Mutations in a-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 a-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 a-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. Keywords: a-synuclein, Lewy body inclusion, Parkinson's disease, 26/20S proteasome, ubiquitin C-terminal hydrolase L1 and parkin.
Levodopa is the most effective symptomatic agent in the treatment of Parkinson's 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.
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 microM) 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 microM), 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.
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