺ treatment. In addition, downregulation of BAG5 in PC12 cells enhanced apoptosis and decreased the expression of these proteins after MPP⁺ treatment.

Discussion: The data suggest that BAG5 inhibits MPP⁺-induced apoptosis through both endogenous and mitochondria-mediated pathways of apoptosis. Through this mechanism, the upregulation of BAG5 levels may occur through its anti-apoptotic activity in PD.     

Purine-induced alterations of dopamine metabolism in rat pheochromocytoma PC12 cells

Studies with cerebrospinal fluid from subjects with Parkinson's disease suggest that purine abnormalities may be present in this disorder. The effects of purines on dopamine metabolism have not been characterized, though adenosine is known to inhibit dopaminergic neurotransmission. In this study, dopamine, its precursor 3,4-dihydroxyphenylalanine (DOPA), and its degradation products 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were measured in rat pheochromocytoma PC12 cells following 24-h incubation with 5, 50, and 500 microM adenosine, adenine, guanosine, guanine, hypoxanthine, xanthine, and uric acid. Incubation with adenosine increased DOPA, DOPAC, and HVA, while adenine treatment decreased DOPA. Guanosine (500 microM) decreased DOPA, dopamine, and DOPAC, while lower concentrations increased DOPAC and HVA. Incubation with guanine decreased dopamine, and xanthine decreased dopamine and DOPAC. Hypoxanthine and uric acid exerted minimal effects. These results indicate that purines exert a variety of effects on dopamine metabolism. The influence of purine metabolism on the dopaminergic deficit in the Parkinsonian brain merits further investigation.     

Reserpine prevents hydroxyl radical formation by MPP in rat striatum

The present study investigated the blockage of dopamine (DA) oxidation by reserpine. 1-Methyl-4 phenylpyridinium ion (MPP+) increased the release of DA and the formation of hydroxyl radical ( r22. OH). The r22. OH generated by DA when captured as the hydroxylated derivative of salicylic acid was measured by the high-performance liquid chromatographic-electrochemical (HPLC-EC) procedure. MPP+ concentration for half-maximal effect of DA producing release (EC50) was 5.2 mM. The maximum attainable concentration of dialysate DA (Emax) by MPP+ was 7.7 microM. However, the EC50 and Emax values with reserpinized animal were 5.2 mM and 1.2 microM, respectively. When high concentration of pargyline (10 mM) were infused in MPP+ (5 mM)-pretreated animals, a marked elevation of DA and r22. OH formation was observed. The level of DA and 2, 3-DHBA formations was drastically reduced, as compared with the MPP+-only treated group. Although the levels of MPP+-induced DA and 2,3-DHBA formation after pargyline treatment increased, pargyline failed to increase either the level of MPP+-induced DA or 2,3-DHBA in the reserpinized group. When DA was administered to the MPP+-pretreatment group, a marked elevation was observed, showing a positive linear correlation DA and r22. OH formation trapped as 2,3-DHBA (R2=0.978) in the dialysate. When corresponding experiment were performed with iron (II), the same results were obtained: a positive linear correlation between the release of DA and 2,3-DHBA (R2=0.989) in the dialysate. These results indicated that reserpine-induced DA depletion may reduce MPP+-induced r22. OH formation.     

Oxidative stress induced by MPTP and MPP: selective vulnerability of cultured mouse astrocytes

Oxidative stress has been implicated in the pathogenesis of Parkinson's disease. In the present study, reactive oxygen species (ROS) formation and antioxidant enzyme superoxide dismutase (SOD) activities were examined in cultured cortical, striatal and mesencephalic mouse astrocytes after 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) or 1-methyl-4-phenylpyridinium (MPP(+)) treatment. Linear regression analysis showed that control mesencephalic (slope coefficient=0.01) astrocytes had a three-fold (F-test, p<0.05) greater rate of change in ROS production when compared to cortical (0.003) or striatal (0.003) astrocytes. However, when treated with 500 microM MPTP for 120 min, mesencephalic and striatal astrocytes demonstrated a decreased and increased rate of change in ROS production respectively. On the other hand, when treated with 10 microM MPP(+), a significant increase in the rate of change in ROS formation was observed in both mesencephalic and striatal astrocytes, with mesencephalic astrocytes producing a four-fold greater increase when compared to striatal astrocytes. Cortical astrocytes did not show any significant changes in ROS production when treated with MPTP or MPP(+). When astrocytes were treated with MPTP over a 24 h period, striatal astrocytes demonstrated significant increases in SOD activity to 12 h, followed by a return towards control levels after 8 h treatment. In contrast, mesencephalic astrocytes showed trends for a decrease in SOD production as well as a significant decrease in ATP levels by 24 h MPTP treatment. The present results suggested that mesencephalic astrocytes are more vulnerable to oxidative stress when compared to striatal astrocytes, given their greater rates of ROS production at basal and MPP(+) conditions. Striatal astrocytes, on the other hand, may have a more protective capacity against oxidative stress by producing greater SOD activities.     

