Addition of Exogenous α-Synuclein Oligomer up-regulates Divalent Metal Transporter-1 and Ferroportin-1 in BV2 Glial Cell Lines

It has been confirmed that α-synuclein (α-syn) aggregation in neurons or glia and formation of Lewybodies is the remarked pathognomonic hallmark of PD. Neuropathology study have found α-Syn in abnormal aggregation often accompany with iron deposits. At present ,a lot of research confirmed that divalent metal transporter 1(DMT1), is up-regulated and ferroportin-1 is downregulated in the SN of PD and accumulate of iron in this region.Therefore understanding exogenous α-Syn oligomer whether arise this phenomenon in BV2 glial cell lines is necessary.


INTRODUCTION
Parkinson's disease(PD) is a degenerative disease of central nervous system.The physiological performance is muscle rigidity, resting tremor and posture reflection problems and characterized by the loss of dopaminergic neurons in the substantia nigra and form acidophilous inclusion body(lewy body) composed mainly with aggregates α-syn [1][2][3] . Although heredity, environment and oxidative stress play a certain role, but the exact pathogenesis of Parkinson's disease is not fully understood.
α-syn, as the primary component of Lewy bodies (LBs), forms insoluble aggregates that accumulated in neuron and glia of the substantia nigra and cause neurotoxicity [3] .
α-syn was found by Maroteaux in 1988 [4] and in 1997, the protein began to widely research because of the idetification of Lewy bodies (LBs) and Lewy neurites (LNs) [5] .α-Synuclein (α-Syn) is a 140-residue abundant cytosolic neuronal protein enriched in synaptic terminals where it has been reported to be involved in synaptic vesicle trafficking [6] , regulation of synaptic vesicle pool [7][8] . α-syn is aboundantly expressed in neurons [9] and its amino acid sequence can be divided into three parts:  (Figure 1). In α-syn, the NAC domain is the most aggregation prone region but it is mainly protected by the positive and negative charges of the N-and C-termini [１０] .The expression of α-syn in neuron are easier to oligomeization,but not fibrillization,indicate that oligomeric species might be the most toxic forms of α-syn.therefore α-syn oligomers are being notoriety in pathological process of PD [11][12] . Neuropathology study have found α-syn in abnormal aggregation often accompany with iron deposits,it is prominent indicate that α-syn aggregation is related with iron deposits [13] .Our lab study in recent years have been proved real high iron can lead to dopaminergic neuron death in substantia nigra par compacta.In brain,iron transport is connected with two transferrins divalent metal transporter 1(DMTl),the mainly protein of iron import; Ferroportin-1 (FPN1),the only known transmembrane protein for cellular iron efflux [14] . The increasing of intracellular iron due to the high expression of DMT1 and low expression of FPN1.In this paper,the dominating objective is to know whether aggregates α-syn oligomer can change two transferrins DMT1 and FPN1,consequently cause the iron deposits,thereby lead to dopaminergic neuron death and PD.

Design
In vitro cytological study.

Time and setting
Experiments were completed at the National Key  37℃constant temperature table oscillation incubation 2d～5d,oscillation frequency 280r/min [15] .

Main outcome measures
Changes in DMT1 and FPN1 expressions after α-syn oligomer intervention.

Statistical analysis
Data are presented as mean±SEM. SPSS 17.0 software was applied for statistics analysis. The intergroup comparisons were made by one-way analysis of variance and Tukey test. P < 0.01 and P < 0.001 were considered to be statistically or greatly statistically significant.

RESULTS
The preparation of α-syn oligomer was detected by 10% sodium dodecyl sulfate-polyacrylamide gels and then transferred to polyvinylidene fluoride membranes.
After treated with 3μmol/L α-syn oligomer for 24 h, there was a 1-fold increase in FPN1 protein level in BV2 glial cell lines (P < 0.001; Figure 4).

DISCUSSION
Parkinson's disease(PD) is the second most frequent neurodegenerateive disorder and the most famous α-synucleinopathy,which is increased the iron levels in the substantia nigra of Parkinson's disease patients,but the potential interlink between both molecule changes has not been fully understand [16] .The accumulation of iron in the substantia nigra has been associated with the oxidative stress,by mean of Fention eaction,iron increase the production of free radicals and can thus contribute to neurotoxicity [17] ,. Interestingly, the presence of increased iron levels may not only enhance radical burden but also foster aggregation of α-synuclein [18] .Several investigations have focused on the role of iron to promote α-syn oligomerization and aggregation [19] .Several observations support the hypothesis that mismetabolism of iron in the SN promotes aggregation of α-syn. Levels of iron are significantly higher in the SN relative to other brain regions, increasing the susceptibility of this region to iron-mediated oxidative stress [20] . Elevated levels of total iron and a shift in the equilibrium of iron to the oxidized state have been reported in the SN of sporadic and familial cases of PD [21] .Redox-active iron has been detected in association with a-synuclein aggregates in Lewy bodies [22] , a phenotype that is likely to promote oxidization and further aggregation of α-syn and other proteins [23] .Aggregation of α-syn is inhibited by the iron chelator desferrioxamine, supporting this hypothesis [24] . α-syn over-expression in the brain led cell more sensitive to the toxic effects of iron,therefore,speculated that excessive α-syn may add the iron level of cell. Recent studies have found that α-syn is a ferrireductase ,can restore the Fe3 + of the cell into Fe2+,therefoe it is provide the posibility that α-syn involved in the iron metabolism,that is say itself a kind of iron metabolism protein,its abnormal expression inevitably involved in the iron metabolism of cell [25] . Our study provides a better understanding of the exogenous α-syn oligomer role in the iron metabolism and the expression of transferrin in the BV2 glial cell lines. Glial cells are important in supporting neuronal survival, synaptic functions and local immunity [26] ,therefore glial cells might be crucial for the initiation and progression of different neurodegenerative diseases, including PD. We could conclusively demonstrate that α-syn oligomers is capable to incerase transmembrane transferrin protein DMT1 and FPN1 expression by a dose-dependent manner in the BV2 glial cell lines.As a consequence,we guess that exogenous α-syn oligomer may be a ferrireductase,regulate the Fe3 +into Fe2+. Due to the higher expressin of iron import protein DMT1, the level of Fe2+ is elevated,causing the iron deposits and oxidative stress and eventually generation cytotoxicity,therefore increase the expressin of iron export protein FPN1 and export iron out of cell.So,we hypothesize exogenous α-syn oligomer involved in the microglia cell iron metabolism as well as the pathological process of PD,although precies molecule mechanism remain unclear.