Neuroprotective Mechanism Of Overexpression Of Mitochondrial Ferritin On 6-hydroxydopamine Induced Dopaminergic SH-SY5Y Cell Damage

Parkinson’s disease (PD) is an age-related neurodegenerative disease characterized by specific dopaminergic neurons degeneration. It can be mimicked by some Parkinsonian neurotoxins, such as 6-hydroxydopamine (6-OHDA) in experimental models. Previous studies suggest that neuronal iron homeostasis disruption and oxidative stress are closely related to the pathogenesis PD. Cytosolic ferritins sequester and store iron and, consequently protects cells against potential iron-mediated free radical damage. Mitochondrial ferritin (MtFt) is a newly identified mitochondrial protein with high similarity with ferritin heavy chain (H-ferritin) and all amino acids in the ferroxidase center are conserved. Although its function is still elusive, recent immunhistochemistry study showed the MtFt specifically expressed in neurons of brain cortex and spinal cord, which require high metabolic activity and oxygen consumption. Previous studies suggested that overexpression of MtFt caused dramatic redistribution of cellular iron, iron deficiency phenotype in cytosol and sensitizing cancer cells against butyl-hydrogen peroxide induced oxidative stress damage. To examine the role of MtFt in 6-OHDA-induced PD cell model, we overexpressed mouse MtFt in dopaminergic neuronal cell lineSH-SY5Y establishing the MtFt-SY5Y and P10-3-SY5Y(blank vector as control) cell lines. In our exprement,we chose SH-SY5Y, MtFt-SY5Y and P10-3-SY5Y cells induced by 6-OHDA as modle to investigate the apoptosis feature using MTT,flow cytometry,fluorescence microscopy,cell immunity fluorescence and western blot. The results were as follow(1) we transfected MtFt gene into SH-SY5Y neurons successfuliy and established MtFt-SY5Y and P10-3-SY5Y cell lines.we found that overexpression MtFt in SH-SY5Y cells might inhibit the cell growth which maybe caused by depletion in intracytoplasm.(2) overexpression of MtFt restrain the increase of ROS, calcium ion and lipid peroxidation,meanwhile restraining the decrease of mitochondrial membrane potential. The indes about these markers had significant difference from control groups(p<0.01)(3) interestingly, MtFt activized the iNOS causing the incrase of ON significantly. The NO in MtFt-SY5Y cells added two times compared with P10-3-SY5Y and SH-SY5Y cells,which had significantly difference(p<0.01).(4) MtFt protected the activity of complexI from 6-OHDA.After treatmented P10-3-SY5Y with 6-OHDA,its activity of complexI decreased 70%,but 20% for MtFt-SY5Y cells.statistics showed there was significantly difference between them(p<0.01).(5) We demonstrated that MtFt inhibited the release of cytochrome C using the cell immunofluorescence and wetern blot.(6) MtFt repressed the increase of LIP significantly caused by 6-OHDA.After exposure to 6-OHDA,the LIP increased 150% for P10-3-SY5Y cells,but 50% for MtFt-SY5Y compared with control group. statistics showed there was significantly difference between them(p<0.01).(7) We demonstrated that MtFt inhibited the apoptosis of neurons induced by 6-OHDA using cell nucleus stain and flow cytometry.the apoptosis rate of SH-SY5Y and P10-3-SY5Y cells were respectively 70% and 67%,but 13% for MtFt-SY5Y cells after exposure to 6-OHDA. statistics showed there was significantly difference between them(p<0.01).(8) Wester blot showed that Bax,Bcl-2、ferritin、TfR and NF-kB had no significant changes wether exposure to drug or not.inversely,these items had significant changes exposure to drug compared with control group. Conclusion:In this paper, we first time transfected MtFt gene into neurons SH-SY5Y,using the PD model induced by 6-OHDA to search the function of MtFt and relationship between iron metabolism,oxidative stress and PD.Our results showed first time that MtFt played an impotant role in oxidative damage to mitochondrial induced by 6-OHDA.the mechanism was that overexpression inhibited the Fenton reaction by regulating the iron metabolism,decreasing the production of ROS,maintaining the mitochondrial membrane potential,keeping the balance of intracellular Ca2+,preventing the increase of LIP and release of cytochrome C,inhibiting the expression of promotive apoptosis proteins and keeping the expression of antiapoptosis proteins.Although the expression of iNOS was upregulated and NO increased by MtFt,the production of peroxynitrite would decrease because of decrease of ROS,which inhibited the apoptosis pathway of ROS/NO caused by 6-OHDA result in cell protection.This study showed that regulating the expression of MtFt can prevent PD,and may repress the apoptosis caused by neurotoxin to protect cell from damage.the precent results will be an important purpose for people to understand the pathologies and curing of PD. MtFt is a endogenous protein and its expression has no sideeffect to body.so understanding the regulative mechanism of MtFt will be a new effective strategy for preventing and curing PD.