The Role And Regulation Mechanism Of Autophagy In Neuronal Senescence Induced By Cadmium

Autophagy is a physiological and evolutionarily highly conserved phenomenon responsible for the removal of protein macromolecule and damaged organelles by the lysosome.Autophagy can help cell escape cell lesion,damage and degeneration via degrading abnormal substances in the condition of metabolic stress.Thus,autophagy plays an important role in cell survival.Cd is a high toxic industrical and environmental pollutant,which can do harm to the health of animal and human beings.It has been shown that Cd could get into brain tissues via permeating the blood brain barrier and affect the function of nervous system.Clinical studies showed that Cd caused neurobehavioral dificit,which is a etiological factor of cognitive dysfunction in children and neurodegenerative disease.In vitro study revealed that neuronal autophagy induced by activating ERK and class ? PI3K/Beclin-1/Bcl-2 pathway protected neuronal cells from Cd toxicity,however the specific mechanism remains unclear.Recent studies indicated that neuronal senescence is the main cause of neurodegeneration disease.However,it still remains unclarified whether neuronal senescence is responsible for the cytotoxicity of cadmium and how autophagy interacts with senescence in neuronal cells.By using the PC 12 cells and rat cerebral cortical neurons as a neuronal model,the present study surveyed the specific mechanism underlying autophagic pathway activated by Cd and explored the effect of autophagy in Cd-induced senescence and oxidative damage.This study will offer new theoretic evidences for future clarifying the mechanism in neurotoxicity of cadmium.1.Cd induced senescence in neuronal cellsIn order to study the effect of Cd on neuronal senescence,this study used PC12 cells as a neuronal model.We monitored the growth curve of PC 12 cells under the condition of different concentration Cd(0,2.5,5,10 ?mol/L).Results showed that 10 ?mol/L Cd declined the cell index(CI)of PC12 cells compared with control group.Cd also induced decrease in CI in a dose and time-dependent manner within the certain consistency and time,which indicated that Cd has cytotoxic effect on PC 12 cells.Then we used PC 12 cells and primary neurons as a neuronal model.After treating PC12 cells or primary neurons with different concentration of Cd(0,5,10,20?mol/L)for 24 h respectively,we counted the positive cells of ?-galactosidase in PC12 cells;Gene expression of inflammatory factors IL1?,IL6 were detected in primary neurons by using qRT-PCR.The expression of senescence related proteins P53,P21,P16 were measured in PC 12 cells by using westemblot.After treating PC 12 cells or primary neurons with 10 ?mol/L Cd for different time(0,2,4,6,8,12,24 h),we performed westernblot to assess the expression of P53,P21,P16 proteins.To investigate the effect of Cd on the skeleton of neuronal cells,F-actin structure was observed by staining with phalloidine in PC 12 cells and primary neurons.The results showed that treatment with 10 ?mol/L Cd increased the number of positive?-galactosidase-expressed PC 12 cells obviously compared with control group(P<0.01).Structural damage of F-actin was observed after treating with 10 ?mol/L Cd for 24 h in both PC12 cells and primary neurons.The expression of P53,P21 and P16 was increased by Cd(P<0.01)in a dose and time-dependent manner in PC12 cells while the gene expression of ILla,IL6 was also increase by Cd(P<0.01)in a dose-dependent manner in primary neurons.In sum,these results suggest that Cd induced senescence in PC 12 cells and primary neurons.2.The role of autophagy in neuronal senescence induced by CdIn order to investigate the role of autophagy in neuronal senescence induced by Cd,in this study,PC 12 cells and primary neurons were pretreated with 100 nmol/L rapamycin for 24 h,followed by treatment with cadmium for another 24 h.The number of the?-galactosidase-expressed PC 12 cells was counted.The results showed that in comparison with the Cd group for 24 h,RAP-mediated autophagy activation decreased the number of?-galactosidase positive cells induced by Cd(P<0.01);Western blot analsis was performed to measure the expression of P53,P21 and P16 protein.We found that the protein expression in PC 12 cells with the co-treatment of RAP and Cd decreased compared with Cd group for 24 h(P<0.01).The gene expression of ILla,IL6in primary neurons was detected by qRT-PCR.The data showed that the ILla,IL6 gene expression induced by Cd was inhibited by RAP(P<0.01).PC 12 cells were pretreated with 5 ?mol/L CQ for 0.5 h or transfected with Atg5 siRNA and then followed by treatment with Cd for another 6 h.The growth curve of the cells was measured by using RTCA,which showed that CQ-mediated autophagy inhibition decreased CI compared with Cd group for 6 h.The result of westernblot revealed