The Regulation And Mechanisms Of Uric Acid On Autophagy And α-synuclein Degradation In PC12 Cells

PART 1 An experimental research on the effect of uric acid on α-synuclein protein in PC12 cellsObject In PC12 cells, to evaluate the role of uric acid(UA) in α-synuclein aggregation.Methods we first overexpressed α-syn in the lentivirus transfected WT-PC12 cells(WT cells). After dose-dependent treatment of UA(0-200μmol/L) for 12 h, We analyzed the cell viability and detected α-syn. Rotenone was used for establishment of PD model.When treated with 0-500 nm rotenone for 12 h, the content of α-syn was detected. In the rotenone model, UA was pre-treated of for 1h, 12 hours later, α-syn levels were detected.Next, UA was treated in PC12 cells for 6h, then evaluated the α-syn transcription by PCR.To evaluate whether UA can change the half-life of α-syn. Cycloheximide(CHX)was used to inhibit protein α-synthesis for 0-12 h in CHX group and CHX+UA group.Results we found that 200μM UA can efficiently reduce α-syn. This result was further verified in the rotenone treated cell model. The clearance of α-syn was not due to a reduction in m RNA for that PCR detection shows no difference in expression levels ofα-syn. compare to CHX group, the degradation velocity of α-syn in UA treated WT cells wasfaster, which provides the evidence that UA promoting the degradation of α-syn.Conclusions Our study demonstrated that UA accelerates the degradation of α-syn.PART 2 The regulation and mechanisms of uric acid on autophagy in PC12cellsObject In PC12 cells, to evaluate the potential molecular mechanisms of how UA induced autophagy.Methods UA(0-200μmol/L)was added in PC12 cells,12 h later we detected LC3 II and p62 by western blot. e GFP-LC3 was transfected in PC12 cells, after treatment with100μmol/L UA and 200μmol/L UA, evaluate the LC3 puncta in contrast with cells transfected with an empty vector. Rotenone was added in PC12 cells which was used to establish the PD model., then LC3 II was detected. In the rotenone cell model, p62、LC3and e GFP-LC3 puncta were observe in control group, rotenone(50nmol/L) group, UA(100μmol/L)+rotenone(50nmol/L) group and UA(200μmol/L)+rotenone(50nmol/L)group. chloroquine(CQ)was used as tool medicine, LC3 II was detected in control group,CQ(30μmol/L) group, UA(200μmol/L)group and CQ(30μmol/L)+UA(200μmol/L)group. Western blot was used to detect the level of proteins in m TOR dependent pathway,m TOR, p70S6 K and their phosphorylation。Results Culture with UA(0-200μmol/L), a dose-dependent up-regulation of LC3 was detected, at the same time, a reduction in the p62 was observed. in the e GFP-LC3 transfected PC12 cells, control with cells transfected with an empty vector, 200μM UA treatment dramatically increased the number of e GFP-LC3 puncta. 50 n M rotenone treatment for 12 h showed a significant decay in LC3 II levels,in the rotenone cell model,100μM and 200μM UA were given 1h before rotenone treatment, we find that rotenone induced upregulation of LC3 and downregulation of p62 were both reversed by UA.Immunofluorescence analysis revealed that contrast with 50 n M rotenone treated cells,pre-treatment of UA increased e GFP-LC3 puncta formation. Incubation with 30μM CQ enhanced the LC3-II levels, both of. phosphorylation of m TOR and p70s6 k began to decrease, after UA treatment for 15 min. At 30 min, UA has the greatest effect.Conclusions UA promotes autophagy via a m TOR-dependent pathway,...