Study On The Screening Of Transcription Factor TFEB Activators And Its Function

Transcription factor EB(TFEB),a member of the MiT family,plays an important role in cellular homeostasis by regulating the expression of autophagy-related or lysosome-related genes.Increasing evidence has indicated that TFEB-mediated autophagy-lysosomal pathway(ALP)is related to the pathogenesis and progress of diseases,such as neurodegenerative disease,cancer and metabolic disorders.Enhancing autophagy and lysosomal function by activating TFEB provides a novel strategy for the treatment of these diseases.Therefore,screening of smallmolecular TFEB activators and studying its mechanism are valuable for the treatment of neurodegenerative disease,cancer and metabolic disorders.In this study,screening of TFEB activators from small molecule libraries was performed using TFEB-GFP cell model,and their mechanism of action was studiedby the molecular cytobiology methods.The main results were as follows:(1)Two TFEB activators named Trifluoperazine 2HCl and Desloratadine were found by using HeLa cells expressing TFEB-GFP.Trifluoperazine 2HCl and Desloratadine treatment can significantly induce TFEB nuclear translocation.The same results were obtained from the subcellular fractionation and Western blot.(2)The nuclear translocation of endogenous TFEBwere detected in HeLa and PC 12 cells after Trifluoperazine 2HCl and Desloratadine treatment.The results indicated that these TFEB activators could significantly induce endogenous TFEB nuclear translocation.(3)Western blot and fluorescence microscope were performed to detect the molecular mechanism of TFEB activation by Trifluoperazine 2HCl and Desloratadine.The results showed that two compounds did not decrease the levels of p70S6k phosphorylation,and calcium chelator BAPTA-AM did not inhibit the nuclear translocation of TFEB,suggesting that the regulation of TFEB activation were independent of mTOR and calcium signal.Moreover,Desloratadine increased the phosphorylation of AMPKα,indicating that Desloratadine might induce TFEB activation through the AMPK signaling pathway.(4)The effects of these two compounds on autophagy were detected by using HeLa cells stably expressing GFP-LC3.Western blot showed that drug treatment resulted in the increase of GFP-LC3 puncts and LC3-Ⅱ levels.To address the effects of two compounds on lysosome function,Western blot and immunofluorescence assays were performed in HeLa and PC 12 cells.The results showed that the levels of LAMP2,CTSB and CTSD were increased after activators treatment.These data indicated that Trifluoperazine 2HCl and Desloratadine promote autophagy and lysosomal biogenesis.(5)MTT was used to detect the role of small-molecule activators against Aβ toxicity in vitro.The results showed that Trifluoperazine 2HCl and Desloratadine could significantly protect Nerve-like cell PC-12 from Aβ toxicity.In lipid metabolism model,Trifluoperazine 2HCl and Desloratadine treatment could significantly promote the degradation of lipid droplet.In summary,Trifluoperazine 2HCl and Desloratadine inducethe nuclear translocation of TFEB,promoting autophagy and lysosomes biogenesis.Both drugs could protect Nerve-like cell PC-12 from Aβ toxicity and promote the degradation of lipid droplet.Our data reveal the molecular mechanism by which Trifluoperazine 2HCl and Desloratadine activate TFEB,and provide a theoretical basis for the drug development of neurodegenerative disease and metabolic disorders.