The contribution of arachidonic acid (AA) release and metabolism to the toxicity that results from glutathione (GSH) depletion was studied in rat mesencephalic cultures treated with the GSH synthesis inhibitor L-buthionine sulfoximine. Our data show that GSH depletion is accompanied by increased release of AA, which is phosholipase A2 (PLA(2)) dependent. Exogenous AA is toxic to GSH-depleted cells. This toxicity is prevented by inhibition of lipoxygenase activity, suggesting participation of toxic byproducts of AA metabolism. Hydroxyperoxyeicosatetraenoic acid (HPETE), one of the primary products of AA metabolism by lipoxygenase is also toxic to GSH-depleted cells, whereas hydroeicosatetraenoic acid (HETE) is not. Cell death caused by GSH depletion is prevented by: (i) replenishment of GSH levels with GSH-ethyl ester; (ii) inhibition of PLA(2) activity; (iii) inhibition of lipoxygenase activity; and (iv), treatment with ascorbic acid. These data suggest that the following events likely contribute to cell death when GSH levels become depleted. Loss of GSH results in increased release of AA, which is PLA(2) dependent. Metabolism of arachidonic acid via the lipoxygenase pathway results in generation of oxygen free radicals possibly produced during conversion of HPETE to HETE, which contribute to cellular damage and death. Our study suggests that limiting AA release and metabolism may provide benefit in conditions with an existing depletion of GSH, such as Parkinson's disease.

Impairment in the capacity of the ubiquitin-proteasome pathway to clear unwanted proteins has been implicated in the cell death that occurs in Parkinson's disease (PD). In support of this concept, defects in proteasomal structure and function, as well as protein aggregates and increased levels of oxidized proteins are found in the substantia nigra of PD patients. We have previously demonstrated that inhibition of proteasome activity in mesencephalic cultures induces degeneration of dopaminergic neurons coupled with the formation of proteinaceous intracellular inclusions. In this study we examined the effect of proteasome inhibition on cultured dopamine neurons when combined with oxidative stress and protein misfolding, in order to better simulate the condition in PD. We demonstrate that two structurally unrelated inhibitors of proteasome activity, lactacystin and carbobenzoxy-L-leucul-L-leucyl-L-leucinal (MG132), cause dose-dependent cell loss that preferentially affects dopaminergic neurons. Conditions that promote protein damage and misfolding such as oxidative stress, heat shock, and canavanine also induce neuronal degeneration with preferential loss of dopamine neurons and cell death is markedly increased when any of these is combined with a proteasome inhibitor. These studies demonstrate a synergistic effect between conditions that promote the formation of damaged proteins and those in which proteasomal function is impaired, and provide further support for the notion that cell loss in PD could be related to a defect in protein handling.

Levodopa is the "gold standard" for the symptomatic treatment of Parkinson's disease (PD). There is a theoretical concern, however, that levodopa might accelerate the rate of nigral degeneration, because it undergoes oxidative metabolism and is toxic to cultured dopaminergic neurons. Most in vivo studies do not show evidence of levodopa toxicity; levodopa is not toxic to normal rodents, nonhuman primates, or humans and is not toxic to dopamine neurons in dopamine-lesioned rodents or nonhuman primates in most studies. However, the potential for levodopa to be toxic in vivo has not been tested under conditions of oxidative stress such as exist in PD. To assess whether levodopa is toxic under these circumstances, we have examined the effects of levodopa on dopamine neurons in mesencephalic cultures and rat pups in which glutathione synthesis has been inhibited by L-buthionine sulfoximine. Levodopa toxicity to cultured dopaminergic neurons was enhanced by glutathione depletion and diminished by antioxidants. In contrast, treatment of neonatal rats with levodopa, administered either alone or in combination with glutathione depletion, did not cause damage to the dopamine neurons of the substantia nigra or changes in striatal levels of dopamine and its metabolites. This study provides further evidence to support the notion that although levodopa can be toxic to dopamine neurons in vitro, it is not likely to be toxic to dopamine neurons in vivo and specifically in conditions such as PD.