that either 3-MA or Atg5 siRNA transfection-mediated autophagy inhibition can promote the expression of P53,P21 and P16 protein.These results demonstrate that Cd-induced neuronal senescence can be reversed by autophagy.3.The impact of ER stress on neuronal autophagy induced by CdTo explore the impact of ER stress on autophagy induced by Cd,the present study used PC 12 cells and primary neurons as neuronal model.PC 12 cells and primary neurons were exposured to 10 ?mol/L for various times(0,2,4,6,8,12,24 h),the expression of ER stress related proteins Bip,ATF4,CHOP and P-eIF2a were examined by western blot.The results showed that the levels of CHOP protein increased in a time-dependent manner while other proteins reached a peak 6 h and then gradually decreased.PC12 cells or primary neurons was cultured with or without the ER stress inhibitor mithramycin for 6 h,western blot analsis was performed to measure the expression of Bip and LC3 protein.Immunofluorescence was performed to observe the expression and distribution of Bip.PC12 cells stably transfected with EGFP-RFP-LC3B were treated as mentioned above,LC3 puncta were observed under a confocal microscope system.The data showed that mithramycin-mediated ER stress inhibition decreased the levels of Bip and LC3 ? proteins(P<0.01)as well as LC3 puncta(P<0.01).The study indicates that Cd induced ER stress in PC 12 cells and primary neurons,which plays an positive role in the regulation of autophagy.4.Bip/AMPK is involved in autophagy activation induced by CdIn order to explore the role of ER chaperone Bip in Cd-induced autophagy activation in neuronal cells,Bip siRNA was used to transfected into PC12 cells to inhibit the expression of Bip.Negative transfection(NC)group cell or Bip siRNA transfection(siBip)group cells were treated with 10 ?mol/L Cd for different time(0,2,6,24 h).The number of the?-galactosidase-expressed PC12 cells was counted by using ?-galactosidase detection kit.Bip,LC3 and P21 protein expression were detected by westernblot.The results showed that compared with NC group,the number of ?-galactosidase-expressed cell was significantly increased by Cd in siBip group(P<0.01).Western blot analysis of PC12 cells transfected with Bip siRNA identified a marked decrease in Bip,P-AMPK protein expression,downregulated LC3?/LC3I conversion but enhanced expression of P21 compared with NC in the presence of Cd.PC 12 cells were treated with Cd in the presense or absence of Bip inhibitor BAPTA,the effect of Bip downregulation on autophagy was evaluated by EGPF-RFP-LC3 puncta formation using confocal microscopy.Either siBip or BAPTA decreased Cd-induced LC3 puncta formation(P<0.01).To confirm the role of Bip in Cd-induced AMPK activation,we exposed the cells to different concentration(0,5,10,20 ?mol/L)of Cd,we found that the expression of P-AMPK protein increased in a dose-dependent manner.We exposed BAPTA to PC 12 cells or primary neurons before treating with Cd.The results showed that BAPTA-mediated Bip inhibition decreased the protein expression of P-AMPK compared with Cd group.Importantly,NC or siBip cells were treated with Cd for 6 h,westernblot was performed to detect the expression of P-AMPK,P-AKT,P-S6K protein,which showed that the depletion of Bip by Bip siRNA apparently abolished the activation of AMPK,but enhanced the expression of P-AKT and P-P70s6k.All the data indicates that Bip plays an important role in the regulation of autophagy and senescence induced by Cd via the activation of AMPK in neuronal cells.5.ROS is involved in both neuronal autophagy and senescence induced by CdIn this study we explored the role of ROS in neuronal autophagy and senescence induced by Cd by using PC 12 cells and rat cortical neurons as neuronal model.PC 12 cells or primary neurons were treated with 10 ?mol/L Cd in the presense or absence of ROS inhibitor NAC(100 nmol/L)for 6 h or 24 h,the data showed that the expression of LC3II was not decreased or increased by NAC-mediated inhibition of ROS at 6 h.The primary neurons were treated with 20?mol/L Cd in the presence or absence of 100 nmol/L NAC.Westernblot analysis was performed to detect the expression of LC3 II,Atg5,Atg7 proteins.The results showed that the up-regulation of these autopahgy related proteins induced by Cd at 6 h was blocked by NAC.In order to confirm ROS was involved in Cd-induced senescence,PC 12 cells or primary neurons were cultured with 10 ?mol/L Cd in the presence or absence of NAC.The results showed that the expression of P21 protein,the number of P-galactosidase-expressed PC 12 cells as well as the gene expression of IL1?,IL6 in primary neurons was decrease by NAC comprared with Cd group at 24 h(P<0.01).The data of the study demonstates that ROS serves an important role in Cd-induced autopahgy in the early stage and is involved in senescence,which depends on the concentration of Cd.