The Mechanism Of Autophagy Induced By VAPB Deficiency

Aim:Autophagy plays an important role in neurodegenerative diseases.This study examines whether vesicle-associated membrane protein-associated protein B(VAPB)plays a role in autophagy.Methods:VAPB was silenced by transfecting with two sets of si-RNAs in three cell lines including HEK293 T,He La and SH-SY5 Y.The protein levels of LC3 and Beclin 1 were detected with immunoblot analysis.FLAG-VAPB was transfected into HEK293 T cells,and then the levels of LC3 and Beclin 1 were detected.VAPB was knocked down in HEK293,and then GFP-LC3 and FLAG-P62 were transfected into HEK293 cells.The co-localization of GFP-LC3 and FLAG-P62 was detected by immunofluorescence assays.The expression of VAPB was silenced in HEK293 T cells,and the levels of Beclin 1 m RNA were detected by real-time quantitative PCR(RT-q PCR).Futhermore,Beclin 1 was silenced in VAPB knockdown HEK293 T cells,and then the levels of LC3 protein were detected.In m Cherry-LC3 stable transfected HEK293 cells,Beclin 1 was further silenced in VAPB knockdown cells,and then the number of m Cherry-LC3 puncta was detected.Finally,the level of autophagy flux was detected in VAPB deficiency and m Cherry-EGFP-LC3 B overexpressed He La cells.Dots of Cherry and GFP were detected using fluorescence microscopy.GFP-HTT60,m Cherry-LC3 and FLAG-P62,or RFP-SOD1-G93 A,GFP-LC3 and FLAG-P62 were transfected in HEK293 cells,and then the co-localization of these proteins was detected with immunofluorescence assays.In VAPB knockdown HEK293 cells,GFP-HTT60 or RFP-SOD1-G93 A was transfected and the proteasome inhibitor MG132 or autophagy inhibitor Baf A1 was administrated.The aggregation of these proteins was detected using fluorescence microscopy.Results:The loss of VAPB significantly increased the protein levels of LC3? and Beclin 1 in HEK293 T,He La and SHSY-5Y,while the overexpression of FLAG-VAPB decreased the protein levels of LC3? and Beclin 1 in HEK293 T.In HEK293 cells that VAPB was knocked down,the co-localization of GFP-LC3 and FLAG-P62 was increased,accompanied by an increase of the m RNA levels of Beclin 1.The increase of LC3? resulting from the loss of VAPB was blocked by the knockdown of Beclin 1.In He La cells that VAPB was knocked down and m Cherry-EGFP-LC3 B was transfected,the ratio of red fluorescence to yellow fluorescence increased,which indicates increases of the autophagic flow.Moreover,in above cells after 4 hours of 100 n M Baf A1 treatment,the number of yellow fluorescence dots increased compared with the control,which was consistent with the result that loss of VAPB initiated the autophagy.We found that GFP-HTT60,m Cherry-LC3 and FLAG-P62,or RFP-SOD1-G93 A,GFP-LC3 and FLAG-P62 were co-localizated in HEK293 cells,indicating that the aggregates of GFP-HTT60 or RFP-SOD1-G93 A were co-localizated with autophagosomes.In HEK293 cells that VAPB was knocked down,the number of aggregates of GFP-HTT60 and RFP-SOD1-G93 A was decreased in the transfected cells,indicating an increased degradation of GFP-HTT60 or RFP-SOD1-G93 A.The aggregation degradation caused by VAPB deficiency was induced in the MG132 treated cells,but blocked in the Baf A1 treated cells,which indicated that the degradation of GFP-HTT60 or RFP-SOD1-G93 aggregation was related to the autophagy induced by VAPB deficiency.Conclusion:Knockdown of VAPB up-regulates Beclin 1 at a transcriptional level,then induces autophagy.And the autophagy induced by loss of VAPB is dependent on Beclin 1.VAPB deficiency enhances autophagy flow and promotes the degradation of GFP-HTT60 and RFP-SOD1-G93 A protein.