Two distinct mechanisms are involved in 6-hydroxydopamine- and MPP⁺- induced dopaminergic neuronal cell death: Role of caspases, ROS, and JNK

In this study, we examined the possibility that MPTP and 6- hydroxydopamine (6-OHDA) act on distinct cell death pathways in a murine dopaminergic neuronal cell line, MN9D. First, we found that cells treated with 6-OHDA accompanied ultrastructural changes typical of apoptosis, whereas MPP⁺ treatment induced necrotic manifestations. Proteolytic cleavage of poly(ADP-ribose)polymerase by caspase was induced by 6-OHDA, whereas it remained uncleaved up to 32 h after MPP⁺ treatment and subsequently disappeared. Accordingly, 6-OHDA- but not MPP⁺-induced cell death was significantly attenuated in the presence of a broad-spectrum caspase inhibitor, N-benzyloxy-carbonyl-Val-Ala-Asp-fluomethylketone (Z-VAD-fmk). As measured by fluorometric probes, the level of reactive oxygen species (ROS) significantly increased after 6-OHDA treatment. In contrast, the level of dihydroethidium-sensitive ROS following MPP⁺ treatment remained unchanged while a slight increase in dichlorofluorescin-sentive ROS was temporarily observed. As demonstrated by immunoblot analysis, the level of superoxide dismutase was down-regulated following 6-OHDA treatment, whereas it remained unchanged after MPP⁺ treatment. Cotreatment of cells with antioxidants such as N-acetylcysteine or Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP, cell-permeable superoxide dismutase mimetic) rescued 6-OHDA- but not MPP⁺-induced cell death, whereas inclusion of catalase or N(G)-nitro-L- arginine had no effect in both cases. In addition, 6-OHDA induced ROS- mediated c-Jun N-terminal kinase (JNK) activation that was attenuated in the presence of N-acetylcysteine or MnTBAP but not catalase or Z-VAD-fmk. In contrast, MPP⁺ has little effect on JNK activity, indicating that ROS and/or ROS-induced cell death signaling pathway seems to play an essential role in 6-OHDA-mediated apoptosis but not in MPP⁺-induced necrosis in a mesencephalon-derived, dopaminergic neuronal cell line.