Nitric oxide enhances MPP(+)-induced hydroxyl radical generation via depolarization activated nitric oxide synthase in rat striatum

We examined the effect of N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, on extracellular potassium ion concentration ([K(+)](o))-enhanced hydroxyl radical (.OH) generation due to 1-methyl-4-phenylpyridinium ion (MPP(+)) was examined in the rat striatum. Rats were anesthetized, and sodium salicylate in Ringer's solution (0.5 nmol/microl per min) was infused through a microdialysis probe to detect the generation of.OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the striatum. Induction of KCl (20, 70 and 140 mM) increased MPP(+)-induced.OH formation trapped as 2,3-dihydroxybenzoic acid (DHBA) in a concentration dependent manner. However, the application of L-NAME (5 mg/kg i.v.) abolished the [K(+)](o) depolarization-induced.OH formation with MPP(+). Dopamine (DA; 10 microM) also increased the levels of DHBA due to MPP(+). However, the effect of DA after application of L-NAME did not change the levels of DHBA. On the other hand, the application of allopurinol (20 mg/kg i.v., 30 min prior to study), a xanthine oxidase (XO) inhibitor was abolished the both [K(+)](o)- and DA-induced.OH generation. Moreover, when iron(II) was administered to MPP(+) then [K(+)](o) (70 mM)-pretreated animals, a marked increase in the level of DHBA. However, when corresponding experiments were performed with L-NAME-pretreated animals, the same results were obtained. Therefore, NOS activation may be no relation to Fenton-type reaction via [K(+)](o) depolarization-induced.OH generation. The present results suggest that [K(+)](o)-induced depolarization augmented MPP(+)-induced.OH formation by enhancing NO synthesis.     

Heat shock proteins protect cultured fibroblasts from the cytotoxic effects of MPP

1-Methyl-4-phenylpyridinium (MPP⁺), the cytotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been shown to be toxic to a variety of cell types in culture. The addition of media containing 1 mM MPP+ to cultures of Chinese hamster ovary (CHO) fibroblasts led to the gradual depletion of cellular ATP stores and subsequent cell death. A 12-min heat shock of the cells at 45°C, 3 h prior to the addition of MPP⁺-containing media, significantly attenuated cell death. Heat shock pretreatment led to an increased synthesis of all the major heat shock proteins (HSPs) in CHO cells. Further, the addition of the protein synthesis inhibitor, cycloheximide, prevented the protective effect of heat shock pretreatment, indicating that this protection was dependent upon new protein synthesis. In additional experiments, a rat fibroblast cell line which has been stably transfected with, and constitutively expresses a cloned human HSP-70 gene, was found to be more resistant to the cytotoxic effects of MPP⁺ than the parental fibroblast cell line. These results indicate that HSPs are protective toward the deleterious effects of MPP+ and that their synthesis represents an important parameter in the neurotoxicity of MPTP.     

Targeting reactive oxygen species,reactive nitrogen species and inflammation in MPTP neurotoxicity and Parkinson’s disease

Parkinson’s disease (PD) is the second most frequent neurodegenerative disorder after Alzheimer’s disease. The main clinical features of PD include tremor, bradykinesia, rigidity and postural instability. The primary pathology of PD is degeneration of dopaminergic neurons in the substantia nigra pars compacta, resulting in loss of the nigrostriatal pathway and a reduction of dopamine contents in the striatum. The biochemical and cellular changes that occur following the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are remarkably similar to that seen in idiopathic PD. Recent evidence shows that oxidative stress contributes to the cascade leading to dopaminergic cell degeneration in PD. However, oxidative stress is intimately linked to other components of neurodegenerative process, such as nitric oxide stress and inflammation. Recently, there is convincing evidence for the involvement of nitric oxide that reacts with superoxide to produce peroxynitrite and ultimately hydroxyl radical production. In view of these new insights, however, the role of reactive nitrogen species, reactive oxygen species and inflammation against MPTP neurotoxicity is not fully understood. In this review, we discuss the possible role of reactive nitrogen species, reactive oxygen species and inflammation in the dopaminergic neurons against MPTP neurotoxicity.     

