The Role Of Autophagy-lysosome Pathway Impairment In PBDE-47-Induced Aβ Pathology In PC12 Cells

Alzheimer’s disease(AD)is a progressive neurodegenerative disease.The main pathological features include extracellular insoluble β-amyloid(Aβ)plaques,and the intracellular neurofibrillary tangles(NFTs)formed by overexphosphonated tau protein.Since the etiology and mechanism of AD are still unknown,there is no effective therapy available to cure AD.Emerging studies show that autophagy-lysosome pathway impairment plays an important role in the early development of AD.At present,accumumalting evidence shows that environmental factors such as exposure to some pollutants would increase the risk of AD in addition to genetic factors.As a new type of environmental persistent organic pollutant,polybrominated diphenyl ethers(PBDEs)have attracted much attention for their neurotoxicity.Our recent studies have shown that2,2’,4,4’-tetrabromodiphenyl ether(PBDE-47),the representative homologues of PBDEs,induces autophagosomes accumulation and lysosomal function damage in PC12 cells,a well-defined in vitro neuronal model.However,it is not clear whether PBDE-47 would also result in AD pathology in neurons via triggering autophagy-lysosome pathway impairment.Objective: The purpose of this study was to investigate the effect of PBDE-47 onβ-amyloid protein(Aβ)pathology in PC12 cells,and to explore the role of autophagy-lysosome pathway impairment behind it.Methods:(1)To study the effects of PBDE-47 on Aβ pathology and autophagy-lysosome pathway in PC12 cells.PC12 cells were treated with different concentrations of PBDE-47(1 μmol/L,10 μmol/L,20 μmol/L)for 24 h.Western blot was used to detect β-amyloid protein(Aβ)pathology-related proteins such as amyloid precursor protein(APP),C-terminal fragment beta(β-CTF)and C-terminal fragment alpha(α-CTF),autophagy-lysosome pathway-related proteins such as autophagy related protein 7(ATG7),autophagy selective substrate p62/SQSTM1,microtubule associated protein 1 light chain 3(LC3)-II,lysosomal associated membrane protein 1(LAMP1)and Cathepsin B(Cat B).Enzyme linked immunosorbent assay(ELISA)was used to detect the level of Aβ42.Immunofluorescence was used to observe the colocalization of Aβ42 with autophagosomal marker LC3 and lysosomal marker LAMP1.(2)To investigate the role of autophagy changes in PBDE-47-induced Aβpathology in PC12 cells,we used adenovirus overexpressing ATG7(Ad-ATG7)or small interfering RNA(si RNA)against ATG7(si RNA-ATG7)to promote or inhibit autophagy before PBDE-47 treatment.(3)To explore the effect of PBDE-47 on the degradation of Aβ pathology-related proteins,cycloheximide(CHX)was used and(or not)combined with PBDE-47 treatment for 0,0.5,1,2,4 and 8 h.(4)To clarify the role of lysosomal degradation in PBDE-47-induced Aβ pathology,adenovirus overexpressing Cat B(Ad-Cat B)combined with lysosomal inhibitor chloroquine(CQ)was used in PC12 cells prior to PBDE-47 treatment.(5)To address the role of overall autophagy-lysosome pathway,Ad-ATG7 and Ad-Cat B were combined to enhance the entire autophagy-lysosome pathway function in PC12 cells before PBDE-47 treatment.As a supplement,adenovirus overexpression of autophagy-lysosome pathway nuclear transcription factor EB(Ad-TFEB)combined with CQ treatment were also used.Western blot,ELISA and immunofluorescence technologies were used to examine the state of autophagy-lysosome pathway,the levels of Aβ pathology-related proteins and the accumulation of Aβ42 in autophagosome and lysosome.Results:(1)Compared with control group,the protein levels of ATG7,LAMP1 and Cat B were decreased,but p62,LC3-II,APP,β-CTF,α-CTF and Aβ42 protein levels were increased in PBDE-47 treatment groups(all P < 0.05);the positive staining of Aβ42 and LC3 was increased,while the positive staining of LAMP1 was decreased,and the colocalization of Aβ42 with LC3 and LAMP1 after PBDE-47 treatment was increased.Collectively,these results suggest that PBDE-47 is able to cause autophagy-lysosome pathway impairment and Aβ pathology in PC12 cells.Moreover,PBDE-47 increases the accumulation of Aβ42 in autophagosome and lysosome.