The Effects Of Trehalose As An Autophagy Enhancer On Untransfected And Transfected PC12 Cells With Human WT Or A53T Mutant SNCA Gene

Parkinson's disease (PD) is a common neurodegenerative disease, but its exactpathogenesis is still unknown so far.α-synuclein, a main component of Lewy bodyof the familial and sporadic PD patient's brain, has been demonstrated to beimplicated in formation of Lewy body and pathogenesis of PD. The aggregation ofα-synuclein is being considered as one of the main cause to the apoptosis indopaminergic neurons, leading to the manifestation of bradykinesia, tremor, andrigidity. How to inhibit the formation of aggregatedα-synuclein or enhance itsdegradation is an issue with significant potential for clinical application. During thepast several years, trehalose has been found to be able to prevent the aggregation ofthe key proteins responsible for other neurodegenerative diseases includingAlzheimer's disease and Huntington's disease. Trehalose was also reported that itcould enhance the autophagy of the doxycycline-inducible PC12 cell lines transfectedwith A53T mutant SNCA and increase the degradation of A53T mutantα-synuclein.In the current study, we constructed PC12 cells lines stably expressing wild type andA53T mutantα-synuclein by using the lentivirus expression vectors ofpLenti6/V5-SNCA-WT and pLenti6/V5-SNCA-A53T and establish an in vitro modelfor PD by applying proteasome inhibitor(500nM MG132), to investigate whethertrehalose can enhance autophagy of untransfected and transfected PC12 cells withhuman WT or A53T mutant SNCA gene, and whether trehalose can accelerate thedegradation of WT and A53T mutantα-synuclein. Furthermore, we also investigatedthe possible effects of the enhanced autophagy induced by trehalose on the cellviability. PartⅠConstruction of The Lentivirus Expression Vector of Wild Type andA53T Mutant Alpha-synuclein and Its Transfection in PC12 CellsAim: To construct PC12 cells which can stably express human wild type andA53T mutantα-synuclein. Methods: The lentivirus containing wild type and A53Tmutantα-synuclein was developed and confirmed by DNA sequencing. Then, it wastransfected into PC12 cells. To identify whether the transfected PC12 cells can stablyexpress theα-synuclein, we detectedα-synuclein-V5 merged protein by using cellularimmunofluorescence staining, confirmed mutant gene sequence by cloning SNCAgene in transfected PC12 cells and sequencing it, measured the level ofα-synucleinexpression in PC12 cells by performing Western Blot, and estimated cell viability byMTT. Results: The lentivirus of pLenti6/V5-SNCA-WT andpLenti6/V5-SNCA-A53T were successfully constructed,α-synuclein-V5 mergedprotein were detected in more than 95% gene-transfected PC12 cells, and thesequence of SNCA-WT and SNCA-A53T gene were confirmed in gene-transfectedPC12 cells by DNA sequencing, the over-expression of SNCA gene were detected ingene-transfected PC12 cells by using Western Blot, and no significant differenceswere detected among the growth curves of three kinds of PC12 cells. Conclusion: Wenot only constructed the lentivirus expression vectors of pLenti6/V5-SNCA-WT andpLenti6/V5-SNCA-A53T, created PC12 cells lines stably expressing wild type andA53T mutantα-synuclein, furthermore, we proved that over-expression of humanWT or A53T mutantα-synuclein had no obvious effects on the cell viability of PC12cells, which lays a good foundation for studying its role in pathogenesis of PD in thefurther research.PartⅡTrehalose Enhances Autophagy of Untransfected and Transfected PC12Cells with Human WT or A53T Mutant SNCA GeneAim: To study whether trehalose can enhance autophagy of untransfected andtransfected PC12cells with human WT or A53T mutant SNCA gene. Methods: Wetreated the cells with trehalose at different concentrations (10mM, 50mM and 100mM)for 24 hours, then detected the change of the number of autophagosome andautolysosome by immunofluorescence and transmission electron microscopy, andanalyzed the expression of LC3Ⅱto investigate whether trehalose could facilitate the autophagy. The expression ofα-synuclein was also analyzed by Western blotting