Catherine Mytilineou

Levodopa in the treatment of Parkinson's disease: Current controversies

Levodopa is the most effective symptomatic agent in the treatment of Parkinsons disease (PD) and the “gold standard” against which new agents must be compared. However, there remain two areas of controversy: (1) whether levodopa is toxic, and (2) whether levodopa directly causes motor complications. Levodopa is toxic to cultured dopamine neurons, and this may be a problem in PD where there is evidence of oxidative stress in the nigra. However, there is little firm evidence to suggest that levodopa is toxic in vivo or in PD. Clinical trials have not clarified this situation. Levodopa is also associated with motor complications. Increasing evidence suggests that they are related, at least in part, to the short half‐life of the drug (and its potential to induce pulsatile stimulation of dopamine receptors) rather than to specific properties of the molecule. Treatment strategies that provide more continuous stimulation of dopamine receptors provide reduced motor complications in MPTP monkeys and PD patients. These studies raise the possibility that more continuous and physiological delivery of levodopa might reduce the risk of motor complications. Clinical trials to test this hypothesis are underway. We review current evidence relating to these areas of controversy.

Toxic and Protective Effects of l-DOPA on Mesencephalic Cell Cultures

Abstract: The autoxidation of L‐DOPA or dopamine (DA) and the metabolism of DA by monoamine oxidase generate a spectrum of toxic species, namely, hydrogen peroxide, oxy radicals, semiquinones, and quinones. When primary dissociated cultures of rat mesencephalon were incubated with L‐DOPA (200 μM) for 48 h, the number of tyrosine hydroxylase‐positive neurons (DA neurons) was reduced to 69.7% of control values, accompanied by a decrease in [3H]DA uptake to 42.3% of control values; the remaining DA neurons exhibited reduced neurite length and overall deterioration. Lack of simultaneous change in the number of neurons stained with neuron‐specific enolase indicated that toxicity was relatively specific for DA neurons. At the same time, the level of GSH, a major cellular antioxidant, rose to 125.2% of control values. Thus, exposure of mesencephalic cultures to L‐DOPA results in both damaging and antioxidant actions. Ascorbate (200 μM), an antioxidant, prevented the rise in GSH. The effect of ascorbate on GSH points to an oxidative signal to initiate the rise in GSH content. On the other hand, neither inhibition of monoamine oxidase with pargyline nor addition of superoxide dismutase or catalase to the culture medium prevented the rise in GSH level or the loss in [3H]DA uptake. The latter results tend to exclude the products of monoamine oxidase activity or the presence of hydrogen peroxide or superoxide in the medium as responsible agents for the rise in GSH or neuronal toxicity. In cultures treated with L‐buthionine sulfoximine (L‐BSO), an inhibitor of GSH synthesis, l‐DOPA prevented cell death by L‐BSO.

Glial Cell Line-Derived Neurotrophic Factor Exerts Neurotrophic Effects on Dopaminergic Neurons In Vitro and Promotes Their Survival and Regrowth After Damage by 1-Methyl-4-Phenylpyridinium

Abstract: 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 Parkinsons disease, which is characterized by dopaminergic cell loss.

Glutathione depletion and oxidative stress.

Oxidative stress is believed to contribute to the pathogenesis of Parkinsons disease. One of the indices of oxidative stress is the depletion of the antioxidant glutathione (GSH), which may occur early in the development of Parkinsons disease. To study the role of GSH depletion in the survival of dopamine neurons we treated mesencephalic cultures with the GSH synthesis inhibitor L-buthionine sulfoximine. Our studies have shown that the depletion of GSH causes a cascade of events, which ultimately may result in cell death. An early event following GSH depletion is a phospholipase A(2)-dependent release of arachidonic acid. Arachidonic acid can cause damage to the GSH-depleted cells through its metabolism by lipoxygenase. The generation of superoxide radicals during the metabolism of arachidonic acid is likely to play an important role in the toxic events that follow GSH depletion.

Deprenyl protects dopamine neurons from the neurotoxic effect of 1-methyl-4-phenylpyridinium ion.

Abstract: 1‐Methyl‐4‐phenylpyridinium ion (MPP+) is the product of the metabolic oxidation of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) by mono‐amine oxidase (MAO). MPP+ is toxic to 3,4‐dihydroxy‐phenylethylamine (dopamine, DA) neurons in explant cultures of rat embryonic midbrain. Addition of 2.5 μM MPP+ to the feeding medium for 6 days results in significant reduction of the DA levels in the cultures (to 19% of control) as well as in the uptake of [3H]DA (to 32% of control). When the cultures are treated with the MAO inhibitor deprenyl (10 μM) 24 h prior to and during exposure to MPP+, the DA neurons are protected from the toxicity of the drug. In the combined deprenyl plus MPP+ treatment, the levels of DA in the cultures remain at the control range and the [3H]DA uptake is reduced to only 73% of control. These results indicate that MAO is involved in the toxicity of MPP+ on DA neurons.

Basic Fibroblast Growth Factor Stimulation of Glial Cells Protects Dopamine Neurons from 6‐Hydroxydopamine Toxicity: Involvement of the Glutathione System

Abstract: 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.

6,7-Dihydroxytetrahydroisoquinoline: Uptake and Storage by Peripheral Sympathetic Nerve of the Rat

6,7-Dihydroxy-1,2,3,4-tetrahydroisoquinoline is a pharmacologically active alkaloid that can be formed by condensation of dopamine with formaldehyde. We used fluorescence microscopy to study in vitro the uptake and storage of this compound by sympathetic nerves of the rat iris. Rats were treated with reserpine or with the methyl ester of α-methyl-p-tyrosine in order to deplete the endogenous catecholamine stores. Accumulation of the alkaloid was about onetenth that of norepinephrine. Uptake was completely blocked by 10-5M desmethylimipramine. These results offer some explanation for the sympathomimetric properties of the alkaloid. Similar results can be expected for similar tetrahydroisoquinolines that may be formed in vivo from endogenous catecholamines during ingestion of alcoholic beverages.

l‐(−)‐Desmethylselegiline, a Metabolite of Selegiline [l‐(−)‐Deprenyl], Protects Mesencephalic Dopamine Neurons from Excitotoxicity In Vitro

Abstract: Selegiline [l‐(−)‐deprenyl], a monoamine oxidase B inhibitor, has been used in the treatment of Parkinsons 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.

Impaired oxidative decarboxylation of pyruvate in fibroblasts from patients with Parkinson's disease

SummaryWhether or not a reported deficiency in brain mitochondrial complex I activity in Parkinsons disease represents a defect encompassing other organs or tissues has been a source of some controversy. We have examined mitochondrial respiration in fibroblasts from patients with Parkinsons disease by measuring the oxidative decarboxylation of [2-14C]pyruvate and [1,4-14C]succinate. We report that oxidation of pyruvate but not succinate was significantly reduced in fibroblasts from Parkinson patients when compared to healthy controls. These observations support the view that a widespread deficit in mitochondrial respiration exists in Parkinsons disease. Fibroblast cultures, moreover, are a source of affected proliferating cells, which can be used for in vitro studies of the nature of the respiratory defect and for testing of pharmacological interventions to correct the deficiency.

Inhibition of proteasome activity sensitizes dopamine neurons to protein alterations and oxidative stress

Summary.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.