Dopamine Selectively Sensitizes Dopaminergic Neurons to Rotenone-Induced Apoptosis

Among various types of neurons affected in Parkinson’s disease, dopamine (DA) neurons of the substantia nigra undergo the most pronounced degeneration. Products of DA oxidation and consequent cellular damage have been hypothesized to contribute to neuronal death. To examine whether elevated intracellular DA will selectively predispose the dopaminergic subpopulation of nigral neurons to damage by an oxidative insult, we first cultured rat primary mesencephalic cells in the presence of rotenone to elevate reactive oxygen species. Although MAP2⁺ neurons were more sensitive to rotenone-induced toxicity than type 1 astrocytes, rotenone affected equally both DA (TH⁺) neurons and MAP2⁺ neurons. In contrast, when intracellular DA concentration was elevated, DA neurons became selectively sensitized to rotenone. Raising intracellular DA levels in primary DA neurons resulted in dopaminergic neuron death in the presence of subtoxic concentrations of rotenone. Furthermore, mitochondrial superoxide dismutase mimetic, manganese (III) meso-tetrakis (4-benzoic acid) porphyrin, blocked activation of caspase-3, and consequent cell death. Our results demonstrate that an inhibitor of mitochondrial complex I and increased cytosolic DA may cooperatively lead to conditions of elevated oxidative stress and thereby promote selective demise of dopaminergic neurons.     

Ultrastructural characterization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced cell death in embryonic dopaminergic neurons

Developing neuronal populations undergo significant attrition by natural cell death. Dopaminergic neurons in the substantia nigra pars compacta undergo apoptosis during synaptogenesis. Following this time window, destruction of the anatomic target of dopaminergic neurons results in dopaminergic cell death but the morphology is no longer apoptotic. We describe ultrastructural changes that appear unique to dying embryonic dopaminergic neurons. In primary cultures of mesencephalon, death of dopaminergic neurons is triggered by activation of glutamate receptors sensitive to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and differs ultrastructurally from both neuronal apoptosis or typical excitotoxicity. AMPA causes morphological changes selectively in dopaminergic neurons, without affecting other neurons in the same culture dishes. Two hours after the onset of treatment swelling of Golgi complexes is apparent. At 3 h, dopaminergic neurons display loss of membrane asymmetry (coinciding with commitment to die), as well as nuclear membrane invagination, irregular aggregation of chromatin, and mitochondrial swelling. Nuclear changes continue to worsen until loss of cytoplasmic structures and cell death begins to occur after 12 h. These changes are different from those described in neurons undergoing either apoptosis or excitotoxic death, but are similar to ultrastructural changes observed in spontaneous death of dopaminergic neurons in the natural mutant weaver mouse.     

Is Bax a mitochondrial mediator in apoptotic death of dopaminergic neurons in Parkinson's disease?

Bax is a proapoptotic member of the Bcl-2 family of proteins. It is believed to exert its action primarily by facilitating the release of cytochrome c from the mitochondrial intermembrane space into the cytosol, leading to caspase activation and cell death. Because alterations in mitochondrial respiratory function, caspase activation and cell death with morphologic features compatible with apoptosis have been observed post mortem in the brain of patients with Parkinson's disease, we tried to clarify the potential role of Bax in this process in an immunohistochemical study on normal and Parkinson's disease post-mortem brain and primary mesencephalic cell cultures treated with MPP(+). We found that Bax is expressed ubiquitously by dopaminergic (DA) neurons in post-mortem brain of normal and Parkinson's disease subjects as well as in vitro. Using an antibody to Bax inserted into the outer mitochondrial membrane as an index of Bax activation, no significant differences were observed between control and Parkinson's disease subjects, regardless of the mesencephalic subregion analysed. However, in Parkinson's disease subjects, the percentage of Bax-positive melanized SNpc neurons containing Lewy bodies, suggestive of DA neuronal suffering, was significantly higher than the overall percentage of Bax-positive neurons among melanized neurons. Furthermore, all melanized SNpc neurons in Parkinson's disease subjects with activated caspase-3 were also immunoreactive for Bax, suggesting that Bax anchored in the outer mitochondrial membrane of melanized SNpc neurons showing signs of neuronal suffering or apoptosis is increased compared with DA neurons that are apparently unaltered. Surprisingly, MPP(+) treatment of tyrosine hydroxylase (TH)-positive neurons in primary mesencephalic cultures did not cause redistribution of Bax, although cytochrome c was released from the mitochondria and nuclear condensation/fragmentation was induced. Taken together, these findings suggest that in the human pathology, Bax may be a cofactor in caspase activation, but our in vitro data fail to indicate a central role for Bax in apoptotic death of DA neurons in an experimental Parkinson's disease paradigm.     

