The Role Of AMPK/ULK1 Axis-modulated Mitophagy In PBDE-47-induced Neurotoxicity And The Intervention Effect Of Melatonin

Polybrominated diphenyl ethers(PBDEs)are a class of widely used brominated flame retardants,and their developmental neurotoxicity has attracted much attention,but the specific mechanism has not been fully elucidated.It is well known that PBDEsinduced neurotoxicity is closely related to mitochondrial dysfunction,and mitophagy plays an important role in maintaining mitochondrial homeostasis.Mitophagy is the process of removing damaged or excess mitochondria,and is essential for the quality control of mitochondria in the cells.When mitochondria are damaged,mitochondrial membrane potential(MMP)is lost,inhibiting the activity of mitochondrial membrane transport enzyme,so PTEN induced putative kinase 1(PINK1),the outer mitochondrial membrane protein,can not be normally transported to the inner mitochondrial membrane for degradation,accumulating on the outer mitochondrial membrane,thereby recruiting Parkin in the cytoplasm to the damaged mitochondria.Parkin is an E3 ubiquitin ligase,whose enzyme activity is activated under the action of PINK1,and marks damaged mitochondria by ubiquitination of mitochondrial outer membrane protein.The autophagy receptor protein p62 recognizes ubiquitination-labeled mitochondria and binds to the autophagosome membrane protein microtubule-associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ),forming mitophagosomes,subsequently fusing with lysosomes,thereby achieving the degradation of mitochondria.Mitophagy is regulated by a variety of signaling pathways.When the ratio of adenosine monophosphate(AMP)to adenosine triphosphate(ATP)increases,the Thr-172 of AMP-activated protein kinase(AMPK)is phosphorylated,activating AMPK.Activated AMPK can directly phosphorylate and activate unc-like kinase 1(ULK1),the autophagy initiation key protein,to induce autophagy.However,how PBDEs affect mitophagy in nerve cells,and the related molecular mechanisms have not been reported.In addition,there is evidence that melatonin can play a neuroprotective role by regulating the process of autophagy.Therefore,this study aimed to investigate the role of AMPK/ULK1 axis-modulated mitophagy in 2,2’,4,4’-tetrabromobiphenyl ether(PBDE-47)neurotoxicity,and whether melatonin can antagonize PBDE-47 neurotoxicity by regulating AMPK/ULK1 signaling pathway,thus providing theoretical basis for further elucidating the mechanism of PBDE-47 neurotoxicity and its prevention.Part 1.The role of AMPK/ULK1 axis-modulated mitophagy in PBDE-47-induced neurotoxicityObjective: This study was to explore the role of mitophagy regulated by the AMPK/ULK1 axis in PBDE-47 neurotoxicity,and provided the basis for further clarifying the mechanism of PBDE-47 neurotoxicity.Methods:(1)In vivo experiments.48 newborn Sprague-Dewley(SD)male rats were randomly divided into four groups according to the body weight and litter: control group(corn oil),1.0 mg/kg,10 mg/kg,and 20 mg/kg PBDE-47-treated group.On the postnatal day 10(PND 10;brain development spurt),a single gavage of PBDE-47 was administered.After the rats were kept to PND 60,the Morris water maze(MWM)test was used to test the learning and memory ability of the rats;the open field test(OFT)was used to observe the changes of autonomous behavior of the rats;the transmission electron microscopy(TEM)was employed to evaluate the ultrastructural changes of striatum nerve cells;Western blot technique was used to detect the key proteins of apoptosis cleaved poly ADP ribose polymerase(cleaved PARP),active cysteine containing aspartate specific protease-3(active caspase-3),mitochondrial number marker protein cytochrome c oxidase IV(COX IV),mitophagy-related proteins PINK1 and Parkin,autophagy-related protein 7(ATG7),LC3-Ⅱ,p62,autophagy upstream AMPK/ULK1 axis related proteins phosphorylated AMPK(p-AMPK)and phosphorylated ULK1(p-ULK1)levels in striatum tissue.