The Protective Effect And Mechanism Of Ndfip1 In MPTP/MPP~+-induced Parkinson’s Disease Model

Objective:Parkinson’s disease(PD)is a common neurodegenerative disease whose pathogenesis might be linked to factors such as aging,iron deposition and proteasome dysfunction,but the exact mechanism has not been fully clarified.Nedd4 family-interacting protein 1(Ndfip1)is an adapter protein that bind to the E3 ubiquitin ligase Nedd4 to mediate the ubiquitination and degradation of the substrate protein.It was reported that there were high levels of Ndfip1 in the surviving cortex neurons of rats following transient focal cerebral ischemia or traumatic brain injury,suggesting that high expression of Ndfip1might have a potential neuroprotection.In this experiment,1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced PD mice model and 1-methyl-4-phenylpyridinium ion(MPP~+)-induced SH-SY5Y cells was selected to investigate the protective effects and possible mechanisms of Ndfip1.Methods:In this experiment,mice were pretreated with recombinant adeno-associated virus(AAV)with high expression of Ndfip1 in bilateral substantia nigra(SN)by stereotaxic apparatus and then injected intraperitoneally with MPTP.The pole test and the open field test were used to investigate the motor function of mice;Immunofluorescence techniques were used to detect the number of tyrosine hydroxylase(TH)positive cells in the SN of mice;Western blot was used to measure the protein expression of TH and Voltage dependent anion-selective channel(VDAC).In vitro experiments,SH-SY5Y cells were infected with recombinant adenovirus following exposure to MPP~+,western blot was used to detect the protein expression of Ndfip1,Nedd4,VDAC and ferroptosis related protein;the TMRE kit was used to investigate the changes of mitochondrial membrane potential.Results:1.Compared with the control group,MPTP mice showed behavioral disorders(P<0.001;P<0.05),reduced number of TH~+cells and TH protein level in the SN(P<0.01;P<0.001) and decreased Ndfip1 protein expression(P<0.05),suggesting that MPTP induced dopaminergic neuronal damage in the SN of PD mice accompanied by a downregulation of Ndfip1 protein expression.2.The movement coordination of AAV.EGFP/MPTP mice was decreased than the control group(P<0.001;P<0.05),while high levels of Ndfip1 could improve MPTP-induced movement disorder of mice(P<0.001;P<0.01).3.After MPTP treatment,the number of TH~+cells and the expression of TH protein in the SN of mice were significantly decreased,compared with the control mice(P<0.001),which could be inhibited by high level of Ndfip1(P<0.05),suggesting that high expression of Ndfip1 effectively inhibited MPTP-induced degeneration of dopaminergic neurons.4.The AAV.Ndfip1/MPTP mice had less levels of VDAC1/2 protein than the AAV.EGFP/MPTP mice(P<0.05).Compared with the control mice,the levels of VDAC1/2 related to TH was reinforced remarkably in the AAV.EGFP/MPTP mice(P<0.001),which was obviously inhibited by high level of Ndfip1(P<0.001).5.After MPP~+treatment,the expression of Ndfip1 in SH-SY5Y cells decreased significantly(P<0.001),while the expression of Nedd4(P<0.01)and VDAC1/2(P<0.01,P<0.001)increased compared to the control group.6.Following the treatment of Ad.Ndfip1 and MPP~+in SH-SY5Y cells,the protein expression of Nedd4 was increased in the Ad.Ndfip1/MPP~+cells compare with Ad.EGFP/MPP~+cells(P<0.05),suggesting that high levels of Ndfip1 promoted the expression of Nedd4 protein in SH-SY5Y cells.7.The TMRE fluorescence intensity of MPP~+and Ad.EGFP/MPP~+cells was significantly lower than that of control cells(P<0.001),which could be antagonized by high level of Ndfip1(P<0.05),suggesting that high levels of Ndfip1 inhibited MPP~+-induced loss of mitochondrial membrane potential.8.Compared with the control group,the Ad.EGFP/MPP~+cells exhibited higher VDAC1and VDAC2 levels(P<0.01),which could be inhibited by Ad.Ndfip1 infection(P<0.05),suggesting that high levels of Ndfip1 inhibited MPP~+-induced increase of VDAC1 and VDAC2 protein.9.Results showed that the Ad.EGFP/MPP~+cells had apparently increased expression of Long-chain acyl-Co A synthetase 4(ACSL4)(P<0.05)and decreased Ferroptosis suppressor protein 1(FSP1)expression(P<0.01)than the control group.High levels of Ndfip1 alleviated MPP~+-induced upregulation of ACSL4 protein(P<0.05)but could not affect the expression of FSP1.Conclusion and Significant:In conclusion,this study explored the neuroprotective effect and the underlying mechanisms of high expression of Ndfip1 on MPTP-induced PD mice model and MPP~+-induced PD cell model.It was confirmed that high expression of Ndfip1could improve the motor dysfunction,antagonize the damage of dopaminergic neurons in the SN of MPTP-induced PD mice.Meanwhile,downregulation of Ndfip1,phosphorylated Nedd4 and upregulation of VDAC1/2 might be involved in MPP~+-induced neurotoxicity as evidenced by the vitro experiments.Further study showed that high expression of Ndfip1increased the expression of Nedd4 and inhibited MPP~+-induced increase of VDAC1/2expression and mitochondrial dysfunction.In addition,high expression of Ndfip1prevented MPP~+-induced increase in ACSL4 protein expression,suggesting that Ndfip1might exert its protective effect by regulating ferroptosis.Our results provide new evidence for the neuroprotective effect of Ndfip1 on PD and provide new strategies for prevention and treatment of PD.