Studies On The Role And Mechanisms Of Cadmium-induced Autophage And MTOR Signaling Activation In Neuronal Cell Death

The present study, by means of cell culture, transmission electron microscopy, laser scanning confocal microscope and western blotting analysis, etc., investigated the participating mechanism of autophage patyway in Cd-induced neuronal apoptosis. The effects of3-MA and/or rapamycin on Cd-induced autophage and the relation of the effects to Cd-activated mTOR pathway contributing to neuronal apoptosis were primarily discussed, and the relation of Cd-induced autophage and mTOR pathway activation to intracellular free of Ca2+([Ca2+]i) elevation in neuronal cells was observed. The results were summarized as follows:1. Studies on the relation action of cadmium-induced autophage to activated mTOR pathway and its action in apoptosis in neuronal cellsThe PC12cells, seeded in6-well flat-bottomed plates, were treated with different concentrations of Cd (0,2.5,5,10and20μM) and with0.1%serum starvation for12h, or with20μM Cd for0-24h, or were exposed to Cd (10and20μM) for12h following pretreatment with3-MA and/or rapamycin (Rap). Transmission electron microscopy, laser scanning confocal microscope, and morphological observation were used to evaluate the manifestation of Cd-induced autophagy and its relation to apoptosis in neuronal cells; Western Blotting detected the protein changes of LC3Ⅰ/Ⅱ and Akt/mTOR pathway. The results showed that exposure of Cd (10and20μM) PC12cells for12h induced autophagy formation. Cd-activated autophagy was associated with neuronal apoptosis, which was supported by the findings that Cd induced LC3-Ⅱ increase in a concentration-and time-dependent manner in neuronal cells, and pretreatment with3-MA partially inhibited Cd-induced LC3-Ⅱ expression and rescued neuronal apoptosis. Cd activation of autophagy and mTOR pathway may participate in neuronal apoptosis in a parallel-accompanied fashion. Our data clearly indicate Cd-induced paralleling activation of autophagy and mTOR pathway plays an important action in neuronal apoptosis together.2. Studies on the action and mechanisms of calcium ion in cadmium-induced activation of autophage and mTOR pathway in neuronal cellsThe PC12cells, seeded in6-well flat-bottomed plates, were cultured at37℃, containing5%CO2incubator. The next day, Cells were exposed to Cd (10and20μM) for12h following pretreatment with20μM BAPTA/AM,100μM of EGTA,100μM 2-APB or10μM KN93for1h. Western Blotting was used to evoluate the changes of LC3Ⅰ/Ⅱ protein, as well as Akt, S6K1and4E-BP1phosphorylation. We fond that BAPTA/AM, EGTA and2-APB inhibited the increase of LC3-Ⅱ expression, Akt phosphorylation, mTOR-mediated S6K1and4E-BP1phosphorylation in Cd-induced neuronal cells, indicating that Cd-induced intracellular Ca2+([Ca2+]i) elevation is related to extracellular Ca2+influx and endoplasmic reticulum (ER) Ca2+release, which is associated with the activation of autophagy and Akt/mTOR pathway in neuronal cells. We noticed that KN93significantly inhibited Cd-induced LC3.-Ⅱ increase and the phosphorylations of Akt and mTOR-mediated S6K1and4E-BP1were obviously blocked by KN93in neuronal cells, suggesting that Cd may cause CaMKII activation by [Ca2+]i elevation, which is associated with the activation of autophagy and Akt/mTOR pathway in neuronal cells. The findings reveal that Ca2+signaling may play an important role in Cd in the activation of autophagy and Akt/mTOR pathway in Cd-induced neuronal cells.