Overexpression Of PGC-1?Influence Mitochondrial Unfolded Protein Response(mtUPR) Induced By MPP~+ In SH-SY5Y Cells

BackgroundParkinson's disease(PD)is a common dyskinesia disease in the elderly,its pathological changes are the degeneration of dopaminergic neurons in substantia nigra.More and more studies have shown that mitochondrial dysfunction,such as the abnormalities of mitochondrial quality control,oxidative stress,ubiquitination system and other mechanisms,lead to the pathological changes in PD.In recent years,mitochondrial protein quality control(PQC)abnormalities,especially early mitochondrial unfolded protein response(mtUPR)may be directly or indirectly involved in the occurrence and development of PD,and its mechanism is unclear.Objective1.To observe the changes of mtUPR induced by MPP~+in SH-SY5Y cells at different time points.2.To investigate whether mtUPR is affected by overexpression of PGC-1?.3.To investigate the effect of PGC-1?/LRPPRC signal transduction on mtUPR.Methods1.Establishment of cell model:overexpression of cell model and PD model was established,according to the optimal adenovirus infection(MOI=100)and MPP~+working concentration(1000 umol/L).2.Immunoblotting and Real-Time PCR were used to detect the expression of YME1L1,HSPA9,CLPP and HSPE1 at different time points.3.Western bloting and Real-Time PCR was to detect the expression of PGC-1?and LRPPRC.Similarily,the expression of proteases(YME1L1,CLPP)and molecular chaperones(HSPA9,HSPE1)were detected by immunoblotting and Real-Time PCR.4.The distribution of LRPPRC in different experiment groups was observed by fluorescence confocal microscopy.Results1.The results of immunoblotting and Real-Time PCR found that:when SH-SY5Y cells were treated with MPP~+,compared with other groups,the protein and transcription levels of the proteases(YME1L1,CLPP)and molecular partners(HSPA9,HSPE1reached the highest in the group of 24 hours.This achieved the activation of mtUPR.2.Fluorescence microscopy showed that when the empty virus and overexpressing of PGC-1?adenovirus infected cells,more than 90%cells were infected at MOI=100,and only less cells died,which was suitable for the infection.3.Immunoblotting and Real-Time PCR showed that in the groups of overexpression of PGC-1?at SH-SY5Y cells,the proteases(YME1L1,CLPP)and molecular partners(HSPA9,HSPE1)were significantly increased in protein and transcription levels when compared with the empty virus group and blank control group,which indicated that PGC-1?overexpression activated the mtUPR,and PGC-1?had a protective effect on SH-SY5Y cells.4.Western bloting and Real-Time PCR showed that the protein and transcription levels of PGC-1?and LRPPRC were significantly improved in the groups of PGC-1?overexpression.Fluorescence confocal results showed that in normal circumstances,LRPPRC almost distributed in the nucleus,mitochondria and cytoskeleton of SH-SY5Y cells,and after MPP~+intervention LRPPRC was transferred to the nucleus.When PGC-1?was overexpressed,LRPPRC was further reduced in the nucleus compared to the MPP~+treated group,which indicated that PGC-1?overexpression may play a protective role to mitochondrial through LRPPRC.Conclusions1.The Parkinson's disease(PD)model was established by MPP~+intervention in SH-SY5Y cells,and the mitochondrial unfolded protein reaction(mtUPR)was also successfully activated.2.The expression of mtUPR proteases(YME1L1,CLPP)and molecular chaperones(HSPA9,HSPE1)almost reached the highest levels at 24 hours after MPP~+intervention,and mtUPR was the most effective at this time point.3.When PGC-1?expression increased,the nuclear transcription factors of proteases(YME1L1,CLPP)and molecular chaperones(HSPA9,HSPE1)which parcipated in mtUPR also improved,suggested that overexpression of PGC-1?may activate mtUPR,reducing oxidative stress injury induced by MPP~+.4.PGC-1?overexpression increased the protein and transcription levels of LRPPRC,which were consist with the trend of mtUPR proteases and chaperone;fluorescence confocal observation found that after MPP~+intervention and overexpression of PGC-1?,LRPPRC had the tendence of transferring from the nucleus.We hypothesized that PGC-1?may activate mtUPR through LRPPRC signaling to maintain mitochondrial homeostasis.