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

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.