Studies On Molecular Mechanisms Of Cadmium-Induced Apoptosis Through Akt-Beclin1 Mediating Blockage Of Autophagic Flux In Neuronal Cells

The present study,using cellular and molecular biology techniques and methods including cell culture,MDC and DAPI staining,fluorescent protein labeling,RNA interference,Western Bloting,etc.,and employing PC12 cells and primary murine neurons as experimental objects,systematically investigated the relationships among cell apoptosis,formation of autophagosomes and autophagic flux in neuronal cells induced by cadmium(Cd),and deeply dissected the role and mechanisms of Akt-Beclin 1 pathway in Cd-induced blockage of autophagic flux contributing to neuronal apoptosis.The detailed results were summarized as follows: 1.Cd induction of autophagy is involved in blockage of autophagic flux in neuronal cellsPC12 cells and primary murine neurons,or PC12 cells and primary murine neurons infected with Ad-GFP-LC3,were treated with 10 and 20 μM Cd for 8,12 and 24 h,followed by analyzing MDC fluorescence intensity by MDC staining and counting changes of GFP-LC3 puncta.PC12 cells and primary murine neurons were treated with different concentrations of Cd(0-20 μM)for 8 h or with 10 μM Cd for 0-24 h,and then detected the expression of LC3-Ⅰ/Ⅱ and p62 using Western blotting.The results showed that Cd evoked the increase of MDC fluorescence intensity in PC12 cells and primary murine neurons.There existed increased large GFP-LC3 puncta in Cd-exposed cells.Western blotting exhibited that Cd elevated expression of LC3-Ⅱ and p62 in a concentration-and time-dependent manner in the cells.Our results suggest that Cd induction of autophagy is involved in blockage of autophagic flux in neuronal cells.2.Cd induction of autophagy results in apoptosis in neuronal cellsBecause 3-MA is a common inhibitor for inhibiting formation of autophagosome,we investigated the effects of 3-MA on MDC fluorescence intensity,manifestation of large GFP-LC3 puncta and apoptosis in neuronal cells induced by Cd.By generating lentiviral shRNAs to LC3-Ⅰ/Ⅱ for down-regulation of LC3-Ⅰ/Ⅱ in PC12 cells,the changes of MDC fluorescence intensity and apoptosis were observed in the cells in response to Cd.The results showed that 3-MA inhibited Cd-induced MDC fluorescence intensity and the formation of large GFP-LC3 puncta.Consistently,3-MA also attenuated Cd-induced apoptosis in the cells.Similarly,down-regulation of LC3-Ⅰ/Ⅱ significantly decreased Cd-induced MDC fluorescence intensity and apoptosis in neuronal cells.The data imply that Cd induction of autophagy results in apoptosis in neuronal cells.3.Cd blocks autophagic flux contributing to accumulation of autophagosomes and apoptosis in neuronal cellsPC12 cells were infected by recombinant adenovirus expressing tandem mCherry-GFP-LC3(Ad-mCherry-GFP-LC3),followed by analyzing manifestation of autophagic flux in the cells induced by Cd;Using chloroquine(CQ),a common inhibitor for blockage of autophagic flux,the role of status of autophagic flux in Cd-induced autophagy contributing to apoptosis in neuronal cells were further confirmed.The results showed that Cd elicited more co-localization of mCherry and GFP-LC3 in the cells,suggesting Cd blockage of autophagic flux in neuronal cells.Inhibition of autophagic flux by CQ observed that CQ further elevated Cd-induced MDC fluorescence intensity,the cleavages of caspase-3 and PARP,and strengthened Cd-triggered neuronal apoptosis.These results indicate that Cd blocks autophagic flux contributing to accumulation of autophagosomes and apoptosis in neuronal cells.4.Cd is involved in blockage of autophagic flux contributing to neuronal apoptosis via activating Akt-Beclin 1 pathway inducing autophagyExpression of Akt and Beclin 1 was detected in Cd-exposed neuronal cells using Western blotting.By generating lentiviral shRNAs to Beclin 1 for down-regulation of Beclin 1 in neuronal cells,the changes of MDC fluorescence intensity and apoptosis were observed in the cells induced by Cd.Using Akt inhibitor or Ad-dn-Akt for inhibiting Akt activity,the manifestations of Cd-induced MDC fluorescence intensity,large GFP-LC3 puncta,autophagic flux and apoptosis in the cells were investigated.The results exhibited that Cd induced phosphorylation of Akt(Ser473)and Beclin 1(Ser295).Down-regulation of Beclin 1 attenuated Cd-induced MDC fluorescence intensity,cleaved-caspase-3 and apoptosis in the cells.Akt inhibitor relieved Cd-evoked the increase of MDC fluorescence intensity and protected from Cd-induced neuronal apoptosis.Expression of dn-Akt inhibited Cd-induced phosphorylation of Beclin 1,formation of large GFP-LC3 puncta,and cell apoptosis.Of importance,Expression of dn-Akt reversed Cd blockage of autophagic flux in neuronal cells.The data suggest that Cd is involved in blockage of autophagic flux contributing to neuronal apoptosis via activating Akt-Beclin 1 pathway inducing autophagy.