(2)In vitro experiments.After treatment with different concentrations of PBDE-47(1.0 μmol/L,10 μmol/L,20 μmol/L)for 24 h in PC12 cells,the ultrastructural changes were observed by TEM;MMP level was detected by flow cytometry;ATP level was detected by adenosine triphosphate(ATP)detection kit;the levels of mitochondrial number marker protein COX Ⅳ,PINK1,Parkin,ATG7,LC3-II,p62 proteins and pAMPK,p-ULK1 proteins were detected by Western blot technique.In order to further clarify the role of mitophagy in PBDE-47 neurotoxicity,PC12 cells were transfected with adenovirus expressing Parkin(Ad-Parkin)or null adenovirus for 24 h,then treated with 20 μmol/L PBDE-47 or dimethylsulfoxide(DMSO)for 24 h;using ATG7 as the target,adenovirus overexpressing Atg7(Ad-Atg7)or small interfering RNA(si RNA)silencing Atg7(si RNA-Atg7)for 24 h,then the cells were treated with PBDE-47 or DMSO for another 24 h.To further reveal the regulatory effect of AMPK/ULK1 axis on mitoophagy in neuronal cell damage induced by PBDE-47,using AMPK as the target,the cells were pre-treated with AMPK inhibitor Compound C or AMPK agonist Acadesine(AICAR)for 2 h,and then treated with PBDE-47 or DMSO for 24 h.Used Western blot technique to detect the related protein levels of AMPK/ULK1 axis,mitophagy and apoptosis;ATP detection kit to detect ATP concentration;cell counting kit 8(CCK8)to detect cell survival rate.Results:(1)In vivo experiments.The MWM test showed that compared with the control group,the latency and swimming distance were increased significantly;the proportion of time and distance in the target quadrant were significantly reduced;in addition,the number of the rat traversed the platform was also significantly reduced in the PBDE-47-treated groups(P < 0.05).OFT found that the proportion of moving distance and time in the central area were significantly reduced in the PBDE-47-treated groups in comparison to the control group(P < 0.05).TEM observed increased autophagy vesicles,obvious mitochondrial structure damage and reduced mitochondrial number in the PBDE-47-treated nerve cells compared with the control group.Western blot results showed that in the PBDE-47-treated group,the expression of cleaved PARP and active caspase-3 proteins were significantly elevated in comparison to the control group,and the expression of COX Ⅳ protein was significantly reduced.In addition,the expression of PINK1,Parkin and ATG7 proteins were also significantly reduced,but LC3-II and p62 protein levels were eleated,and p-AMPK and p-ULK1 protein expression levels were also increased significantly(P < 0.05).(2)In vitro experiments.TEM observed increased autophagic vesicles,reduced and damaged mitochondria in PBDE-47-treated cells.Compared with the control group,the levels of COX Ⅳ protein,MMP and ATP in PBDE-47-treated group were significantly reduced;the expression levels of PINK1,Parkin and ATG7 proteins were significantly reduced;the levels of LC3-Ⅱ and p62 proteins were significantly enhanced;p-AMPK and p-ULK1 protein expression levels were also significantly increased(P < 0.05).Compared with PBDE-47-treated group,PINK1,Parkin,LC3-Ⅱ and p62 protein levels were increased;COX Ⅳ protein expression and ATP levels were decreased;cleaved PARP and active caspase-3 protein expression were increased and cell survival rate was decreased in PBDE-47+Ad-Parkin-treated group(P < 0.05).The results of the PBDE-47+AdATG7-treated group were similar,except the no significant change in the level of LC3-Ⅱ protein.In contrast,ATG7,PINK1,Parkin and LC3-Ⅱ protein levels were reduced;p62,COX Ⅳ protein levels as well as ATP level were increased;cleaved PARP and active caspase-3 protein expression levels were significantly reduced;cell survival rate was increased in PBDE-47+si RNA-ATG7-treated group(P < 0.05).p-AMPK and p-ULK1 protein expression were decreased;PINK1,Parkin,ATG7,LC3-Ⅱ and p62 protein expression were increased significantly;COX Ⅳ protein and ATP levels were decreased significantly;cleaved PARP,active caspase-3 protein expression and cell death were further increased in PBDE-47+Compound C-treated group(P < 0.05).As for PBDE-47+AICAR-treated group,p-AMPK and p-ULK1 protein expression were significantly increased;ATG7,PINK1,Parkin,p62 protein levels were decreased;COX Ⅳ protein and ATP levels were significantly enhanced;cleaved PARP,active caspase-3 protein expression and cell death were significantly reduced(P < 0.05);however,LC3-Ⅱ protein level did not change significantly.Conclusion: On the one hand,PBDE-47 can inhibit the formation of PINK1/Parkin mediated mitophagy in nerve cells,on the other hand,it can also hinder the degradation of autophagosomes,blocking the mitochondrial degradation,resulting in mitochondrial damage and triggering apoptosis,eventually reducing cell survival;targeting activation rather