Studies On The Role And Molecular Mechanisms Of Neuronal MTOR Signaling Inhibition In Development Of Experimental Parkinson’s Disease

The present study, using RNA interference, Western blotting, cell culture, flow In this paper, PC12cells and primary neurons and mice were chosen to establish in vitro and in vivo models of Parkinson’s disease. By means of cell and molecular biology techniques such as gene cloning, RNA interference, Western blotting, immunohistochemical analysis, etc., we deeply studied the role of neuronal mTOR signaling inhibition in development of Parkinson’s disease (PD), and the relation and mechanisms of PTEN, PDK1, Akt and AMPK functional status to mTOR signaling inhibition contributing to neuronal apoptosis in PD progression were discussed. The results were summarized as follows:1. mTOR signaling inhibition induced neuronal apoptosis in PD developmentPC12cells and primary neurons were chosen and treated with different concentrations of6-OHDA (0-240μmol/L) for12h, or with120μmol/L6-OHDA for different time (0-24h), or with MPP+(1mmol/L) and rotenone (1μmol/L) for12and/or24h, respectively. Overexpression of wild-type mTOR or constitutively active S6K1by adenovirus interference, or downregulation of4E-BP1by lentiviral shRNA interference were conducted in PC12cells. We showed that6-OHDA induced inhibition of mTOR and mTOR-mediated S6K1and4E-BP1phosphorylation in a concentration-and time-dependent manner, leading to cell death. MPP+and rotenone exhibited similar effects. Three inducers caused activation of caspase-3and its downstream PARP proteins in neuronal cells. Overexpression of mTOR, constitutively active S6K1and downregulated4E-BP1expression partially prevented neuronal apoptosis in PD models. Our findings suggest that inhibition of mTOR pathway induces neuronal cell apoptosis in PD development.2. PDK1and Akt were involved in mTOR signaling inhibition contributing to neuronal apoptosis in PD developmentPC12and primary neurons were chosen and treated with different concentration of6-OHDA (0-240μmol/L) for12h, or with120μmol/L6-OHDA for different time (0-24h), or with MPP+(1mmol/L) and rotenone (1μmol/L) for12and/or24h, respectively. Overexpression of Akt by adenovirus interference was conducted in PC12cells. The results showed that6-OHDA induced inhibition of PDK1and Akt phosphorylation in a concentration-and time-dependent manner, the effects were also observed in neuronal cells treated with MPP+and rotenone. Three inducers resulted in phosphorylated Akt inhibition implicated in neuronal apoptosis. Overexpression of Akt prevented prevented neuronal apoptosis induced by6-OHDA, MPP+and rotenone. Our data have demonstrated that PDK1and Akt are involved in inhibition of mTOR pathway, leading to neuronal apoptosis in PD development.3. Activation of AMPK mediated mTOR signaling inhibition contributing to neuronal apoptosis in PD developmentPC12and primary neurons were chosen and treated with different concentrations of6-OHDA (0-240μmol/L) for12h, or with120μmol/L6-OHDA for different time (0-24h), or with MPP+(1mmol/L) and rotenone (1μmol/L) for12and/or24h. PC12cells were pretreated with compound C for2h to inhibit AMPK activity. We revealed that6-OHDA induced neuronal AMPKa activation in a concentration-and time-dependent manner. Similarly, activation of AMPKa due to MPP+and rotenone was seen in neuronal cells. Compound C prevented activation of AMPKa and apoptosis induced by6-OHDA, MPP+and rotenone in neuronal cells. We found that inhibition of AMPKαactivity due to compound C attenuated inhibition of mTOR pathway and apoptosis induced by6-OHDA, MPP+and rotenone in neuronal cells. Our findings suggest that AMPK activation is involved in mTOR signaling inhibition, resulting in neuronal apoptosis in PD development.4. Akt/AMPK mediated inhibition of mTOR pathway contributing to neuronal apoptosis in brain of mouse PD modelSixty healthy adult male C57BL mice were chosen and randomly divided into normal control group (treated with0.9%physiological saline) and experiment group (treated with MPTP). MPTP (30mg/kg body weight) was dissolved in0.9%physiological saline (2mg/mL) and administered by intraperitoneal injection each day. The entire experiment lasted5days. Randomly taked5mice from each group for biain homogenates at24h after the1st,3nd,5th days of injection and the5th,10th,20th days after the last injection with MPTP, and brain tissue samples of5mice, respectively, form normal control MPTP groups was collected and marked as Day-5, Day-3, Day-1, Day5, Day10and Day20. Using Western blotting, changes of PDK1, Akt, AMPK, mTOR and its downstream molecules as well as caspase-3and PARP were assayed. We found mTOR signaling inhibition leading to neuronal apoptosis in the brain of mouse PD model, diseaseed PDK1and Akt phosphorylation, and increased of AMPKa phosphorylation. These findings support the notion that Akt inhibiton and AMPK activation are associated with inhibition of mTOR pathway, leading to neuronal apoptosis in brain of mouse PD model.