Abstract: Objective α-Synuclein(α-Syn) is a 140 amino acid protein, which is strongly implicated in the pathogenesis of Parkinson's disease.The amino acid sequence of this protein can be divided into three distinct domains: a highly conserved amino terminal(residues 1～60) with four KTKEGV repeats,which mediates its binding to phospholipid membranes;A central hydrophobic NAC domain(residues 61～95) with two additional KTKEGV repeats,which makes up the highly amyloidogenic part of the molecule,and a carboxyl terminal acidic tail(residues 96～140),which is composed primarily of glutamate and aspartate residues that appears critical for the chaperone-like activity of α-Syn.Previous studies have shown that this protein can be secreted from the cells and detected in the culture medium and cerebrospinal fluid.Whether or not the extracellular α-Syn may affect neuronal function remains unknown.We have previously reported that α-Syn,when applied extracellularly,can enter into MES 23.5 dopaminergic cells and promote proliferation of the cells.However,β-Syn,another member of synuclein family,which is highly identical to α-Syn in N-terminal but different from α-Syn in NAC domain and C-terminal,showed no effect on cell proliferation.These findings indicate that the effect of α-Syn on cell proliferation is highly dependent on its molecular structure.In this study,we will study the transportation of three functional fragments of α-Syn into dopaminergic neuronal cells and their effects on the proliferation of the cells.Methods Human recombinant α-Syn and its three functional fragments: N-terminal(α-Syn 1～65 amino acid),NAC(α-Syn 60～95 amino acid) and C-terminal(α-Syn 96～140 amino acid) were expressed in E.coli by a prokaryotic expression system.The proteins expressed were then passed through a column of Glutathione Sepharose 4B,purified by gel filtration and reverse chromatograph,respectivly.The correction of the full-length α-Syn and its three functional fragments were identified by Western blot analysis using specific antibodies against different parts of α-Syn.The full-length α-Syn and its three functional fragments were then added to the culture medium of MES 23.5 dopaminergic neuronal cells.The entry and subcellular localization of the proteins were identified using immunofluorecent labeling.The effect of the proteins on cellproliferation was measured with cell growth curve drawn by cell counting.Results Western blot analysis using specific antibodies againstdifferent parts of α-Syn demonstrated the correction of the full-length α-Syn and its three functional fragments.After applied to the culture medium,the full-length α-Syn and its three functional fragments were shown to enter into the cells.However,their subcellular localization in the cell was different.The full-length α-Syn was distributed in both cytoplasm and nucleus with its amount higher in the nucleus than in the cytoplasm.The N-terminal fragment was presented in the cytoplasm and processes of the cells.The NAC fragment was only seen in the cytoplasm. The C-terminal fragment was mainly observed in the nucleus.Growth curve showed that the full-length α-Syn and its NAC and C-terminal fragments promoted cell proliferation.In contrast,the N-terminal fragment had no effect on cell proliferation.Conclusion The extracellularly applied recombinant α-Syn and its three functional fragments can enter into MES23.5 dopaminergic cells with different subcellular localizations.The promotion effect of α-Syn on cell proliferation may depend on its NAC and C-terminal amino acid sequences.The full-length α-Syn and its NAC and C-terminal fragments promoted cell proliferation,while the N-terminal fragment had no effect on cell proliferation.These results suggest that the NAC and C-terminal of α-Syn may be responsible for its role in cell proliferation.

Key words: α-Synuclein, cell proliferation, dopaminergic neuronal cells