Experimental Study Of Iron-induced Alpha-synuclein Aggregation In The Pathogenesis Of Parkinson's Disease

Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease affecting 1.7% of population over the age of 65 in China. PD is pathologically charactized by a selective loss of dopaminergic neurons in the substantia nigra (SN) and the subsequent dopamine (DA) depletion in the striatum. The pathological hallmark of PD are intracellular inclusions, termed Lewy bodies (LBs), which are mainly composed by a mixture of proteins including aggregated alpha-synuclein, iron, proteasome subunits, ubiquitin, heat shock proteins (HSPs), etc. Increased levels of alpha-synuclein mRNA in the midbrain of patients are both found in sporadic PD and familial PD.The deposition of these components to form the LBs may provide us some new insights into the mechanisms underlying neurodegeneration in PD.Mounting evidence demonstrate that iron plays a crucial role in the pathogenesis of PD. Increased iron content and LBs formation are found in degenerative dopaminergic neurons in PD. Studies have found that iron could promote alpha-synuclein aggregation, but the exact mechanism is largely unknown. Heme oxygenase-1 (HO-1),also known as HSP32,is present in LBs.A recent study reports that over-expression of HO-1 triggers intracellular proteasomal degredation of alpha-synuclein. A new class of activator protein-1 (AP-1)related sequences found in the promoter region of genes encoding HO-1 was termed antioxidant responses elements (ARE), which are tightly regulated by the redox-sensitive transcription factor, NF-E2-regulated factor 2 (Nrf2).Nrf2/HO-1 pathway has been proved to be an important intracellular anti-oxidative mechanism. Whether Nrf2/HO-1 pathway is involved in iron-induced alpha-synuclein aggregation is not clear.Using MTT assay, flow cytometry, PCR, siRNA, thioflavin S staining, electron microscopy, and other methods, in the present study we investigate the possible mechanism underlying iron-induced alpha-synuclein aggregation, as well as the subsequent toxic events by exposing SK-N-SH cells to iron. The results are as follows:1.Alpha-synuclein over-expression had no effects on SK-N-SH cells viability, while there was a 10% reduction in the cell viability with alpha-synulein knockdown. Ferric iron (100μmol/L,500μmol/L,1 mmol/L and 10 mmol/L) incubation for 24 h resulted in a significant decrease in the cell viabiltiy in cells with alpha-synuclein overexpression, while a significant restoration in cell viability was observed in cells with alpha-synuclein knockdown compared to that of vector control treated with ferric iron.2.Ferrous iron(100μmol/L) incubation for 4 h or ferric iron(100μmol/L,1 mmol/L) for 24 h resulted in a significant reduction in mitochondrial transmembrane potential (ΔΨm) and increase in reactive oxygen species (ROS) generation in SK-N-SH cells with alpha-synuclein over-expression, while a significant restoration ofΔΨm and less ROS generation were observed in cells with alpha-synuclein knockdown compared to that of vector control treated with ferrous or ferric iron.3.Ferrous iron(100μmol/L) incubation for 4 h could exacerbate ferrous iron-induced apoptosis in SK-N-SH cells with alpha-synuclein over-expression, indicated by a significant Bcl-2 mRNA down-regulation and apoptotic body formation, while no apoptosis was observed in cells with alpha-synuclein knockdown compared to that of vector control treated with ferrous iron.4.Obvious aggregates of alpha-synuclein were observed in cells with alpha-synuclein over-expression treated with ferrous iron(100μmol/L) for 4 h or ferric iron(100μmol/L,1 mmol/L) for 24 h. Long fibrils around the cell nuclear and the swollen mitochondria were observed in cells with alpha-synuclein over-expression. No aggregates were observed in cells with alpha-synuclein knockdown.5.Ferrous iron(100μmol/L) induced time course expression regularity of Nrf2 and HO-1. During this process, iron could down-regulate Nrf2 and HO-1 mRNA levels.6.Ferrous iron(100μmol/L) incubation for 4 h could down-regulate Nrf2 and HO-1 mRNA levels, which were aggravated with alpha-synuclein over-expression while brought to the basal levels in SK-N-SH cells with alpha-synuclein knockdown.7.Increased HO-1 expression could promote alpha-synuclein mRNA degradation in SK-N-SH cells.The above results suggest that iron could exert its toxicity by decreasingΔΨm, increasing ROS generation, inducing apoptosis and promote alpha-synuclein aggregation by down-regulating Nrf2 and HO-1 mRNA levels.The aggregated alpha-synuclein could further exacerbate iron-induced toxicity and aggravated iron-induced down-regulation of Nrf2 and HO-1 mRNA levels, causing a vicious cycle, leading ultimately to cell death. Our findings provide a new insight into the mechanism that the interaction between iron and alpha-synuclein that damages dopaminergic neurons and thus show some light on the development of potential clinical approaches.