Critical role of ASK1 in the 6‐hydroxydopamine‐induced apoptosis in human neuroblastoma SH‐SY5Y cells

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Abstract
6‐Hydroxydopamine (6‐OHDA)‐induced apoptosis in dopaminergic neuronal cells is a common cell model of Parkinson's disease (PD). The role of apoptosis signal‐regulating kinase 1 (ASK1) in this model has not been well studied. We observed significant activation of ASK1, p38 and JNK, as well as apoptosis in human dopaminergic neuroblastoma SH‐SY5Y cells exposed to 6‐OHDA. Over‐expressing kinase‐dead mutant ASK1(K709M) or knock‐down of endogenous ASK1 by its small interfering RNA (siRNA) greatly suppressed activation of these kinases and apoptosis in the cells. It was found that the activation of p38 and JNK was suppressed to almost the same extent as that of ASK1 in the ASK1‐knock‐down cells, suggesting that activated ASK1 is almost totally responsible for activation of p38/JNK. It was also observed that the 6‐OHDA‐induced cell apoptosis could be effectively prevented by over‐expressing the dominant‐negative mutant of p38 or p38 inhibitor SB203580, demonstrating that activation of p38/JNK signalling is required for initiating the programmed cell death. Furthermore, suppression of the 6‐OHDA‐generated reactive oxygen species (ROS) by pre‐incubation of cells with N‐acetyl‐l‐cysteine effectively inhibited the 6‐OHDA‐induced activation of ASK1, p38 and JNK, and protected the cells from apoptosis. This study clearly shows the route from ROS generation by 6‐OHDA to initiation of p38/JNK signalling via activation of ASK1 in the studied PD model.