Role Of TFEB In Autophagosome-lysosome Fusion Disorder In PC12 Cells Induced By PBDE-47

Objective: 2,2’,4,4’-tetrabromodiphenyl ether(PBDE-47)can induce neurotoxicity mainly with behavioral and cognitive impairment,but its mechanism of toxic effect has not been fully elucidated.On the basis of the findings that the neurotoxicity of PBDE-47 was related to abnormal autophagy caused by it,this study was intended to further investigate the effect of PBDE-47 on the fusion process between the autophagosome and lysosome during autophagy process in rat adrenal medulla pheochromocytoma PC12 cells,and discuss the role of transcription factor EB(TFEB)which can regulate autophagy and lysosomal biosynthesis in this process,so as to provide scientific evidence and theoretical basis for further study on the mechanism and prevention of neurotoxicity of PBDE-47.Methods: We used PBDE-47(1 μmol/L,10 μmol/L,20 μmol/L)to treat PC12 cells,24 h later,the morphology and number of autolysosomes in cells were observed by transmission electron microscope.After microtubule-associated protein 1 light chain3(LC3)was labeled with Alexa Fluor 488 green fluorescence,and lysosomal associated membrane protein 1(LAMP1)was labeled with Alexa Fluor 594 red fluorescence,the fusion of autophagosome and lysosome was evaluated by laser confocal microscope to observe the two co-localized yellow fluorescent spots.Western blot was used to detect the protein expression levels of the key proteins of autophagosome-lysosome fusion,they include syntaxin 17(STX17),synaptosomal-associated protein 29(SNAP29),vesicle-associated membrane protein 8(VAMP8).Western blot was also used to detect the protein expression levels of TFEB,autophagy-related gene 14(ATG14)and Ras-related protein 7(Rab7),the last two proteins can regulate the autophagosome-lysosome fusion core factor STX17-SNAP29-VAMP8 complex.To verify the role of TFEB in the fusion process of autophagosome and lysosome of PC12 cells induced by PBDE-47,cells were transfected with adenovirus overexpressing TFEB,24 h later,the protein expression levels of STX17,SNAP29,VAMP8,ATG14 and Rab7 were observed by Western blot.The fusion changes of autophagosome and lysosome were observed by laser confocal microscope after transfected with adenovirus Ad-mCherry-GFP-LC3 B,which can detect the autophagy state.The co-localization changes of LC3 and LAMP1 were observed with confocal microscope by immunofluorescence method.The changes of morphology and quantity of autolysosomes were observed by transmission electron microscope.Results: A large number of autolysosomes were observed in the cells of the control group under transmission electron microscope,and the morphology and number of autolysosomes in the 1 μmol/L PBDE-47 group were similar to the control group,but the number of autolysosomes in the 10 μmol/L PBDE-47 treated group was significantly reduced.The number of intracellular autolysosomes decreased and their volume increased in the 20 μmol/L PBDE-47 treated group.The results showed that with the increase of the dose of PBDE-47,the green fluorescent spots labeled LC3 in the cytoplasm increased,the yellow spots labeled LC3 and LAMP1 co-localization decreased.Western blot results showed that compared with the control group,the protein expression levels of STX17,SNAP29,VAMP8,Rab7 and TFEB in 10 μmol/L and 20 μmol/L PBDE-47 treated groups were significantly decreased(P < 0.05),and the protein expression levels of ATG14 in 1 μmol/L,10 μmol/L and 20 μmol/L PBDE-47 treated groups were markedly decreased(P < 0.05).After TFEB was overexpressed after 24 h,Western blot results showed that compared with adenovirus null+PBDE-47 treated group,the protein expression levels of SNAP29,VAMP8,ATG14 and Rab7 in TFEB overexpression+PBDE-47 treated group were observably increased(P < 0.05).Ad-mCherry-GFP-LC3 B transinfection and immunofluorescence co-localization results also found that compared with the adenovirus null+PBDE-47 treated group,TFEB overexpression+PBDE-47 treated group indicated that the yellow autophagosome fluorescence spots were decreased,the spots can indicate the fusion of autophagosomes and lysosomes was blocked,and the co-localization of LC3 and LAMP1 was enhanced.At the same time,transmission electron microscope also showed that,compared with the adenovirus null+PBDE-47 treated group,the number of autolysosomes in TFEB overexpression+PBDE-47 treated group was increased and the volume of autolysosomes returned to normal.Conclusions: A certain dose of PBDE-47 can inhibit the expression levels of autophagosome-lysosome fusion related proteins and TFEB in PC12 cells,thus inhibiting the process of autophagosome and lysosome fusion,resulting in decreased number and impaired functions of autolysosomes.TFEB overexpression can alleviate the barrier of autophagosome and lysosome fusion caused by PBDE-47 by increasing autophagosome-lysosome fusion related proteins,thus increasing the number of autolysosomes and improving their functions.