Ndfip1Attenuats6-OHDA-induced Cellular Iron Accumulation Via Degradation Of DMT1in Dopaminergic Cells

Parkinson’s disease (PD) is a common neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantial nigra pars compacta (SNpc) and the formation of filamentous intraneuronal inclusions known as Lewy bodies (LBs), with clinical symptoms of rigidity, resting tremor and bradykinesia. Although the etiology of PD remains unknown, increasing evidence suggests that iron plays a key role in PD. Misregulation of some iron transporters might be involved in this process. Divalent metal transporter1(DMT1) is a widely expressed transmembrane protein. Increased DMT1expression was found in the SN of PD patients and animal models, suggesting that DMT1might be involved in the nigral iron accumulation in PD.Nedd4family interacting protein1(Ndfip1), also known as Nedd4WW-domain binding protein5(N4WBP5), is an adaptor protein for Nedd4-mediated ubiquitination. It was reported that Ndfip1-mediated protein ubiquitination might be a possible survival strategy in neuronal injury. It has been found that Ndfipl plays an essential role in regulating DMT1and iron homeostasis in human cortical neurons. However, whether Ndfip1has effect on nigral iron accumulation of PD and its mechanisms are still not elucidated. Therefore, real time PCR, western blots, the technique of laser scanning confocal microscopy, flow cytometry and other methods were applied to investigate the effect of Ndfip1on6-hydroxydopamine (6-OHDA)-induced iron accumulation in MES23.5dopaminergic cells and the possible mechanisms involved in this process. The results were as follows:1. Ndfip1protein level were decreased in the lesioned side of SN in6-OHDA-induced PD rats (P<0.05, compared with control).2. Ndfip1mRNA and protein level were decreased in MES23.5cells after10μmol/L6-OHDA treatment (P<0.05, compared with control).3. Ndfip1protein level was increased in pCMV6-XL5-Ndfip1transfected cells. Over expression of Ndfip1decreased DMT1(+IRE) protein level in MES23.5cells (P<0.01, compared with control). 4. DMT1(+IRE) protein level was increased in MES23.5cells after10μmol/L6-OHDA treatment for24h (P<0.05, compared with control); while the up-regulation of DMT1(+IRE) induced by6-OHDA was reversed in cells with Ndfip1over-expression (P<0.05, compared with6-OHDA treatment group).5. Ferrous iron influx was increased in MES23.5cells after10μmol/L6-OHDA treatment for24h (P<0.01, compared with control); while the increased iron influx induced by6-OHDA was reversed in cells with Ndfipl over-expression (P<0.01, compared with6-OHDA treatment group).6.100μmol/L ferrous iron incubation for4h resulted in an increase in reactive oxide species (ROS) generation and a reduction of the mitochondrial transmembrane potential (ΔΨm) in MES23.5cells (P<0.01, compared with the control); while this increase of ROS generation and decrease of ΔΨm were reversed in cells with Ndfipl over-expression (P<0.05, compared with the iron treatment group).7. Increased iron influx by10μmol/L6-OHDA treatment resulted in an increase in ROS generation and a reduction of ΔΨm in MES23.5cells (P<0.05, compared with the iron treatment group); while over-expressed Ndfipl reversed this increase of ROS generation (P<0.05, compared with the6-OHDA/iron treatment group) and decrease of ΔΨm (P<0.01, compared with the6-OHD A/iron treatment group).The above results suggest that the expressions of Ndfipl decreased in6-OHDA-induced PD models and6-OHDA treated MES23.5cells. Over-expression of Ndfipl could mediate degradation of the iron importer DMT1(+IRE) via ubiquitin-proteasome pathway, then decrease DMT1(+IRE) protein level in6-OHDA treated MES23.5cells and reduce DMT1(+IRE)-mediated iron influx. This further suppressed iron-induced ROS generation and decrease of ΔΨm, and then protect the cells. The present study provides more experimental evidence of the interaction of DMT1and Ndfip1and new targets for prevention and treatment of PD.