Involvement of caspase-like proteinases in apoptosis of neuronal PC12 cells and primary cultured microglia induced by 6-hydroxydopamine.

Activation of proteolytic enzymes, including the caspase family of proteinases, is a feature characteristic of the apoptotic program. In the present study, we examined a potential role of intracellular proteinases in the death of neuronal PC12 and primary cultured rat microglial cells induced by 6-hydroxydopamine (6-OHDA). In both neuronal PC12 and microglial cells, 6-OHDA (10-200 microM) induced apoptosis in a dose-dependent manner as judged by the DNA break. The 6-OHDA was ineffective in Bcl-2-overexpressing neuronal PC12 cells. Pretreatment of these cells with two caspase inhibitors, acetyl-Try-Val-Ala-Asp-aldehyde and acetyl-Asp-Glu-Val-Asp-aldehyde, prevented the 6-OHDA-induced apoptosis. Pepstatin A and leupeptin, potent inhibitors of aspartic and cysteine proteinases, respectively, partly inhibited the apoptosis of microglia but not neuronal PC12 cells. In contrast, GBR12935, a dopamine uptake inhibitor, significantly inhibited the apoptotic death of neuronal PC12 cells but not microglia. These results suggest that mechanisms by which 6-OHDA induces apoptosis in these two cell types are distinct; 6-OHDA incorporated into neuronal PC12 cells and its metabolites may activate the caspase-like enzymes, whereas oxidative metabolites of the agent produced extracellularly may activate the caspase and the endosomal/lysosomal proteolytic systems in microglia.