α-突触核蛋白功能片段向多巴胺能神经细胞内的转运及其对细胞增殖的影响

Transportation of Functional Fragments of α-Synuclein into Dopaminergic Neuronal Cells and Their Effects on Proliferation of the Cells

目的研究α-突触核蛋白(α-Syn)3个功能片段的促多巴胺能神经细胞增殖作用并且观察其亚细胞分布。方法原核表达重组人野生型α-Syn及其3个功能片段:N-末端、NAC片段和C-末端。向MES 23.5多巴胺能神经细胞的培养基中添加α-Syn及其3个功能片段,观察α-Syn全长及其3个功能片段对细胞的增殖的影响的生长曲线,用细胞免疫荧光检测α-Syn及其功能片段向细胞内的进入和分布。结果α-Syn全长及其NAC片段和C-末端均可促进细胞的增殖,但其N-末端片段对细胞增殖无影响。α-Syn及其3个功能片段均可以进入MES23.5多巴胺能神经细胞,3个功能片段在细胞内的亚细胞分布不同:α-Syn全长分子分布于胞质和胞核,但胞核中的含量多于胞质;N-末端片段主要分布于胞质和突起;NAC片段分布于胞质;C-末端片段主要分布于细胞核中。结论α-Syn对细胞增殖的促进作用与其NAC片段以及C-末端片段的氨基酸排列顺序有关。α-Syn及其功能片段均可进入多巴胺能神经细胞内,但其亚细胞分布不同。

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 MES23.5 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 cell proliferation was measured with cell growth curve drawn by cell counting. Results Western blot analysis using specific antibodies against different 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 extracellalarly 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.