The Roles Of Autophagy Impairment And Excessive Apoptosis In PBDE-47-induced Neurotoxicity

Part 1: Developmental exposure to PBDE-47 induced autophagy impairment and apoptosis in hippocampal tissue of female offspring ratsObjective: Polybrominated diphenyl ethers(PBDEs)cause neurotoxicity mainly characterized by behavioral and cognitive impairment.However,the underlying mechanisms are still unclear.We aimed to explore the roles of autophagy and apoptosis in 2,2',4,4'-tetrabromodiphenylether(PBDE-47)-induced neurotoxicity of female offspring Sprague-Dawley(SD)rats and provide scientific evidences and theoretical basis for illuminating the potential mechanisms.Methods: Forty eight female SD rats and twenty four male SD rats(Grade SPF,two-month old)were kept for one-week acclimation.Then the female rats were divided into four groups randomly as the control group(corn oil),0.1,1.0,and 10 mg/kg·day PBDE-47 groups.Weight the rats and gavage them with PBDE-47 according to the weight at 8:00-10:00 A.M.daily.After ten-day exposure,the treated female rats mate with untreated male rats at 2:1 randomly.The exposure was last until the weaning of offspring(except the parturition day).The offspring rats were re-caged according to sex and exposure dose after weaning and kept until postnatal day 88(three months).All rats were given free access to standardized granular food and tap water.The Morris water maze(MWM)task was used to assess spatial learning and memory abilities.Nissl staining was performed to observe the neuronal change.Transmission electron microscopy(TEM)was used to visualize the ultrastructure of hippocampus.Immunohistochemical experiments were conducted to detect the location and expression of apoptosis key protein,active cysteine containing aspartate specific protease-3(caspase-3),as well as the key proteins of autophagy,microtubule-associated protein 1 light chain 3(LC3)and the selective autophagy substrates,p62.Western blot analysis was performed to detect the protein levels of apoptosis key proteins,active caspase-3 and cleaved poly(ADP-ribose)polymerase(PARP)as well as the autophagy key proteins,LC3,p62 and autophagy-related protein 7(ATG7).Results: The results showed that in the place navigation test of MWM task,compared with the control group,the latency and swimming distance were significantly increased in 0.1 mg/kg·day PBDE-47-treated rats on the 4th day(P < 0.05);in addition,the longer latency of 1.0 mg/kg·day PBDE-47-treated group were also observed on the 4th day(P < 0.05);however,the swimming speed of 1.0 and 10 mg/kg·day PBDE-47-treated rats were significantly decreased starting from the 2nd day(P < 0.05).For the spatial probe test of MWM task,1.0 and 10 mg/kg·day PBDE-47-treated rats showed reduced swimming time in the target quadrant than the control rats(P < 0.05),with an additional significant decreased swimming distance in the target quadrant of 10 mg/kg·day PBDE-47 treated rats(P < 0.05).Nissl staining showed that the control hippocampal neurons with abundant Nissl bodies,were arranged regularly in the hippocampal CA3 region.In contrast,PBDE-47-treated rats showed that neurons were in disordered arrangement with lost neurons and less stained Nissl bodies.TEM showed that increased autophagosome-like double-membrane vesicles were observed in PBDE-47-treated groups when compared with the control group which has normal neuronal morphology,round cell nucleus and uniformly distributed chromatin.Immunohistochemical staining showed that active caspase-3,LC3 and p62 all located in the neuronal cytoplasm and the positive staining was notably increased in PBDE-47-treated rats in the hippocampal CA3 region.Western blot showed that compared with the control group,the levels of apoptosis key proteins active caspase-3 and cleaved PARP,as well as the autophagic key protein LC3-II,p62 were dramatically increased in 1.0 and 10 mg/kg·day PBDE-47-treated groups,while the levels of ATG7 were significantly decreased in the same exposure doses.Besides,the levels of LC3-II were also increased in the 0.1 mg/kg·day PBDE-47-treated rats.Conclusion: Developmental exposure to PBDE-47 induces memory impairments in female offspring SD rats.PBDE-47 exposure causes the loss of hippocampal neurons.In addition,PBDE-47 exposure induces increased apoptosis level.Meanwhile,PBDE-47 exposure inhibits the autophagosome formation and blocks the autophagic degradation,inducing autophagy impairment.Taken together,autophagy impairment is involved in PBDE-47-induced neurotoxicity in SD rats.Part 2: The roles of autophagy impairment and excessive apoptosis in PBDE-47-induced toxicity in PC12 cellsObjective: To explore the roles of autophagy impairment and excessive apoptosis in PBDE-47-induced toxicity in PC12 cells,as well as their relationship in this process,and provide novel insight for the mechanism of PBDE-47-induced neurotoxicity.Methods: Different doses of PBDE-47(0,1,10,20 ?mol/L)treated PC12 cells for 24 h,DMSO as the control group.Flow cytometry was performed