Loss of caspase-2-dependent apoptosis induces autophagy after mitochondrial oxidative stress in primary cultures of young adult cortical neurons

Mitochondrial dysfunctions have been associated with neuronal apoptosis and are characteristic of neurodegenerative conditions. Caspases play a central role in apoptosis; however, their involvement in mitochondrial dysfunction-induced neuronal apoptosis remains elusive. In the present report using rotenone, a complex I inhibitor that causes mitochondrial dysfunction, we determined the initiator caspase and its role in cell death in primary cultures of cortical neurons from young adult mice (1-2 months old). By pretreating the cells with a cell-permeable, biotinylated pan-caspase inhibitor that irreversibly binds to and traps the active caspase, we identified caspase-2 as an initiator caspase activated in rotenone-treated primary neurons. Loss of caspase-2 inhibited rotenone-induced apoptosis; however, these neurons underwent a delayed cell death by necrosis. We further found that caspase-2 acts upstream of mitochondria to mediate rotenone-induced apoptosis in neurons. The loss of caspase-2 significantly inhibited rotenone-induced activation of Bid and Bax and the release of cytochrome c and apoptosis inducing factor from mitochondria. Rotenone-induced downstream activation of caspase-3 and caspase-9 were also inhibited in the neurons lacking caspase-2. Autophagy was enhanced in caspase-2 knock-out neurons after rotenone treatment, and this response was important in prolonging neuronal survival. In summary, the present study identifies a novel function of caspase-2 in mitochondrial oxidative stress-induced apoptosis in neurons cultured from young adult mice.     

Subcellular expression and neuroprotective effects of SK channels in human dopaminergic neurons

Small-conductance Ca²⁺-activated K⁺ channel activation is an emerging therapeutic approach for treatment of neurological diseases, including stroke, amyotrophic lateral sclerosis and schizophrenia. Our previous studies showed that activation of SK channels exerted neuroprotective effects through inhibition of NMDAR-mediated excitotoxicity. In this study, we tested the therapeutic potential of SK channel activation of NS309 (25 μM) in cultured human postmitotic dopaminergic neurons in vitro conditionally immortalized and differentiated from human fetal mesencephalic cells. Quantitative RT-PCR and western blotting analysis showed that differentiated dopaminergic neurons expressed low levels of SK2 channels and high levels of SK1 and SK3 channels. Further, protein analysis of subcellular fractions revealed expression of SK2 channel subtype in mitochondrial-enriched fraction. Mitochondrial complex I inhibitor rotenone (0.5 μM) disrupted the dendritic network of human dopaminergic neurons and induced neuronal death. SK channel activation reduced mitochondrial membrane potential, while it preserved the dendritic network, cell viability and ATP levels after rotenone challenge. Mitochondrial dysfunction and delayed dopaminergic cell death were prevented by increasing and/or stabilizing SK channel activity. Overall, our findings show that activation of SK channels provides protective effects in human dopaminergic neurons, likely via activation of both membrane and mitochondrial SK channels. Thus, SK channels are promising therapeutic targets for neurodegenerative disorders such as Parkinson''s disease, where dopaminergic cell loss is associated with progression of the disease.     