toevaluate whether trehalose could enhance the degradation of WT and A53T mutantα-synuclein. The effects of trehalose on cell viability were judged by MTT assay.Results: Both 50mM and 100mM trehalose significantly increased the number ofautophagosome and autolysosome, up-regulated the expression of LC3Ⅱ(P＜0.01) ,and enhanced the degradation of A53T mutantα-synuclein (50mM trehalose group,P＜0.05; 100mM trehalose group, P＜0.01 ) , but had no obvious effects on theexpression of WTα-synuclein (P＞0.05) . When the cells were pretreated with 2mM3-MA for 3 hours, the autophagy induced by trehalose was inhibited. Both 50mM and100mM trehalose significantly decreased the cell viability of untransfected andtransfected PC12cells with human A53T mutant SNCA gene (P＜0.01) , only 100mMtrehalose reduced the cell viability of transfected PC12cells with human WT SNCAgene (P＜0.05. The reduction of cell viability of transfected PC12cells with human WTSNCA gene induced by trehalose was less than that of the other two types ofPC12cells (P＜0.05) Conclusion: Trehalose could enhance the autophagy ofuntransfected and transfected PC12cells with human WT or A53T mutant SNCA gene,accelerate the degradation of A53T mutantα-synuclein and decrease the cell viabilityof all the three above types of PC12 cells. Trehalose exerted no obvious effects on theexpression of WTα-synuclein. The reduction of cell viability of transfected PC12cells with human WT SNCA gene induced by trehalose was less than that of the othertwo types of PC12 cells.PartⅢThe Effects of Trehalose on The PD cell Models Induced by ProteasomeInhibitorAim: To investigate whether trehalose can enhance the autophagy of the PD cellmodels induced by proteasome inhibitor. Methods: We treated the cells withMG132(500nM), or MG132(500nM) and trehalose of different concentration(10mM,50mM,100mM) for 24 hours, then detected the change of the number ofautophagosome and autolysosome by immunofluorescence, and analyzed theexpression of LC3Ⅱby Western blotting to investigate whether trehalose couldenhance the autophagy of the PD cell models induced by proteasome inhibitor. Theexpression ofα-synuclein was also analyzed by Western blotting to investigatewhether trehalose could facilitate the degradation of the aggregated WT and A53Tmutantα-synuclein. The effects of trehalose on the viability of the cells treated by MG132 (500nM) were evaluated by MTT assay. Results: MG132(500nM) couldsignificantly inhibit the degradation ofα-synuclein (P＜0.05), increase the number ofautophagosome, lysosome and autolysosome. Compared with MG132(500nM) group,Trehalose could significantly enhance the autophagy of the cells induced byMG132(500nM), obviously upregulate the expression of LC3Ⅱ(P＜0.05) , alsofacilitate the degradation of the aggregated A53Tmutantα-synuclein (50mM trehalosegroup, P＜0.05; 100mM trehalose group, P＜0.01) , but had no effects on thedegradation of the aggregated WTα-synuclein induced by MG132. Trehalosesignificantly decreased the viability of the cells treated by MG132 (P＜0.05)Conclusion: MG132 could facilitate the aggregation ofα-synuclein, compensatorilyenhance autophagy. Trehalose could enhance the autophagy of the cells induced byMG132 and accelerate the degradation of the aggregated A53T mutantα-synuclein,but had no effects on the degradation of the WTα-synuclein. Trehalose significantlydecreased the viability of the cells treated by MG132.Summary1. We successfully constructed PC12 cells lines stably expressing wild type and A53Tmutantα-synuclein.2. Trehalose could enhance the autophagy of untransfected and transfected PC12cellswith human WT or A53T mutant SNCA gene, facilitate the degradation of A53Tmutantα-synuclein.3. Trehalose could decrease the cell viability of all three types of PC12 cells.4. Proteasome inhibitor (MG132) could cause the aggregation ofα-synuclein,compensatorily enhance autophagy.5. Trehalose could enhance the autophagy of the cells induced by proteasomeinhibitor (MG132) and accelerate the degradation of the aggregated A53Tmutantα-synuclein6. Trehalose significantly decreased the viability of the cells treated by proteasomeinhibitor (MG132) .