to evaluate the percentage of apoptotic cells.Terminal deoxynucleotidyl transferase mediated d UTP nick end labeling(TUNEL)assay was conducted to detect the DNA fragmentation.TEM was used to observe the cell ultrastructure.Laser scanning confocal microscopy was used to observe the aggregation of autophagosome marker molecular LC3 fluorescence dots.Western blot was performed to detect the protein levels of apoptosis key proteins,active caspase-3 and cleaved PARP as well as the autophagy key proteins,LC3-II,p62 and ATG7.PC12 cells were treated with 20 ?mol/L PBDE-47 for 3 h,6 h,12 h and 24 h.Western blot analysis was used to evaluate the protein levels of apoptosis key protein,active caspase-3 and autophagy key protein,LC3-II.PC12 cells were treated with PBDE-47(20 ?mol/L)with/without Rapamycin(Rapa)(pre-treated for 24 h),wortmannin(WM)and caspase-3 inhibitor,Ac-DEVD-CHO(DEVD)for 24 h.The protein levels of apoptosis key proteins,active caspase-3,cleaved PARP and autophagy key proteins,LC3-II,p62,ATG7 were detected by western blot.PC12 cells were infected with adenovirus expressing Atg7(Ad-Atg7)for 24 h,then exposed to 20 ?mol/L PBDE-47 for another 24 h.Besides,cells were transfected with si RNA-Atg7 for 24 h before treated with PBDE-47.The protein levels of apoptosis and autophagy key proteins were detected by western blot.Moreover,the immunofluorescence colocalization between autophagosome marker LC3 fluorescence dots and TUNEL-positive cells were observed by laser scanning confocal microscopy.The cell viability was examined by CCK-8 assay after all kinds of intervention treatment.Results: The percentages of total apoptotic cells were significantly increased in 10 and 20 ?mol/L PBDE-47-treated group,with an additional significant increase in the percentage of early apoptotic cells in 20 ?mol/L PBDE-47-treated group(P < 0.05).TUNEL assay showed that treatment with 20 ?mol/L PBDE-47 notably increased the number of TUNEL-positive cells(%).The increased number of typical double-membrane autophagosomes was visualized upon PBDE-47 treatment compared with the control group.Confocal microscopy revealed that PBDE-47 induces the accumulation of LC3 fluorescence dots in PC12 cells.Moreover,compared with the control group,10 ?mol/L and 20 ?mol/L PBDE-47 treatment dramatically increased the protein levels of active caspase-3 and cleaved PARP,as well as LC3-II and p62(P < 0.05);however,the protein levels of ATG7 were dramatically decreased.The protein levels of LC3-II were significantly increased from 3 h after PBDE-47 treatment and this increase was sustained up to 24 h,while the levels of active caspase-3 were elevated dramatically starting from 12 h until 24 h after PBDE-47 treatment(P < 0.05).Compared with the PBDE-47-treated group,Rapa further increased the levels of active caspase-3 and cleaved PARP;WM decreased the levels of active caspase-3 and cleaved PARP;DEVD reduce the levels of LC3-II.Atg7 overexpression significantly increased cleaved PARP;while,Ad-Atg7 decreased the level of active caspase-3 when compared with PBDE-47 combined Ad-Null treatment group.The levels of active caspase-3 and especially cleaved PARP were dramatically decreased after DEVD administration,in comparison to PBDE-47 in combination with Ad-Null or Ad-Atg7 treatment rats.si RNA-Atg7 combined with PBDE-47 treatment reduced the level of active caspase-3 and cleaved PARP compared with the si RNA-NC+PBDE-47 group.Atg7 overexpression increased the number of TUNEL-positive cells containing LC3 fluorescence dots following PBDE-47 treatment,while the number of PBDE-47-induced TUNEL-positive cells with less LC3 fluorescence dots accumulation was notably decreased after si RNA-Atg7 interference.Besides,the results of CCK-8 showed that compared with the PBDE-47 group,Rapa+PBDE-47 further decreased the cell viability;WM+PBDE-47 increased the cell viability;DEVD+PBDE-47 also increased the cell viability;however,WM combined with DEVD did not further increased the cell viability.Adenoviral Atg7 overexpression reduced the cell viability,and si RNA-Atg7 treatment has the opposite results(P < 0.05).Conclusion: PBDE-47 can cause the excessive apoptosis.Moreover,PBDE-47 treatment inhibits the autophagosome formation and blocks the autophagic degradation,inducing autophagy impairment in PC12 cells.PBDE-47-induced impaired autophagy and apoptosis both have time-effect relationship,and autophagy alteration is more sensitive than apoptosis induction in PC12 cells.Stimulation of autophagy aggravates PBDE-47-induced apoptosis and promotes PC12 cells death.Inhibition of autophagy attenuates PBDE-47-induced apoptosis and alleviates PC12 cells death.Blockage of apoptosis mitigates PBDE-47-induced autophagy impairment facilitating PC12 cells survival.In conclusion,autophagy impairment promotes excessive apoptosis,which in turn disrupts autophagy,ultimately resulting in PC12 cell death.As the coupled events,autophagy impairment and excessive apoptosis contribute to PBDE-47-induced PC12 cell neurotoxicity.