The pesticide rotenone induces caspase-3-mediated apoptosis in ventral mesencephalic dopaminergic neurons

In vivo, the pesticide rotenone induces degeneration of dopamine neurons and parkinsonian-like pathology in adult rats. In the current study, we utilized primary ventral mesencephalic (VM) cultures from E15 rats as an in vitro model to examine the mechanism underlying rotenone-induced death of dopamine neurons. After 11 h of exposure to 30 nm rotenone, the number of dopamine neurons identified by tyrosine hydroxylase (TH) immunostaining declined rapidly with only 23% of the neurons surviving. By contrast, 73% of total cells survived rotenone treatment, indicating that TH+ neurons are more sensitive to rotenone. Examination of the role of apoptosis in TH+ neuron death, revealed that 10 and 30 nm rotenone significantly increased the number of apoptotic TH+ neurons from 7% under control conditions to 38 and 55%, respectively. The increase in apoptotic TH+ neurons correlated with an increase in immunoreactivity for active caspase-3 in TH+ neurons. The caspase-3 inhibitor, DEVD, rescued a significant number of TH+ neurons from rotenone-induced death. Furthermore, this protective effect lasted for at least 32 h post-rotenone and DEVD exposure, indicating lasting neuroprotection achieved with an intervention prior to the death commitment point. Our results show for the first time in primary dopamine neurons that, at low nanomolar concentrations, rotenone induces caspase-3-mediated apoptosis. Understanding the mechanism of rotenone-induced apoptosis in dopamine neurons may contribute to the development of new neuroprotective strategies against Parkinson's disease.     

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.     

Novel imine antioxidants at low nanomolar concentrations protect dopaminergic cells from oxidative neurotoxicity

Strong evidence indicates that oxidative stress may be causally involved in the pathogenesis of Parkinson's disease. We have employed human dopaminergic neuroblastoma cells and rat primary mesencephalic neurons to assess the protective potential of three novel bisarylimine antioxidants on dopaminergic cell death induced by complex I inhibition or glutathione depletion. We have found that exceptionally low concentrations (EC₅₀ values ∼20 nM) of these compounds (iminostilbene, phenothiazine, and phenoxazine) exhibited strong protective effects against the toxicities of MPP⁺, rotenone, and l -buthionine sulfoximine. Investigating intracellular glutathione levels, it was found that MPP⁺, l -buthionine sulfoximine, and rotenone disrupted different aspects of the native glutathione equilibrium, while the aromatic imines did not further influence glutathione levels or redox state on any baseline. However, the imines independently reduced protein oxidation and total oxidant flux, saved the mitochondrial membrane potential, and provided full cytoprotection under conditions of complete glutathione depletion. The unusually potent antioxidant effects of the bisarylimines could be reproduced in isolated mitochondria, which were instantly protected from lipid peroxidation and pathological swelling. Aromatic imines may be interesting lead structures for a potential antioxidant therapy of Parkinson's disease and other disorders accompanied by glutathione dysregulation.     

Bioenergetic and oxidative effects of free 3-nitrotyrosine in culture: selective vulnerability of dopaminergic neurons and increased sensitivity of non-dopaminergic neurons to dopamine oxidation

Protein bound and free 3-nitrotyrosine (3NT) levels are elevated in neurodegenerative diseases and have been used as evidence for peroxynitrite generation. Intrastriatal injection of free 3NT causes dopaminergic neuron injury and represents a new mouse model of Parkinson's disease (PD). We are investigating the nature of free 3NT neurotoxicity. In primary ventral midbrain cultures, free 3NT damaged dopaminergic neurons, while adjacent non-dopaminergic neurons were unaffected. Combined treatment with free 3NT and subtoxic amounts of dopamine caused extensive death of non-dopaminergic forebrain neurons in culture. Free 3NT alone directly inhibited mitochondrial complex I, decreased ATP, sensitized neurons to mitochondrial depolarization, and increased superoxide production. Subtoxic concentrations of rotenone (instead of free 3NT) caused similar results. Additionally, free 3NT and dopamine combined increased extraneuronal hydrogen peroxide and decreased intraneuronal glutathione levels more than dopamine alone. Oxidative and bioenergetic processes have been proposed to contribute to neurodegeneration in PD. As free 3NT is a compound that is increased in PD, damages dopamine neurons in vivo and in vitro and has detrimental effects on neuronal bioenergetics, it is possible that free 3NT is an endogenous contributing factor to neuronal loss, in addition to being a marker of oxidative and nitrative processes.     