(2)Compared with PBDE-47 treatment group,the levels of ATG7,p62,LC3-II,LAMP1,Cat B,APP,β-CTF,α-CTF and Aβ42 were increased in PBDE-47 + Ad-ATG7 treatment group(all P < 0.05);the positive staining of Aβ42,LC3 and LAMP1,and the colocalization of Aβ42 with LC3 and LAMP1 were increased after PBDE-47 +Ad-ATG7 treatment;the levels of ATG7,LC3-II,APP,β-CTF,α-CTF and Aβ42 were decreased,but the level of p62 was increased in PBDE-47 + si RNA-ATG7 treatment group(all P < 0.05);the positive staining of Aβ42,LC3 and LAMP1,and the co-localization of Aβ42 with LC3 and LAMP1 were decreased after PBDE-47 +si RNA-ATG7 treatment.Together,these results indicate that promoting autophagy aggravates while inhibiting autophagy alleviates PBDE-47-induced Aβ accumulation in PC12 cells.(3)Compared with control group,the decline rates of Aβ pathology-related proteins APP,β-CTF and α-CTF were decreased,and the half-life of each protein was prolonged in CHX + PBDE-47 treatment group(all P < 0.05).These results suggest that PBDE-47 inhibits the degradation of Aβ pathology-related proteins in PC12 cells.(4)Compared with PBDE-47 treatment group,the levels of p62,LC3-II,APP,β-CTF,α-CTF and Aβ42 were decreased,while the levels of LAMP1 and Cat B were increased in PBDE-47 + Ad-Cat B treatment group(all P < 0.05);the positive staining of Aβ42 and LC3 was decreased,while the positive staining of LAMP1,and the colocalization of Aβ42 with LC3 and LAMP1 were decreased after PBDE-47 +Ad-Cat B treatment.Compared with PBDE-47 + Ad-Cat B treatment group,the levels of p62,LC3-II,LAMP1,APP,β-CTF and α-CTF were increased,however,the level of Cat B was decreased in PBDE-47 + Ad-Cat B + CQ treatment group(all P < 0.05).Collectively,these results indicate that overexpression of Cat B alleviates PBDE-47-induced Aβ accumulation by enhancing lysosomal degradation.(5)Compared with PBDE-47 treatment group,the levels of LC3-II,β-CTF,α-CTF and Aβ42 were decreased,but the levels of ATG7,p62,LAMP1,Cat B and APP were increased in PBDE-47 + Ad-ATG7 + Ad-Cat B treatment group(all P < 0.05);the levels of TFEB,ATG7,p62,LAMP1,Cat B were increased,however,the levels of LC3-II,APP,β-CTF,α-CTF and Aβ42 were decreased in PBDE-47 + Ad-TFEB treatment group(all P < 0.05).In these two groups,the positive staining of Aβ42 and LC3 was decreased,while the positive staining of LAMP1,and the colocalization of Aβ42 with LC3 and LAMP1 were decreased.Compared with PBDE-47 + Ad-TFEB treatment group,the levels of ATG7 and Cat B were decreased,while the levels of TFEB,p62,LC3-II,LAMP1,APP,β-CTF and α-CTF were increased in PBDE-47 + Ad-TFEB + CQ treatment group(all P < 0.05).Together,these results suggest that the activation of autophagy-lysosome pathway improves PBDE-47-induced Aβ pathology via enhancement of lysosomal degradation function.Conclusions: PBDE-47 is able to impair the autophagy-lysosome pathway by inhibiting autophagosome generation and lysosomal degradation,inducing the accumulation of Aβ pathology-related proteins,thus leading to Aβ pathology in PC12 cells.Enhancement of lysosomal function,or promotion of entire autophagy-lysosome pathway activity alleviates PBDE-47-elicited Aβ pathology in PC12 cells.