Department of Physical Chemistry, Lund University, Lund, Sweden.
J Parkinsons Dis. 2011;1(4):359-71. doi: 10.3233/JPD-2011-11067.
Aggregated α-synuclein in Lewy bodies is one of the hallmarks of Parkinson's disease (PD). Earlier observations of α-synuclein aggregates in neurons grafted into brains of PD patients suggested cell-to-cell transfer of α-synuclein and a prion-like mechanism. This prompted the current investigation of whether α-synuclein passes over model phospholipid bilayers. We generated giant unilamellar vesicles (GUVs) containing a small amount of a lipid-conjugated red emitting dye (rhodamine B) and varied the membrane charge by using different molar ratios of DOPC and DOPS or cardiolipin. We then used confocal fluorescence microscopy to examine how monomer, fibril as well as on-pathway α-synuclein species labeled with a green emitting fluorophore (Alexa488) interacted with the phospholipid bilayers of the GUV. We defined conditions that yielded reproducible aggregation kinetics under basal conditions and with none or moderate shaking. We found that on-pathway α-synuclein species and equilibrium amyloid aggregates, but not α-synuclein monomers, bound to lipid membranes. α-Synuclein was particularly strongly associated with GUVs containing the anionic lipids cardiolipin or DOPS, whereas it did not associate with GUVs containing only zwitterionic DOPC. We found that α-synuclein progressively aggregated at the surface of the GUVs, typically in distinct domains rather than uniformly covering the membrane, and that both lipid and protein were incorporated in the aggregates. Importantly, we never observed transport of α-synuclein over the GUV bilayer. This suggests that α-synuclein transport over membranes requires additional molecular players and that it might rely on active transport.
路易体中的聚集α-突触核蛋白是帕金森病（PD）的标志之一。先前在移植到 PD 患者大脑中的神经元中观察到α-突触核蛋白聚集体的研究表明，α-突触核蛋白可以在细胞间传递，并具有类朊病毒的机制。这促使我们当前研究α-突触核蛋白是否可以穿过模型磷脂双层。我们生成了含有少量脂质偶联的红色发射染料（罗丹明 B）的巨大单层囊泡（GUV），并通过使用不同摩尔比的 DOPC 和 DOPS 或心磷脂来改变膜电荷。然后，我们使用共焦荧光显微镜检查用绿色发射荧光团（Alexa488）标记的单体、纤维以及途径上的α-突触核蛋白与 GUV 的磷脂双层如何相互作用。我们定义了在基础条件下以及无或适度搅拌下可产生可重复聚集动力学的条件。我们发现，途径上的α-突触核蛋白物种和平衡淀粉样蛋白聚集体，但不是α-突触核蛋白单体，与脂质膜结合。α-突触核蛋白与含有阴离子脂质心磷脂或 DOPS 的 GUV 特别强结合，而与仅含有两性离子 DOPC 的 GUV 不结合。我们发现α-突触核蛋白在 GUV 表面逐渐聚集，通常在不同的区域而不是均匀地覆盖膜，并且脂质和蛋白质都包含在聚集体中。重要的是，我们从未观察到α-突触核蛋白在 GUV 双层上的转运。这表明，α-突触核蛋白在膜上的转运需要其他分子，并可能依赖于主动运输。