than inhibition of the AMPK/ULK1 axis can significantly alleviate PBDE-47-induced mitophagy impairment and cell death.Part 2.Intervention effect of melatonin regulating AMPK/ULK1 signaling pathway on PBDE-47-induced neurotoxicityObjective: The purpose of this study was to investigate whether melatonin can antagonize PBDE-47 neurotoxicity by regulating the AMPK/ULK1 signaling pathway, thereby providing a theoretical basis for the prevention and treatment of PBDE-47 neurotoxicity.Methods:(1)In vivo experiments.48 newborn male SD rats were randomly divided into four groups according to body weight and litter: control group,PBDE-47-treated group,PBDE-47+melatonin-treated group and melatonin-treated group.A single gavage of 20 mg/kg PBDE-47 or corn oil was performed on PND 10.From PND 8 to PND 60(the end of the model),10 mg/kg/day melatonin or solvent control(0.9% saline+2% ethanol)was intraperitoneally injected daily from 19:00 to 20:00.The MWM experiment was used to detect the learning and memory abilities of rats;the OFT was used to observe the changes of autonomous behavior of rats;TEM was used to observe the ultrastructural changes of striatum nerve cells;Western blot technique was used to detect the levels of AMPK/ULK1 axis,mitophagy and apoptosis related proteins,p-AMPK,p-ULK1,COX IV,PINK1,Parkin,ATG7,LC3,p62,cleaved PARP and active caspase-3.(2)In vitro experiments.PC12 cells were divided into four groups: control group,PBDE-47-treated group,PBDE-47+melatonin-treated group and melatonin-treated group.Cells were pre-treated with 25 μmol/L melatonin or solvent control(DMSO)for 2 h,and then treated with PBDE-47 or DMSO for 24 h.Used Western blot technique to detect the levels of AMPK/ULK1 axis,mitophagy and apoptosis related proteins;ATP detection kit to test ATP level;CCK8 to detect cell survival rate.To further clarify whether melatonin could ameliorate the toxic effect of PBDE-47 on nerve cells by regulating the AMPK/ULK1 signaling pathway,melatonin and AMPK inhibitor Compound C were cotreated in PC12 cells.Divided PC12 cells into four groups: control group,PBDE-47-treated group,and PBDE-47+melatonin-treated group and PBDE-47+melatonin+Compound C-treated group.Cells were pre-treated with Melatonin and Compound C for 2 h,then co-treated with PBDE-47 for 24 h,subsequently testing the above indicators.Results:(1)In vivo experiments.In the MWM experiment,compared with the PBDE-47-treated group,the latency of was significantly reduced,and the number of crossing the platform was significantly increased in the PBDE-47+melatonin-treated group.In OFT,and the proportion of moving in the central area was significantly increased in PBDE-47+melatonin-treated group in comparison with the PBDE-47-treated group(P < 0.05).TEM observed that compared with the PBDE-47-treated group,the autophagic vesicles were reduced in the striatum nerve cells;the mitochondrial structural damage was reduced;mitochondrial number was increased in the melatonin+PBDE-47-treated group.In addition,compared with the PBDE-47-treated group,the expression of p-AMPK,p-ULK1,COX Ⅳ,Parkin,ATG7 were increased,but the levels of LC3-II,p62,cleaved PARP and active caspase-3 proteins were decreased in the melatonin+PBDE-47-treated group.(2)In vitro experiments.Compared with PBDE-47-treated group,the expression of p-AMPK,p-ULK1,COX Ⅳ,PINK1,Parkin,ATG7 proteins were incresed significantly;ATP level was increased significantly;the levels of LC3-II,p62,cleaved PARP and active caspase-3 proteins were significantly reduced;the cell survival rate was significantly increased in PBDE-47+melatonin-treated group.Compared with the PBDE-47+melatonin-treated group,the expression of p-AMPK,p-ULK1,COX Ⅳ,PINK1,Parkin,ATG7 proteins were significantly reduced;ATP level was also decreased;however,the levels of LC3-II,p62,cleared PARP and active caspase-3 proteins were significantly increased;cell survival rate was significantly reduced in PBDE-47+melatonin+Compound C-treated group.Conclusions: Melatonin can alleviate PBDE-47-induced mitophagy impairment by activating the AMPK/ULK1 axis,reducing autophagosome accumulation,increasing mitochondrial number and function,inhibiting cell apoptosis,and promoting cell survival,thus playing a protective role in PBDE-47 neurotoxicity.Melatonin targeting the AMPK/ULK1 axis may be a potential prevention strategy for PBDE-47 neurodevelopmental toxicity.