The Role Of TFEB-mediated Lysosomal Dysfunction In PBDE-47-induced PC12 Cell Injury

Objective: The neurotoxicity of brominated flame retardants(PBDEs)has received a lot of attention.It’s well documented that PBDEs-induced neurotoxicity is related to autophagy,but the specific mechanism is still unclear.This study is to investigate the effect of PBDE-47,the most toxic and widespread PBDEs,on lysosomal function and autophagic flux in PC12 cells,as well as the role of the lysosomal mastering factor(TFEB)in PBDE-47-induced PC12 cell impairment,providing experimental basis for the mechanism and treatment of PBDEs-induced neurotoxicity.Methods: PC12 cells were treated with different doses of PBDE-47(1,10,20 μmol/L)for 24 h.Western blot was used to detect the expression of cathepsinB(Cat-B)protein in lysosome and cytoplasmic.Lysotracker was used to detect the change of pH caused by PBDE-47.Western blot was used to detect the change of protein expression,include lysosome-associated membrane glycoprotein 1(LAMP1)and Cat-B.After transfect Ad-mCherry-GFP-LC3 B,we use confocal microscopy to observe the fusion of autophagosomes and lysosomes.The effect of PBDE-47 on the expression of Synaptosomal-associated protein 29(SNAP29),Syntaxin 17(STX17)and Vesicle-associated membrane protein 8(VAMP8)proteins in PC12 cells was detected by western blot.Then we mesured TFEB protein expression level and TFEB mRNA expression level in PC12 cells by western blot and qPCR.After overexpression of TFEB,we detected the protein expression level of LAMP1 and Cat-B,and the key proteins of the fusion of lysosome-autophagy.And we also mesured pH after the overexpression of TFEB.Because lysosomal function are related to autophagy flux,we test the protein expression level of autophagy related of protein.Finally,we use CCK8 to mesure the cell viability.Results: Compared with the control group,the expression level of Cat-B protein in the cytoplasm was significantly increased(P < 0.05),while the expression level of Cat-B protein in the lysosome was significantly decreased(P < 0.05).The results of immunofluorescence showed that the red spots in the control group were more and brighter,and the number gradually decreased and became dull with the increase dose of PBDE-47.Compared with the control group,the expression of LAMP1 protein was significantly decreased in the 10 μmol/L and 20 μmol/L PBDE-47 groups(P < 0.05).At the same time,in 1 μmol/L,10 μmol/L and 20 μmol/L PBDE-47 group,the expression level of Cat-B protein was also significantly decreased(P < 0.05).Confocal microscopy showed that the control group showed diffuse yellow fluorescence.With the increase of PBDE-47 concentration,the cell volume decreased,the diffuse yellow light decreased,and the red spots became more.The 20 μmol/L PBDE-47 group appeared obvious yellow spots.Compared with the control group,the expression levels of SNAP29,STX17 and VAMP8 were significantly decreased in the 10 μmol/L PBDE-47 group and the 20 μmol/L PBDE-47 group(P < 0.05).Compared with the control group,the expression of TFEB mRNA in the 10 μmol/L and 20 μmol/L PBDE-47 groups was significantly increased(P < 0.05),while the expression level of TFEB protein was significantly decreased in each PBDE-47 group(P < 0.05).After overexpression of TFEB,the expression levels of lysosome and autophagosome fusion proteins,SNAP29 and VAMP8 in TFEB+PBDE-47 group were significantly increased than those in adenovirus empty+PBDE-47 group(P < 0.05);The expression levels of LAMP1 and Cat-B proteins were also significantly increased(P < 0.05);the expression of ATG7 protein was significantly increased(P < 0.05),and the expression level of LC3 II protein was significantly decreased(P < 0.05).The expression level of p62 protein has not changed significantly.In addition,lysotracker staining also showed that the TFEB+PBDE-47 group had more and brighter red fluorescence than the adenovirus empty+PBDE-47 group.Finally,CCK8 results showed that the survival rate of the TFEB+PBDE-47 group was significantly higher than that of the adenovirus empty +PBDE-47 group(P < 0.05).Conclusions: PBDE-47 can cause lysosomal functional impairment and autophagosome-lysosomal fusion obstacle.Overexpression of TFEB can alleviate lysosomal functional and restore autophagic flux,ultimately improving cell survival.