The Role And Mechanism Of TAp63 In The Pathogenesis Of Parkinson’s Disease By Regulating PI3K/AKT/mTOR Mediated Autophagy

Parkinson’s disease(PD)is a kind of clinical manifestation of diversity,pathogenetic mechanisms are unknown,and the rapid development of nervous system degenerative diseases.At present,it is recognized that the main mechanisms include degeneration of dopaminergic neurons and abnormal aggregation ofα-synuclein.Autophagy,as one of the major degradation pathways,is a process in which cytoplasmic organelle components are degraded through complex membrane movement and then some molecular components are released.To preserve the cells in the cycle and the dynamic balance of the body,regulate the homeostasis of the body.Objective:To investigate the effects of TAp63 expression on cell viability,autophagy protein level,and related pathways in Parkinson’s disease cell model.Methods:The MPP~+induced neuroblastoma cells(SH-SY5Y)were used to construct an in vitro Parkinson’s disease model.SH-SY5Y cells were transfected with no-load and TAp63overexpression lentiviruses,respectively,and divided into 6 groups.Six groups of cells were the control group(Group A,SH-SY5Y group),MPP~+group(Group B,MPP~+treatment group),si-TAp63-NC group(Group C,lentivirus negative control group),si-TAp63-NC-MPP~+group(Group D,lentivirus negative control MPP~+treated group),si-TAp63 overexpression group(Group E),and si-TAp63 over-expression-MPP~+group(Group F).CCK-8 method was used to detect the relative survival rate of cells treated with drug concentrations in different groups.Western Blot(WB)was used to detect the expression levels of PI3K/AKT/m TOR,TAp63,PTEN,and autophagy-related proteins(LC3,p62,and Beclin-1).Using the western blot method and immunofluorescence technique to detect the C,D,E,and F group cell autophagy-related p62,Beclin-1,and protein expression level of LC3.Using the western blot method to detect cell C,D,E,and F groups of PI3K/AKT/m TOR,TAp63,PTEN expression level.Results:1.Compared with the control group,the relative survival rate of SH-SY5Y cells in the MPP~+1.00mmol/L group decreased significantly after 24h of incubation,and there was a concentration dependence.In MPP~+1.25mmol/L group,the cell relative survival rate was less than 50%after 24h,48h,and 72h.2.Compared with Group A,the expression levels of LC3Ⅱprotein(*P<0.05)and Beclin-1 protein(**P<0.01)in the MPP~+treatment group was significantly decreased,whereas the expression level of p62 protein was significantly increased(**P<0.01).The expression level of TAp63 was significantly decreased(*P<0.05).Therefore,MPP~+treatment can down-regulate the expression of LC3Ⅱ,Beclin-1,and TAp63 in SH-SY5Y,and up-regulate the expression of p62.3.Compared with the Group A,the expression levels of p-m TOR/m TOR protein(**P<0.01)and PTEN protein(**P<0.01)in the MPP~+treatment group were significantly increased.However,the expression levels of p-PI3K/PI3K protein(**P<0.01)and p-AKT/AKT protein(***P<0.001)were significantly decreased.4.Compared with the si-NC-MPP~+group,the combined treatment of TAp63 and MPP~+significantly reduced the level of p62(**P<0.01),indicating that the overexpression of TAp63 promoted the autophagic flux.In Figure 2,the expression level of Beclin-1 decreased after MPP~+treatment,while in Figure 4,the overexpression of TAp63 combined with MPP~+treatment significantly increased the expression level(****P<0.0001).5.Immunofluorescence results show that si-NC-MPP~+compared with the si-NC group,LC3 relative fluorescence intensity is reduced,and p62 relative fluorescence intensity increases.Compared with the si-TAp63 group,LC3 relative fluorescence intensity was decreased and the relative fluorescence intensity of p62 was increased in the si-TAp63-MPP~+group.At the same time,compared with the si-NC group,LC3 relative fluorescence intensity in the si-TAp63-MPP~+group was increased,and the relative fluorescence intensity of p62 was decreased.6.Si-NC-MPP~+compared with si-NC group and si-TAp63-MPP~+group compared with si-TAp63,p-m TOR/m-TOR and PTEN expression were significantly increased.The expression levels of p-m TOR/m-TOR and PTEN were decreased in the si-TAp63-MPP~+group compared with MPP~+induction treatment.On the contrary,the expression of p-AKT/AKT and p-PI3K/PI3K increased in the si-TAp63-MPP~+group compared with the si-NC-MPP~+group and decreased in the si-NC-MPP~+group compared with the si-NC group and the si-TAP63-MPP~+group compared with the si-TAp63 group.7.Flow cytometry results showed that the proportion of apoptotic cells in the si-NC-MPP~+group was increased compared with that in the si-NC group,and the proportion of apoptotic cells in the si-TAp63-MPP~+group was decreased compared with that the in the si-TAp63 group.Compared with the si-NC-MPP~+group,the proportion of apoptotic cells in the si-TAp63-MPP~+group was decreased.Conclusions:These results indicated that the expression levels of TAp63,LC3,p-AKT/AKT,p-PI3K/PI3K,and Beclin-1 were decreased in PD,whereas the expression levels of p62,p-m TOR/m TOR,and PTEN were increased.Overexpression of TAp63 can inhibit the down-regulation of autophagy protein LC3 expression,which may change the autophagic flux by regulating the PI3K/AKT/m TOR signaling pathway and play a protective role in neurons.