Rapid purification and mass spectrometric characterization of mitochondrial NADH dehydrogenase (Complex I) from rodent brain and a dopaminergic neuronal cell line

Oxidative stress and mitochondrial dysfunction signify important biochemical events associated with the loss of dopaminergic neurons in Parkinson's disease (PD). Studies using in vitro and in vivo PD models or tissues from diseased patients have demonstrated a selective inhibition of mitochondrial NADH dehydrogenase (Complex I of the OXPHOS electron transport chain) that affects normal mitochondrial physiology leading to neuronal death. In an earlier study, we demonstrated that oxidative stress due to glutathione depletion in dopaminergic cells, a hallmark of PD, leads to Complex I inhibition via cysteine thiol oxidation (Jha et al. (2000) J. Biol. Chem. 275, 26096-26101). Complex I is a approximately 980-kDa multimeric enzyme spanning the inner mitochondrial membrane comprising at least 45 protein subunits. As a prerequisite to investigating modifications to Complex I using a rodent disease model for PD, we developed two independent rapid and mild isolation procedures based on sucrose gradient fractionation and immunoprecipitation to isolate Complex I from mouse brain and a cultured rat mesencephalic dopaminergic neuronal cell line. Both protocols are capable of purifying Complex I from small amounts of rodent tissue and cell cultures. Blue Native gel electrophoresis, one-dimensional and two-dimensional SDS-PAGE were employed to assess the purity and composition of isolated Complex I followed by extensive mass spectrometric characterization. Altogether, 41 of 45 rodent Complex I subunits achieved MS/MS sequence coverage. To our knowledge, this study provides the first detailed mass spectrometric analysis of neuronal Complex I proteins and provides a means to investigate the role of cysteine oxidation and other posttranslational modifications in pathologies associated with mitochondrial dysfunction.     

Protective effects of riluzole on dopamine neurons: involvement of oxidative stress and cellular energy metabolism

Riluzole is neuroprotective in patients with amyotrophic lateral sclerosis and may also protect dopamine (DA) neurons in Parkinson's disease. We examined the neuroprotective potential of riluzole on DA neurons using primary rat mesencephalic cultures and human dopaminergic neuroblastoma SH-SY5Y cells. Riluzole (up to 10 microM:) alone affected neither the survival of DA neurons in primary cultures nor the growth of SH-SY5Y cells after up to 72 h. Riluzole (1-10 microM:) dose-dependently reduced DA cell loss caused by exposure to MPP(+) in both types of cultures. These protective effects were accompanied by a dose-dependent decrease of intracellular ATP depletion caused by MPP(+) (30-300 microM:) in SH-SY5Y cells without affecting intracellular net NADH content, suggesting a reduction of cellular ATP consumption rather than normalization of mitochondrial ATP production. Riluzole (1-10 microM:) also attenuated oxidative injury in both cell types induced by exposure to L-DOPA and 6-hydroxydopamine, respectively. Consistent with its antioxidative effects, riluzole reduced lipid peroxidation induced by Fe(3+) and L-DOPA in primary mesencephalic cultures. Riluzole (10 microM) did not alter high-affinity uptake of either DA or MPP(+). However, in the same cell systems, riluzole induced neuronal and glial cell death with concentrations higher than those needed for maximal protective effects (> or =100 microM:). These data demonstrate that riluzole has protective effects on DA neurons in vitro against neuronal injuries induced by (a) impairment of cellular energy metabolism and/or (b) oxidative stress. These results provide further impetus to explore the neuroprotective potential of riluzole in Parkinson's disease.     

Prostate Apoptosis Response-4 Mediates Trophic Factor Withdrawal-Induced Apoptosis of Hippocampal Neurons

Prostate apoptosis response-4 (Par-4) is the product of a gene up-regulated in prostate cancer cells undergoing apoptosis. We now report that Par-4 mRNA and protein levels rapidly and progressively increase 4-24 h following trophic factor withdrawal (TFW) in cultured embryonic rat hippocampal neurons. The increased Par-4 levels follow an increase of reactive oxygen species, and precede mitochondrial membrane depolarization, caspase activation, and nuclear chromatin condensation/fragmentation. Pretreatment of cultures with 17beta-estradiol, vitamin E, and uric acid largely prevented Par-4 induction and cell death following TFW, demonstrating necessary roles for oxidative stress and membrane lipid peroxidation in TFW-induced neuronal apoptosis. Par-4 antisense oligonucleotide treatment blocked Par-4 protein increases and attenuated mitochondrial dysfunction, caspase activation, and cell death following TFW. Collectively, our data identify Par-4 as an early and pivotal player in neuronal apoptosis resulting from TFW and suggest that estrogen and antioxidants may prevent apoptosis, in part, by suppressing Par-4 production.     

