Comparison of the neurotoxic effects of proteasomal inhibitors in primary mesencephalic cultures.

Impairment of the ubiquitin-proteasome system (UPS) has been implicated in the pathogenesis of Parkinson's disease (PD). Because the neurodegenerative process of PD results in a severe loss of dopaminergic cells, previous in vitro studies have investigated the possibility that these neurons may be particularly vulnerable to proteasomal inhibition. Results of this earlier work are difficult to compare, however, since they were obtained using different proteasomal inhibitors at various concentrations and under diverse culture conditions. Here, four UPS inhibitors, i.e., lactacystin, PSI, epoxomicin and MG-132, were directly evaluated in terms of their ability to damage dopaminergic and GABAergic neurons in primary rat mesencephalic cultures. Using a broad range of concentrations and different incubation lengths, we found that proteasomal inhibitors consistently killed both dopaminergic and GABAergic neurons. The degree of toxicity was slightly different, however, between the two neuronal populations. When measurements of neurotransmitter uptake were used as indicators of neuronal cell viability, the extent of reduction of dopamine uptake caused by proteasomal inhibitors was slightly greater than the decrease in GABA uptake. With PSI the difference in reduction of dopamine vs. GABA uptake was less than 10% and did not reach statistical significance. With the other three inhibitors, dopaminergic cells were up to 20% more affected than GABAergic neurons; this difference reached statistical significance only at specific concentrations and time points. Preincubation of cultures with alpha-methyl-p-tyrosine, an inhibitor of dopamine synthesis, reduced dopamine concentration by 65% but failed to significantly change lactacystin- and MG-132-induced damage to dopaminergic neurons. Data indicate a modest preferential toxicity of proteasomal inhibitors toward dopaminergic cells and thus only in part support the hypothesis that a selective vulnerability to UPS dysfunction underlies the pathogenesis of nigrostriatal degeneration in PD.