Mutations in α‐synuclein, parkin and ubiquitin C‐terminal hydrolase L1, and defects in 26/20S proteasomes, cause or are associated with the development of familial and sporadic Parkinson's disease (PD). This suggests that failure of the ubiquitin–proteasome system (UPS) to degrade abnormal proteins may underlie nigral degeneration and Lewy body formation that occur in PD. To explore this concept, we studied the effects of lactacystin‐mediated inhibition of 26/20S proteasomal function and ubiquitin aldehyde (UbA)‐induced impairment of ubiquitin C‐terminal hydrolase (UCH) activity in fetal rat ventral mesencephalic cultures. We demonstrate that both lactacystin and UbA caused concentration‐dependent and preferential degeneration of dopaminergic neurons. Inhibition of 26/20S proteasomal function was accompanied by the accumulation of α‐synuclein and ubiquitin, and the formation of inclusions that were immunoreactive for these proteins, in the cytoplasm of VM neurons. Inhibition of UCH was associated with a loss of ubiquitin immunoreactivity in the cytoplasm of VM neurons, but there was a marked and localized increase in α‐synuclein staining which may represent the formation of inclusions bodies in VM neurons. These findings provide direct evidence that impaired protein clearance can induce dopaminergic cell death and the formation of proteinaceous inclusion bodies in VM neurons. This study supports the concept that defects in the UPS may underlie nigral pathology in familial and sporadic forms of PD.

Glutathione is an important cellular antioxidant present at high concentrations in the brain. We have previously demonstrated that depletion of glutathione in mesencephalic cultures results in cell death and that the presence of glia is necessary for the expression of toxicity. Cell death following glutathione depletion can be prevented by inhibition of lipoxygenase activity, implicating arachidonic acid metabolism in the toxic events. In this study we examined the effect of glial activation, known to cause secretion of cytokines and release of arachidonic acid, on the toxicity induced by glutathione depletion. Our data show that treatment with the endotoxin lipopolysaccharide activated glial cells in mesencephalic cultures, increased interleukin-1beta in microglia and caused depletion of glutathione. The overall effect of lipopolysaccharide treatment, however, was protection from damage caused by glutathione depletion. Addition of cytokines or growth factors, normally secreted by activated glia, did not modify L-buthionine sulfoximine toxicity, although basic fibroblast growth factor provided some protection. A large increase in the protein content and the activity of Mn-superoxide dismutase, observed after lipopolysaccharide treatment, may indicate a role for this mitochondrial antioxidant enzyme in the protective effect of lipopolysaccharide. This was supported by the suppression of toxicity by exogenous superoxide dismutase. Our data suggest that superoxide contributes to the damage caused by glutathione depletion and that up-regulation of superoxide dismutase may offer protection in neurodegenerative diseases associated with glutathione depletion and oxidative stress.

Abstract: We have examined the role of glial cells in the toxicity that results from inhibition of reduced glutathione (GSH) synthesis by L‐buthionine sulfoximine (BSO) in mesencephalic cell cultures. We show that GSH depletion, to levels that cause total cell loss in cultures containing neurons and glial cells, has no effect on cell viability in enriched neuronal cultures. An increase in the plating cell density sensitizes glia‐containing cultures to GSH depletion‐induced toxicity. This suggests that cell death in this model is the consequence of events that are induced by GSH depletion and are mediated by glial cells. The antioxidant ascorbic acid and the lipoxygenase (LOX) inhibitor nordihydroguaiaretic acid (1‐10 μM) provide full protection from BSO toxicity, indicating that arachidonic acid metabolism through the LOX pathway and the generation of reactive oxygen species play a role in the loss of cell viability. In contrast, inhibition of nitric oxide (NO) synthase affords only partial protection from BSO toxicity, suggesting that increased NO production cannot entirely account for cell death in this model. Our data provide evidence that GSH depletion in the presence of glial cells leads to neuronal degeneration that can be prevented by inhibition of LOX. This may have relevance to the pathogenesis of Parkinson’s disease, where glial activation and depletion of GSH have been found in the substantia nigra pars compacta.