Nicotine administration reduces striatal MPP+ levels in mice

Nicotine administration has previously been shown to attenuate nigrostriatal damage in animal models of Parkinson's disease, including the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mouse. The present experiments were done to determine whether nicotine may be exerting its effects by altering striatal levels of 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of MPTP. Mice were injected with nicotine (0.33-1 mg/kg i.p.) 10 min prior to MPTP (30 mg/kg s.c.) followed by three subsequent doses of nicotine at 15-min intervals according to a dose schedule previously shown to be neuroprotective. The mice were sacrificed 1.5, 4 and 8 h after MPTP administration and striatal MPP+ levels measured. Nicotine administration (0.33-1.0 mg/kg) resulted in a time-dependent decline in striatal MPP+ levels that was significantly enhanced over that in saline injected animals. Experiments done to examine the effect of age showed that the decrease was observed in older (8-10 months) but not young (6-8 weeks) mice, a finding which may explain some of the variability in the effect of nicotine in the MPTP-induced model of nigrostriatal degeneration. In summary, these results suggest that nicotine may exert its neuroprotective action against nigrostriatal degeneration, at least in part, by decreasing striatal MPP+ levels.     

Release of dopamine by perfusion with 1-methyl-4-phenylpyridinium ion (MPP) into the striatum is associated with hydroxyl free radical generation

In Parkinson's disease (PD), the dopamine (DA) neuronal cell death in the nigrostriatal system has been proposed to be mediated by reactive oxygen radicals such as hydroxyl radicals (.OH). This.OH production may cause lipid peroxidation of cell membranes leading to neuronal cell death. This paper report that the DA-selective neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP(+)), (1 nmol/microl per min for 1 h) infusion into the striatum of rats induces elevation of extracellular DA and.OH formation. These elevations seem to induce lipid peroxidation of striatum membranes, as detected by increases in non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) levels. To test the involvement of DA release in the.OH generation and lipid peroxidation, the rats were pretreated with reserpine (5 mg/kg, i.v., 24 h before MPP(+) or without MPP(+)) to deplete presynaptic DA. Reserpine treatment alone did not change the levels of DA or 2,3-DHBA, while the combined treatment with both MPP(+) and reserpine clearly decreased 2,3-DHBA, as well as DA levels, compared to those in the group treated with MPP(+) alone. After injection into reserpinized rats, DA at various doses (2, 5 and 10 microM) small increased 2,3-DHBA levels dose-dependently, as compared to the MPP(+) alone-treated group. These results clearly indicate that MPP(+) perfusion into the striatum increases extracellular DA levels and this increase may concomitantly induce the formation of reactive free oxygen radicals, such as.OH free radicals. These events may contribute, at least in part, to the nigrostriatal neurons cell death after MPP(+).     

6-OHDA诱导PC12细胞凋亡及作用机制

目的 探讨6-羟基多巴胺(6-OHDA)诱导PC12细胞损伤的可能作用机制.方法 不同剂量6-OHDA加入培养的大鼠肾上腺嗜铬细胞瘤细胞(pheochromocytoma cell,PC12)24h后,用四甲基偶氮唑盐法(MTT法)检测细胞的活力,流式细胞仪检测细胞凋亡率以及Bax、Bcl-2的蛋白表达.结果 在加入不同浓度的6-OHDA时PC12细胞活力显著下降,细胞凋亡百分率对照组为8.73±1.09,不同浓度的6-OHDA处理组显著上升,分别为10.97±1.52、25.77±0.95、57.94±1.23,较对照组有显著性差异(P<0.01),并且Bcl-2蛋白表达下降,Bax蛋白表达上升,Bcl-2/Bax比值和正常组比较有显著性差异(P<0.01).结论 6-OHDA能显著诱导PC12细胞损伤,并呈剂量依赖性,其作用机制涉及到促进细胞内bax,以及抑制Bcl-2的表达.     

