Mechanisms of MPP⁺-induced PC12 cell apoptosis via reactive oxygen species

Abstract

Apoptosis of dopaminergic neurons in the nigrostriatal projection plays a crucial role in the pathogenesis of Parkinson’s disease (PD). Although the detailed mechanisms responsible for dopaminergic neuron loss are still under investigation, oxidative stress is identified as a major contributor for neuronal apoptosis. In the current study, we studied the effects of MPP⁺, a substrate that mimics oxidative stress, on neuron-like PC12 cells and the underlying mechanisms. PC12 cells were cultured and treated by 100 μmol/L MPP⁺ for 4, 8, 16, 24 and 48 h, respectively. For drug pretreatment, the PC12 cells were incubated with N-acetyl-l-cysteine (NAC, 5 mmol/L), an antioxidant, SP600125 (20 μmol/L) or PD98059 (100 μmol/L), two pharmacological inhibitors of JNK and ERK1/2, for 1 h before addition of MPP⁺. Cell apoptosis was measured by flow cytometry. The mRNA expression of Cu²⁺/Zn²⁺-SOD, GSH-Px, Bcl-2 and Bax was detected by RT-PCR. The protein expression of p-ERK1/2 and p-JNK was determined by Western blotting. Our results showed that MPP⁺ exposure could induce substantial PC12 cell apoptosis. The pretreatment of SP600125 or PD98059 could effectively reduce the apoptosis rate by reducing the ratio of Bax/Bcl-2 mRNA levels. MPP⁺ exposure also induced high level of reactive oxygen species (ROS), marked by dramatic increase of Cu²⁺/Zn²⁺-SOD and GSH-Px mRNA levels. The elevated ROS was strongly associated with the activation of JNK and ERK1/2 signal pathways after MPP⁺ exposure, since the pretreatment of NAC significantly reduced the upregulation of p-JNK and p-ERK1/2. Finally, the pretreatment of SP600125, but not PD98059, alleviated the increase of Cu²⁺/Zn²⁺-SOD and GSH-Px mRNAs induced by MPP⁺, suggesting that the activation of the JNK signal pathway, but not the ERK1/2 signal pathway, could, in some degree, antagonize the generation of ROS induced by oxidative stress. In conclusion, our results suggest that JNK and ERK1/2 signal pathways, which are activated via ROS, play a crucial role in neuronal apoptosis induced by oxidative stress.

Keywords

MPP⁺ / apoptosis / reactive oxygen species / JNK / ERK1/2 / Cu²⁺/Zn²⁺-SOD / GSH-Px

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