Glial cell line derived neurotrophic factor (GDNF) has been shown to be a potent neurotrophic factor for dopamine neurons in culture and to prevent the loss of substantia nigra dopamine neurons following in vivo lesions with 6-hydroxydopamine (6-OHDA). In this study we used mesencephalic cultures containing both neurons and glia to examine whether GDNF protects dopamine neurons from 6-OHDA toxicity in vitro. Our data show that GDNF does not prevent the loss of dopamine neurons caused by treatment with 6-OHDA in vitro. However, continuous exposure to GDNF increases the high affinity dopamine uptake in cultures treated with 6-OHDA, suggesting that it enhances the growth of damaged dopamine neurons. We also show that in vitro treatment with 6-OHDA causes widespread cell death in mesencephalic cultures, which is not restricted to dopamine neurons. The lack of selectivity of 6-OHDA toxicity when applied in vitro may explain the inability of GDNF to prevent the loss of dopamine neurons in mesencephalic cultures. The stimulation of the growth of 6-OHDA damaged dopamine neurons by GDNF, observed in our study, suggests that it may prove beneficial in the treatment of injured dopamine neurons.

Oxidative stress is thought to play an important role in the pathogenesis of Parkinson's disease (PD). Glutathione (GSH), a major cellular antioxidant, is decreased in the substantia nigra pars compacta of PD patients. The aim of the present study was to investigate whether deprenyl and its desmethyl metabolite, putative neuroprotective agents in the treatment of PD, could protect cultured rat mesencephalic neurons from cell death caused by GSH depletion due to treatment with L-buthionine-(S,R)-sulfoximine (BSO). BSO (10 microM) caused extensive cell death after 48 hr, as demonstrated by disruption of cellular integrity and release of lactate dehydrogenase into the culture medium. Both deprenyl and desmethylselegiline, at concentrations of 5 and 50 microM, significantly protected dopaminergic neurons from toxicity without preventing the BSO-induced loss in GSH. Protection was not associated with monoamine oxidase type B inhibition in that pargyline, a potent MAO inhibitor, was ineffective and pretreatment with pargyline did not prevent the protective effects of deprenyl. Protection was not associated with inhibition of dopamine uptake by deprenyl because the dopamine uptake inhibitor mazindol did not diminish BSO toxicity. The antioxidant ascorbic acid (200 microM) also protected against BSO-induced cell death, suggesting that oxidative events were involved. This study demonstrates that deprenyl and its desmethyl metabolite can diminish cell death associated with GSH depletion.

Selegiline [L-(-)-deprenyl], a monoamine oxidase B inhibitor, has been used in the treatment of Parkinson's disease as a putative neuroprotective agent. Selegiline is metabolized rapidly in the gastrointestinal tract and liver to desmethylselegiline (DMS) and methamphetamine. We have previously shown that selegiline protects dopamine neurons in mesencephalic cultures from toxicity resulting from activation of glutamate receptors. In the present study we examined whether DMS has similar neuroprotective effects. Our data show that DMS protects dopamine neurons from N-methyl-D-aspartate receptor-mediated excitotoxic damage. The efficacy of DMS is greater than that of selegiline, as it can cause protection at lower concentrations and provide significantly greater levels of protection at the same concentrations. Our results suggest that DMS might be the active compound responsible for the neuroprotective properties of selegiline.

Neurotrophic factors have been shown to support the survival and promote the recovery of injured neurons both in vivo and in vitro. Here, we investigated whether glial cell line-derived neurotrophic factor (GDNF) and basic fibroblast growth factor (bFGF) could modify the damage to dopamine (DA) neurons in mesencephalic cultures caused by the neurotoxin 6-hydroxydopamine (6-OHDA). The data show that bFGF, but not GDNF, effectively protected DA neurons from 6-OHDA toxicity. Because bFGF is a glial mitogen, whereas GDNF is not, we tested whether glial cells participated in bFGF neuroprotection. inhibition of glial cell proliferation completely prevented the protective effect of bFGF. Because oxidative events have been associated with 6-OHDA-induced damage, we examined the levels of glutathione (GSH) in control and bFGF-treated cultures. Cultures treated with bFGF had higher levels of GSH, which increased even further in response to 6-OHDA exposure. Control cultures failed to up-regulate GSH levels after 6-OHDA, suggesting a relationship between increased GSH levels and protection from 6-OHDA. Inhibition of glial cell proliferation prevented the rise in GSH in bFGF-treated cultures and abolished the increase after 6-OHDA treatment. Protection from 6-OHDA by bFGF was also diminished when GSH levels were decreased by the GSH synthesis inhibitor L-buthionine sulfoximine. Our study shows that stimulation of glial cells by bFGF allows the up-regulation of antioxidant defenses and supports cell survival during oxidative stress.

