Mechanisms Of Iron-induced Alpha-synuclein Aggregation

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized symptomatically by bradykinesia, resting tremor and rigidity. PD is pathologically characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN) and the presence of alpha-synuclein aggragates in intracellular inclusions known as Lewy bodies (LBs). The incidence of PD is 1.7% in the people over 65 in China. Along with the aging of our society, PD is becoming an overwhelming problem. Since mechanisms underlying PD pathogenesis are not fully understood, all of the efferts used in the clinic today aims to alleviate the symptoms. It is of great importance to studing the mechanisms underlying neurodegeneration in PD.Iron plays a crucial role in PD pathogenesis. Excessive free iron, especially ferrous iron could generate hydroxyl radical (OH-) through Fenton reaction, while ferric iron could be reduced to ferrous iron by Haber-Weiss cycle. The OH·could damage proteins, nucleus and mounts of unsaturated fatty acids, which could finally lead to cell death. Mounting evidence suggest the role of alpha-synuclein in PD pathosenesis. High levels of alpha-synuclein are prone to promote the formation of the aggregates due to a decrease in the lag time and an increase in the rate constant for fibril growth. Neuropathological studies showed that synucleinopathies are generally associated with iron accumulation, which is consistent with a pathological link between iron and alpha-synuclein. It has been found that high concentration of ferrous and ferric iron can promote alpha-synuclein aggregation in vivo and in vitro. Recently a predicated iron responsive element (IRE) was discovered in the 5'- untranslated region (UTR) of alpha-synuclein, providing a possible mechanism through which iron could cause the death of dopaminergic neurons by up-regulating alpha-synuclein expression. To investigate the mechanisms underlying iron-induced alpha-synuclein aggregation,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay, flow cytometry (FCM), thioflavin S staining and immunofluorence staining, reverse transcription polymerase chain reaction (RT-PCR) and western blots analysis were applied in our present study. Effects of the antioxidant vitamine E (VE) on cell viability, intracellular oxidative stress and alpha-synuclein aggregation were observed in iron-treated SK-N-SH cells to investigate the relationship between oxidative stress and alpha-synuclein aggregation. The alpha-synuclein-IRE-Luc luciferase reporter, which contained the predicted IRE of alpha-synuclein, was constructed. Dual luciferase reporter assays were conducted to investigate whether the inserted predicated IRE is a functional element in HEK293 cells. Then we confirm the results in SK-N-SH cells, to observed whether iron could regulate the expression of alpha-synuclein through iron regulatory proteins (IRPs)/IRE system. The results were as follows:1.1 mmol/L ferrous iron or ferric iron treatment for 24 h reduced cell viability and reactive oxygen species (ROS) generation in SK-N-SH neuroblastoma cells (P< 0.05, compared to the control), and alpha-synuclein aggragates were observed compared to the control;2. Pretreatment with 5μmol/L VE, a potent ROS scavenger, could fully block ROS formation and cell viability reduction induced by iron, however the intracellular alpha-synuclein aggregation could only be partially alleviated. The reasults indicated that oxidative stress partially contributes to iron-induced alpha-synuclein aggregation in SK-N-SH cells;3. The luciferase reporter alpha-synuclein-IRE-Luc was successfully constructed and dual luciferase assay was applied in HEK293 cells to detect the function of the inserted predicated IRE in post-transcriptional regulation; Ferroportinl-IRE-Luc was constructed to be the positive control;4.24 h after cotransfection by pRL-TK and pGL3 vectors (pGL3-control, alpha-synuclein-IRE-Luc, ferroportin-IRE-Luc) in the ratio of 1:20, cells were treated with lmmol/L iron or 100μmol/L deferoxamine mesylate (DFO). In iron-treated group, alpha-synuclein-IRE-Luc produced consistently higher luciferase activity compared to that of the control group (P< 0.05), while in DFO treated group, lower luciferase activity was detected (P< 0.05), indicating the inserted predicated IRE is iron responsive;5. Cells were transfected with three plasmid mixture in ratio of 1:20:80 (pRL-TK versus pGL3 versus pSilencerTM1.0-U6-IRP or pCMV6-AC-ACO1). In pSilencerTM1.0-U6-IRP transfected groups, alpha-synuclein-IRE-Luc produced consistently higher luciferase activity compared to that of the control group (P< 0.05), in pCMV6-AC-ACO1 groups, lower luciferase activity was detected (P< 0.05). The results indicated that iron-dependent regulation was mediated by intracellular IRP1 levels.6. SK-N-SH cells were transfected with pSilencerTM1.0-U6-IRP, and alpha-synuclein expression in cells with IRP1 knockdwon was up-regulated compared to that of the control. The above results suggest that iron could induce alpha-synuclein aggregation partially through oxidative stress in SK-N-SH cells, as indicated by the results that although VE could completely blocked ROS generation and oxidative stress, there were still alpha-synuclein aggregation in cells. In accordance with IRE/IREP regulatory system, IRE in the 5'-UTR of alpha-synuclein mRNA was responsive to intracellular iron and IRPs levels alterations, indicated that the predicted IRE in alpha-synculein 5'-UTR is functional, and iron could regulate alpha-synuclein expression through IRE/IRP system. Our findings provid evidence that the predicated IRE in the 5'-UTR of alpha-synuclein is a functional element. Iron could cause the death of dopaminergic neurons by inducing alpha-synuclein expression and aggregation through IRE/IRP system and oxidative stress. The present study provides more experimental evidence to the interaction of iron and alpha-synuclein in PD and provides a new taget for therapitical approaches of PD.