Role of reactive nitrogen and reactive oxygen species against MPTP neurotoxicity in mice

There is growing evidence indicating that reactive nitrogen species (RNS) and reactive oxygen species (ROS) are a major contributor to the pathogenesis and progression of Parkinson's disease. Here we investigated whether edaravone (free radical scavenger), minocycline (inducible nitric oxide synthase, iNOS inhibitor), 7-nitroindazole (neuronal NOS, nNOS inhibitor), fluvastatin (endothelial NOS, eNOS activator) and pitavastatin (eNOS activator) can protect against MPTP neurotoxicity in mice under the same condition. The present study showed that 7-nitroindazole could protect dose-dependently against the striatal dopamine depletions in mice 5 days after MPTP treatment. In contrast, edaravone, minocycline, fluvastatin and pitavastatin did not show the neuroprotective effect on MPTP-induced striatal dopamine depletion. Our immunohistochemical study showed that TH (tyrosine hydroxylase) and DAT (dopamine transporter) immunoreactivity was decreased significantly in the striatum and substantia nigra 5 days after MPTP treatment. The administration of 7-nitroindazole showed a protective effect against the severe reductions in levels of TH and DAT immunoreactivity in the striatum and substantia nigra 5 days after MPTP treatment. Furthermore, our Western blot analyses study showed the remarkable loss of TH protein levels in the striatum 5 days after MPTP treatment. In contrast, 7-nitroindazole prevented a significant loss in TH protein levels in the striatum 5 days after MPTP treatment. On the other hand, GFAP (glial fibrillary acidic protein) immunoreactivity increased significantly in the striatum and substantia nigra, 5 days after MPTP treatment. 7-Nitroindazole ameliorated severe increases in number of GFAP immunoreactive astrocytes in the striatum and substantia nigra 5 days after MPTP treatment. Furthermore, our Western blot analyses study showed the increase of GFAP protein levels in the striatum 5 days after MPTP treatment. 7-Nitroindazole prevented a significant increase in the GFAP protein levels in the striatum 5 days after MPTP treatment. The present results indicate that 7-nitroindazole can protect dose-dependently against the striatal dopamine depletions in mice 5 days after MPTP treatment. In contrast, edaravone, minocycline, fluvastatin and pitavastatin did not show the neuroprotective effect on MPTP-induced striatal dopamine depletions. These findings demonstrate that the overexpression of nNOS may play a major role in the neurotoxic processes of MPTP, as compared to the production of ROS, the overexpression of iNOS and the modulation of eNOS. Thus, our findings provide strong evidence for neuroprotective properties of nNOS inhibitor in this animal model of Parkinson's disease.     

Calcium overload enhances hydroxyl radical generation by 1-methyl-4 phenylpyridinium ion (MPP+) in rat striatum

We examined whether ouabain-induced Ca(2+) overload increases hydroxyl radical (*OH) generation by 1-methyl-4-phenylpyridinium ion (MPP(+)) in rat striatum. These elevations seem to induce lipid peroxidation of striatum of rats, as detected by increases in non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) levels. Ouabain enhanced MPP(+)-induced *OH formation trapped as DHBA. Moreover, when iron (II) was administered to MPP(+) then ouabain (100 micro M)-pretreated animals, a marked elevation in the level of DHBA was observed, as compared with the iron (II)-only-treated animals. These results suggests that Ca(2+) overload might enhance *OH generation by MPP(+) in rat striatum.     

Mechanisms of MPP incorporation into cerebellar granule cells

Exposure of cerebellar granule cells to 1-methyl-4-phenylpiridinium (MPP(+)) results in cell death. We have studied the implication of various membrane transporter systems on MPP(+) neurotoxicity, including the dopamine transporter system (DAT) and cationic amino acid transporters (CAT). We have showed a partial protection against MPP(+) toxicity when the dopamine transporter is inhibited by 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]4-(3-phenylpropyl)piperazinedihydrochloride (GBR-12909). However, almost full protection is only achieved by the simultaneous addition of GBR-12909 and cationic amino acids. These results suggest two ways system of MPP(+) entrance into cerebellar granule cells: the DAT with high activity and the CAT with low activity. We also demonstrated that 5,7-dichlorokynurenic acid (MK-801) failed to protect against MPP(+) exposure, evidencing that N-methyl-D-aspartate (NMDA) receptor is not involved in the MPP(+)-induced cell death.     