Rotenone induces cell death in primary dopaminergic culture by increasing ROS production and inhibiting mitochondrial respiration

Although the definite etiology of Parkinson's disease is still unclear, increasing evidence has suggested an important role for environmental factors such as exposure to pesticides in increasing the risk of developing Parkinson's disease. In the present study, primary cultures prepared from embryonic mouse mesencephala were applied to investigate the toxic effects and underlying mechanisms of rotenone-induced neuronal cell death relevant to Parkinson's disease. Results revealed that rotenone destroyed dopaminergic neurons in a dose- and time-dependent manner. Consistent with the cytotoxic effect of rotenone as evidenced by dopaminergic cell loss, it significantly increased the release of lactate dehydrogenase into the culture medium, the number of necrotic cells in the culture and the number of nuclei showing apoptotic features. Rotenone exerted toxicity by decreasing the mitochondrial membrane potential, increasing reactive oxygen species production and shifting respiration to a more anaerobic state.     

Increased sensitivity of striatal dopamine release to H2O2 upon chronic rotenone treatment

It is believed that both mitochondrial dysfunction and oxidative stress play important roles in the pathogenesis of Parkinson's disease (PD). We studied the effect of chronic systemic exposure to the mitochondrial inhibitor rotenone on the uptake, content, and release of striatal neurotransmitters upon neuronal activity and oxidative stress, the latter simulated by H(2)O(2) perfusion. The dopamine content in the rat striatum is decreased simultaneously with the progressive loss of tyrosine hydroxylase (TH) immunoreactivity in response to chronic intravenous rotenone infusion. However, surviving dopaminergic neurons take up and release only a slightly lower amount of dopamine (DA) in response to electrical stimulation. Striatal dopaminergic neurons showed increased susceptibility to oxidative stress by H(2)O(2), responding with enhanced release of DA and with formation of an unidentified metabolite, which is most likely the toxic dopamine quinone (DAQ). In contrast, the uptake of [(3)H]choline and the electrically induced release of acetylcholine increased, in coincidence with a decline in its D(2) receptor-mediated dopaminergic control. Thus, oxidative stress-induced dysregulation of DA release/uptake based on a mitochondrial deficit might underlie the selective vulnerability of dopaminergic transmission in PD, causing a self-amplifying production of reactive oxygen species, and thereby contributing to the progressive degeneration of dopaminergic neurons.     

Cytochrome c release from rat brain mitochondria is proportional to the mitochondrial functional deficit: implications for apoptosis and neurodegenerative disease

Apoptosis may be initiated in neurons via mitochondrial release of the respiratory protein, cytochrome c. The mechanism of cytochrome c release has been studied extensively, but little is known about its dynamics. It has been claimed that release is all-or-none, however, this is not consistent with accumulating evidence of cytosolic mechanisms for 'buffering' cytochrome c. This study has attempted to model an underlying disease pathology, rather than inducing apoptosis directly. The model adopted was diminished activity of the mitochondrial respiratory chain complex I, a recognized feature of Parkinson's disease. Titration of rat brain mitochondrial respiratory function, with the specific complex I inhibitor rotenone, caused proportional release of cytochrome c from isolated synaptic and non-synaptic mitochondria. The mechanism of release was mediated, at least in part, by the mitochondrial outer membrane component Bak and voltage-dependent anion channel rather than non-specific membrane rupture. Furthermore, preliminary data were obtained demonstrating that in primary cortical neurons, titration with rotenone induced cytochrome c release that was subthreshold for the induction of apoptosis. Implications for the therapy of neurodegenerative diseases are discussed.     