Abstract: l‐Deprenyl is a relatively selective inhibitor of monoamine oxidase (MAO)‐B that delays the emergence of disability and the progression of signs and symptoms of Parkinson's disease. Experimentally, deprenyl has also been shown to prevent neuronal cell death in various models through a mechanism that is independent of MAO‐B inhibition. We examined the effect of deprenyl on cultured mesencephalic dopamine neurons subjected to daily changes of feeding medium, an experimental paradigm that causes neuronal death associated with activation of the NMDA subtype of glutamate receptors. Both deprenyl (0.5–50 µM) and the NMDA receptor blocker MK‐801 (10 µM) protected dopamine neurons from damage caused by medium changes. The nonselective MAO inhibitor pargyline (0.5–50 µM) was not protective, indicating that protection by deprenyl was not due to MAO inhibition. Deprenyl (50 µM) also protected dopamine neurons from delayed neurotoxicity caused by exposure to NMDA. Because deprenyl had no inhibitory effect on NMDA receptor binding, it is likely that deprenyl protects from events occurring downstream from activation of glutamate receptors. As excitotoxic injury has been implicated in neurodegeneration, it is possible that deprenyl exerts its beneficial effects in Parkinson's disease by suppressing excitotoxic damage.

The effect of glial cell line-derived neurotrophic factor (GDNF) on the growth of mesencephalic dopaminergic neurons and on their survival following exposure to the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) was examined in vitro. In cultures developing under normal conditions, GDNF at 1 ng/ml optimally improved the survival and stimulated the growth of dopaminergic neurons without affecting glial growth. In cultures treated with MPP+, GDNF could not prevent toxicity to dopaminergic neurons. The uptake of [3H]dopamine and the number of tyrosine hydroxylase-positive neurons were similarly reduced by MPP+ in the presence or absence of GDNF. However, after removal of MPP+, GDNF protected dopaminergic neurons from the continuous cell death and stimulated the regrowth of dopaminergic fibers damaged by MPP+. We conclude that GDNF supports the growth of normally developing dopaminergic neurons and stimulates their survival and recovery after damage. These findings suggest that GDNF could be useful in the development of therapeutic approaches to Parkinson's disease, which is characterized by dopaminergic cell loss.

Incubation with L-DOPA induced a rise in GSH level in cultures of fetal rat mesencephalon, mouse neuroblastoma (Neuro-2A), human neuroblastoma (SK-N-MC), pig kidney epithelial cells (LLC-PK1), and glia from newborn rat brain, but not C6 glioma cells or neuronal cultures (no glia) from the mesencephalon, The pure neuronal cultures were destroyed by incubation with L-DOPA; added ascorbic acid or superoxide dismutase protected the cells. Washout of L-DOPA after 48 h amplified the rise in GSH content in mixed cultures (neurons plus glia). Examination of structure-activity relationships for elevating GSH levels in responsive cell types revealed that autooxidizable compounds (alpha-methyl-DOPA, dopamine, apomorphine, catechol, and hydroquinone) behaved similarly to L-DOPA, whereas structural analogues that cannot undergo autooxidation (3-O-methyl-DOPA, tyrosine, 2,4-dihydroxyphenylalanine, and resorcinol) failed to elevate GSH levels. Therefore, up-regulation of GSH appears to be a response to a mild oxidative stress. When mixed mesencephalic cultures were exposed to a strong oxidant stress by incubation with tert-butyl hydroperoxide, a loss in viability was seen. Cultures pretreated with L-DOPA or hydroquinone were protected from loss of viability. However, when cultures were pretreated with both L-DOPA and ascorbate, which prevents the rise in GSH level, protection was lost, in accord with the failure to up-regulate GSH, These results show that the up-regulation of cellular GSH evoked by autooxidizable agents is associated with significant protection of cells. Glia play an essential role in the response of mesencephalic cell cultures. An ability to up-regulate GSH may serve a protective role in vivo.