Oxidative metabolite of dopamine, 3,4-dihydroxyphenylacetaldehyde, induces dopamine release from PC12 cells by a Ca2+-independent mechanism

3,4-dihydroxyphenylacetaldehyde (DOPALD), an oxidative metabolite of dopamine (DA), induced dose-dependent DA release from pheochromocytoma (PC12) cells without affecting leakage of lactate dehydrogenase from the cells. DOPALD-induced DA release was independent of extracellular Ca2+ concentration and was not blocked by nifedipine, an L-type Ca2+ channel antagonist. These results indicated a novel intrinsic role of DOPALD in dopaminergic nerve terminals that may take part in the activation of dopamine neurons.     

Morphine reverses the effects of 1-methyl-4-phenylpyridinium in PC12 cells through activating PI3K/Akt

Aim of the study: Parkinson’s disease (PD) is a neurodegenerative disorder. It is caused by the degeneration of dopaminergic neurons and the dopamine (DA) deletion in the substantia nigra pars compacta (SNpc). Morphine elevates the level of dopamine in the mesolimbic dopamine system and plays a role in alleviating PD symptoms. However, the molecular mechanism is still unclear. The aim of the study is to investigate the mechanism on morphine alleviating PD symptoms.

Materials and methods: The viability of PC12 cells was measured by using MTT assay. The expressions of tyrosine hydroxylase (TH), thioredoxin-1 (Trx-1), CyclinD1 and Cyclin-dependent kinase5 (Cdk5) were detected by Western Blot.

Results: In present study, we found that morphine increased the cell viability in PC12 cells. 1-methyl-4-phenylpyridi-nium (MPP+) reduced the cell viability and TH expression, which were reversed by morphine. MPP+ decreased the expressions of Trx-1, CyclinD1, Cdk5, which were restored by morphine. Moreover, the role of morphine in restoring the expressions of Trx-1, CyclinD1 and Cdk5 decreased by MPP+ was abolished by LY294002, phosphatidylinositol-3-kinase (PI3K)/Akt inhibitor.

Conclusions: These results suggest that morphine reverses effects induced by MPP þ through activating PI3K/Akt pathway.     

Neuronal protective and rescue effects of deprenyl against MPP+ dopaminergic toxicity

Summary Intranigral infusion of 1-Methyl-4-phenylpyridinium ion (MPP⁺, 2.1–16.8 nmol) dose-dependently injured nigral neurons as reflected by reduced dopamine levels in the ipsilateral striatum four days after the infusion of this toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Coadministration of deprenyl (4.2 nmol) with MPP⁺ into the substantia nigra protected against MPP⁺-induced moderate (20–50%) but not severe (over 70%) nigral injury as reflected in striatal dopamine reductions. However, supplementary treatment with deprenyl (0.25 mg/kg, s.c., twice daily for 4 days) after intranigral infusion of MPP⁺ significantly rescued nigral neurons from more severe damage caused by a higher MPP⁺ does (8.4 nmol) manifested by a lesser striatal dopamine decrease (–31%) compared to the non-deprenyl treated group (–70%). Thus, in addition to the blockade of bioactivation of MPTP, deprenyl can protect and/or rescue nigral neurons from MPP⁺-induced dopaminergic neurotoxicity. These in vivo data add further evidence to suggest that deprenyl, a putative and clinically unproven neuroprotective agent, may be of value in slowing the progressive nigral degeneration in early Parkinson's disease, but may prove to be less so in its terminal stages.     

Overexpression of Bcl-2 attenuates MPP, but not 6-ODHA, induced cell death in a dopaminergic neuronal cell line

In order to investigate the role of Bcl-2 in dopaminergic cells, we established a dopaminergic neuronal cell line (MN9D) stably expressing human Bcl-2 (MN9D/Bcl-2) or neomycin (MN9D/Neo). Overexpression of Bcl-2 in MN9D cells attenuated cell death due to treatment of mitochondrial electron transport inhibitors includingN-methyl-4-phenylpyridinium, whereas it did not prevent cell death induced by reagents generating reactive oxygen species including 6-hydroxy-dopamine. Moreover, the rate of glucose uptake in MN9D/Bcl-2 was significantly lower than that in MN9D/Neo after MPP⁺treatment. Thus, Bcl-2 may counter aberrations in mitochondrial electron transfer processes by altering energy metabolism within the MN9D cells.