HaFGF_14-154 Promotes Amyloid ?-Peptide Internalization And Clearance Via Clathrin-Mediated Endocytosis Pathway

Objective:The imbalance between production and clearance of amyloid?-peptide?A??may cause Alzheimer's disease?AD?.Astrocytes can not only produce A?,but also uptake and degrade A?.However,the specific mechanisms of these actions aren't known.Using an AD model in vitro,we have explored the mechanism of human acidic fibroblast growth factor(haFGF_14-154)regulating amyloid?-peptide?A??on both internalization and clearance.These findings could provide an effective target for new drugs and highlight the essential role of haFGF_14-154in the therapy on neurodegenerative diseases including AD.Methods:?1?To obtain more than 95%astrocytes,we identified the purity of primary cortex astrocytes from neonatal rats by immunofluorescence?IF?;?2?In order to set up an AD model in vitro,astrocytes were treated by A?_1-42oligomers,together with 10,100 and 1000 ng/m L haFGF_14-154.After that,the expression of APP processing proteins?APP?ADAM10?BACE1?and insulin degrading enzyme,as well as Adam10 in m RNA levels,were detected by Western Blot and RT-q PCR,respectively.?3?To observe the endocytosis of A?,astrocytes were treated for 3 h and 6 h,followed by the co-localization of A?and lysosome marker,LAMP1,which were showed by IF.Also,the expression of endocytic proteins?clathrin,?-adaptin,AP2M1and VPS35?and genes?Ap2m1and Vps35?were seen by Western Blot and RT-q PCR,respectively,which give us an insight on how haFGF_14-154mediating the internalization of A?.?4?To investigate how haFGF_14-154regulating mi R-133a-3p,the expression of mi R-133a-3p were displayed by RT-q PCR.Then the expression of endocytic proteins and genes were detected,following the treatment of mi R-133a-3p mimic or inhibitor on astrocytes,for the purpose of observing the regulation of mi R-133a-3p.Morover,the relationship between mi R-133a-3p and Clta were verified by dual luciferase reporter gene assay.Results:?1?High purity?over 98%?of primary astrocytes were isolated successfully.?2?Compare with A?treatment,haFGF_14-154significantly reduced A?peptides deposition of astrocytes.Western Blot showed that haFGF_14-154markedly increased the expression of ADAM10 and IDE,but decreased the expression of BACE1 and PS1,as well as had little effect on APP.Also,haFGF_14-154may up-regulate Adam10 in m RNA levels.Using IF analysis,we found that astrocytes can also produce A?after A?_1-42treatment.?3?After incubation for 3 h,astrocytes with haFGF_14-154treatment accumulated more A?,compare with A?treatment.However,haFGF_14-154dramatically reduced A?levels after incubation for 6 h,when compare with A?treatment.?4?In comparison with A?treatment,haFGF_14-154obviously enhanced the expression of endocytic proteins?clathrin,?-adaptin,AP2M1and VPS35?in m RNA and protein levels.With regard to endocytic genes,haFGF_14-154 with lower concentration clearly reinforced Ap2m1 and Vps35 in m RNA levels.?5?A?down-regulated the expression of mi R-133a-3p,while haFGF_14-154 reversed this regulation.Notably,mi R-133a-3p could also regulate the expression of endocytic proteins.And then,dual luciferase reporter gene assay represented that Clta is a direct target of mi R-133a-3p.Conclusion:haFGF_14-154mediated the internalization of A?peptides via down-regulating mi R-133a-3p,which have a binding site with Clta.After the uptake of astrocytes,A?peptides were transported from early endosome to lysosome,and finally were hydrolyzed,which through clathrin-mediated pathway.Additionally,some intracellular A?peptides were degraded by IDE.What's more,haFGF_14-154 markedly increased the expression of ADAM10 but decreased the expression of BACE1,which reduced the production of A?peptides by astrocytes.