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.
Department of Biology, Yonsei University College of Science, Seoul, Korea.
W S
WS
Choi
S Y
SY
Yoon
T H
TH
Oh
E J
EJ
Choi
K L
KL
O'Malley
Y J
YJ
Oh
D015655:1-Methyl-4-phenylpyridinium / Q000633:toxicity*; D000818:Animals; D017209:Apoptosis; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D004298:Dopamine / Q000502:physiology*; D015259:Dopamine Agents / Q000633:toxicity*; D004789:Enzyme Activation; D015151:Immunoblotting; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology / Q000502:physiology; D016627:Oxidopamine / Q000633:toxicity*; D017382:Reactive Oxygen Species / Q000378:metabolism*
D015259:Dopamine Agents; D017382:Reactive Oxygen Species; D016627:Oxidopamine; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D020169:Caspases; D015655:1-Methyl-4-phenylpyridinium; D004298:Dopamine