The Role And Underlying Mechanisms Of BAG3 In Regulating Selective Autophagy And α-synuclein Degradation In SNCA^WT-PC12 Cells

Part Ⅰ BAG3 involved in the regulation of autophagy and α-synuclein degradation in SNCAWT-PC12 cells.Objective:To investigate the effect of BAG3 on autophagy and to explore the potential role of BAG3 in α-synuclein degradation in SNCAWT-PC12 cells.Methods:High-differentiated PC 12 cells were used in this study. Lentiviral vector driving the recombinant eukaryotic expression vector that contains wild type α-synuclein (pLentiVENUS-YFP-SNCA) was transduced into PC 12 cells. The expression of α-synuclein level was tested by microscope and Western blot method. MG132, the proteasome inhibitor was used to treat SNC4WT-PC12 cells. The cell viability was determined with the MTT assay. Western blot was used to detect the levels of a-synuclein, p62, microtubule-associated protein 1 light chain 3 (LC3) and BAG3. Reverse-transcription PCR (RT-PCR) was used to determine the mRNA levels of α-synuclein, p62 and Bag3. The small interfering RNA (siRNA) targeting Rat Bag3 was applied to knockdown Bag3 in SNC4WT-PC12 cells. pCMV6-BAG3 plasmid was applied to induce BAG3 overexpression in SNC4WT-PC12 cells to detecte the change of LC3Ⅱ and α-synuclein levels.Results:SNCAWT-PCl2 cells viability was concentration-dependently decreased by MG132. a-synuclein protein levels, but not its mRNA levels, in MG132 treated SNC4WT-PC12 cells showed a concentration-dependent increase. p62, LC3Ⅱand BAG3 protein levels in MG132 treated SNC4WT-PC12 cells showed a dose-dependent and time-dependent increase, accompanied with a concentration-dependent increase of p62 and Bag3 mRNA levels. Bag3 knockdown decreased the LC3Ⅱ protein levels but increased the α-synuclein protein expressions in MG132 treated or untreated SNC4WT-PC12 cells. Conversely, BAG3 overexpression increased the LC3Ⅱ but decreased the a-synuclein protein levels in MG132 treated or untreated SNC4WT-PC12 cells.Conclusion:MG132 results in the dysfunction of α-synuclein degradation in SNCAWT-PC12 cells. BAG3 overexpression can enhance autophagy activity and decrease α-synuclein accumulation, which plays a role in regulating autophagy and α-synuclein degradation in SNCAWT-PC12 cells.Part Ⅱ BAG3 promoted the α-synuclein degradation via selective autophagy in SNC4WT-PC12 cells.Objective:To study the mechanism of BAG3 in regulating α-synuclein degradation and to clarify whether the chaperone Hsp70 is involved in the selective autophagy process.Methods:The small interfering RNA (siRNA) targeting Rat Atg5 was applied to induce autophagy deficiency in SNC4WT-PC12 cells to explore whether the effect of BAG3 on a-synuclein degradation was autophagy dependent. Western blot analysis was used to detect the level of Hsp70. Reverse-transcription PCR (RT-PCR) was used to determine the mRNA level of Hsp70. Co-immunoprecipitation (Co-IP) was used to assess the binding level of BAG3 with Hsp70 and p62 in SNC4WT-PC12 cells.Results:In Atg5-deficient SNC4WT-PC12 cells, BAG3 overexpressing had no effect on alleviating a-synuclein accumulation. MG132 enhanced the protein level of Hsp70 in dose-and time-dependent manners, and the mRNA level of Hsp70 also showed a dose-dependent increase. Both p62 and Hsp70 could interact with BAG3 protein. With the treatment of MG132 or BAG3 overexpression, their bound quantities with BAG3 protein also increased.Conclusion:BAG3 upregulates autophagy to ameliorate α-synuclein accumulation in SNC4WT-PC12 cells. The underling mechanism is related to the interaction of BAG3-Hsp70 complex with p62 which induces selective autophagy.