Studies On The Role And Mechanisms Of MTOR Signaling Pathway During Progress Of MPP~+-induced Oxidative Stress Mediation Of Apoptosis In Neuronal Cells

The present study, using cell culture, fluorescent staining, Western blotting, etc., via in vitro experiments, investigated the changes of mTOR signaling pathway during progress of MPP+-induced neuronal apoptosis, and further discussed the mechanisms of MPP+-induced ROS inhibition of mTOR pathway leading to neuronal apoptosis. This study is aimed at providing theoretical foundation and scientific guidance. The results were summarized as follows:1. Studies on MPP+-induced neuronal apoptosis by inhibition of mTOR pathwayPC12cells or isolated mouse primary neurons were treated with0-1.5mM MPP+for24h, or with1.5mM MPP+for0-24h. Cell viability was evaluated using an MTT assay, cell apoptosis was assayed using DAPI staining, and the phosphorylated expressions of Akt and mTOR-mediated S6K1and4E-BP1proteins were detected by Western blotting. The results showed that MPP+induced significantly morphological change and decreases of cell adhesion and viability in neuronal cells. MPP+obviously elicited neuronal cell apoptosis in a concentration-dependent manner. Consistently, phosphorylations of Akt、S6K1and4E-BP1were inhibited by MPP+in a concentration-and time-dependent fashion. Our data suggest that MPP+induces neuronal apoptosis via inhibition of Akt/mTOR pathway.2. Study on MPP+-induced ROS inhibition of mTOR pathway leading to neuronal apoptosisPC12cells or isolated mouse primary neurons were treated with0-1.5mM MPP+for24h, or with0.75mM MPP+for24h following pretreatment with/without300-700U/ml CAT for1h. ROS level was detected using fluorescent probe CM-H2DCFDA, live cell numbers and cell apoptosis were evaluated using cell counting and DAPI staining, and phosphorylated expressions of Akt and4E-BP1were determined by Western blotting. The results showed that MPP+induced ROS elevation in a concentration-manner in PC12cells and primary neurons. CAT attenuated MPP+-induced ROS generation and apoptosis in PC12cells, suggesting that MPP+-induced ROS may mainly be H2O2. MPP+-induced ROS was associated with inhibition of mTOR signaling pathway, which is supported by the finding that CAT prevented the decreased phosphorylations of MPP+-induced Akt and4E-BP1in primary neurons. The findings indicate that MPP+-induced ROS inhibits mTOR pathway, leading to neuronal apoptosis.