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

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Abstract
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 NG‐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. J. Neurosci. Res. 57:86–94, 1999.