Superoxide dismutase/catalase mimetics are neuroprotective against selective paraquat-mediated dopaminergic neuron death in the substantial nigra: implications for Parkinson disease

Exposure of mice to the herbicide paraquat has been demonstrated to result in the selective loss of dopaminergic neurons of the substantia nigra, pars compacta (SNpc) akin to what is observed in Parkinson disease (PD). In this study, we investigate the efficacy of two synthetic superoxide dismutase/catalase mimetics (EUK-134 and EUK-189) in protecting against paraquat-induced dopaminergic cell death in both the rat dopaminergic cell line 1RB3AN27 (N27) and primary mesencephalic cultures in vitro and in adult mice in vivo. Our data demonstrate that pretreatment with either EUK-134 or EUK-189 significantly attenuates paraquat-induced neurotoxicity in vitro in a concentration-dependent manner. Furthermore, systemic administration of EUK-189 decreases paraquat-mediated SNpc dopaminergic neuronal cell death in vivo. These findings support a role for oxidative stress in paraquat-induced neurotoxicity and suggest novel therapeutic approaches for neurodegenerative disorders associated with oxidative stress such as PD.     

Mechanisms to prevent caspase activation in rotenone-induced dopaminergic neurodegeneration: role of ATP depletion and procaspase-9 degradation

The evidence implicating a mode of cell death that either favors or argues against caspase-dependent apoptosis is available in studies that used experimental models of Parkinson’s disease. We sought to investigate the mechanisms by which release of cytochrome c is not linked to caspase activation during rotenone-induced dopaminergic (DA) neurodegeneration. Unlike caspase activation in 6-hydroxydopamine-treated cells, both MN9D DA neuronal cells and primary cultures of mesencephalic neurons showed no obvious signs of caspase activation upon exposure to rotenone. We found that intracellular levels of ATP significantly decreased at the early phase of neurodegeneration (<~24 h) and therefore external addition of ATP to the lysates obtained at this stage reconstituted caspase-3 activity. At a later phase of cell death (>~24 h), both decreased levels of ATP and procaspase-9 contributed to the lack of caspase-3 activation. Under this condition, calpain and the proteasome system were responsible for the degradation of procaspase-9. Consequently, external addition of ATP and procaspase-9 to the lysates harvested at the later phase was required for activation of caspase-3. Similarly, caspase-3 activity was also reconstituted in the lysates harvested from cells co-treated with inhibitors of these proteases and incubated in the presence of external ATP. Taken together, our findings provided a sequential mechanism underlying how DA neurons may undergo caspase-independent cell death, even in the presence of cytoplasmic cytochrome c following inhibition of mitochondrial complex I.     

The Role of Glutathione in Dopaminergic Neuronal Survival

Abstract: An increased production of reactive oxygen species is thought to be critical to the pathogenesis of Parkinson's disease. At autopsy, patients with either presymptomatic or symptomatic Parkinson's disease have a decreased level of glutathione in the substantia nigra pars compacta. This change represents the earliest index of oxidative stress in Parkinson's disease discovered to this point. This study compares the sensitivity of dopaminergic and nondopaminergic neurons in dissociated mesencephalic cultures to the depletion of glutathione. We have found that dopaminergic neurons are more resistant to the toxicity of glutathione depletion than nondopaminergic neurons. The possibility that dopaminergic neurons have a higher baseline glutathione level than nondopaminergic neurons is suggested by measurements of levels of cellular glutathione in a parallel system of immortalized embryonic dopaminergic and nondopaminergic cell lines. We also examined the role of glutathione in 1-methyl-4-phenylpyridinium toxicity. Decreasing the glutathione level of dopaminergic neurons potentiates their susceptibility to 1-methyl-4-phenylpyridinium toxicity, although 1-methyl-4-phenylpyridinium does not deplete glutathione from primary mesencephalic cultures. Our data suggest that although a decreased glutathione content is not likely to be the sole cause of dopaminergic neuronal loss in Parkinson's disease, decreased glutathione content may act in conjunction with other factors such as 1-methyl-4-phenylpyridinium to cause the selective death of dopaminergic neurons.