Nanobiophysics, MESA+ Institute for Nanotechnology, University of Twente, Enschede, Netherlands.
PLoS One. 2010 Dec 13;5(12):e14292. doi: 10.1371/journal.pone.0014292.
The question of how the aggregation of the neuronal protein α-synuclein contributes to neuronal toxicity in Parkinson's disease has been the subject of intensive research over the past decade. Recently, attention has shifted from the amyloid fibrils to soluble oligomeric intermediates in the α-synuclein aggregation process. These oligomers are hypothesized to be cytotoxic and to permeabilize cellular membranes, possibly by forming pore-like complexes in the bilayer. Although the subject of α-synuclein oligomer-membrane interactions has attracted much attention, there is only limited evidence that supports the pore formation by α-synuclein oligomers. In addition the existing data are contradictory.
METHODOLOGY/PRINCIPAL FINDINGS: Here we have studied the mechanism of lipid bilayer disruption by a well-characterized α-synuclein oligomer species in detail using a number of in vitro bilayer systems and assays. Dye efflux from vesicles induced by oligomeric α-synuclein was found to be a fast all-or-none process. Individual vesicles swiftly lose their contents but overall vesicle morphology remains unaltered. A newly developed assay based on a dextran-coupled dye showed that non-equilibrium processes dominate the disruption of the vesicles. The membrane is highly permeable to solute influx directly after oligomer addition, after which membrane integrity is partly restored. The permeabilization of the membrane is possibly related to the intrinsic instability of the bilayer. Vesicles composed of negatively charged lipids, which are generally used for measuring α-synuclein-lipid interactions, were unstable to protein adsorption in general.
CONCLUSIONS/SIGNIFICANCE: The dye efflux from negatively charged vesicles upon addition of α-synuclein has been hypothesized to occur through the formation of oligomeric membrane pores. However, our results show that the dye efflux characteristics are consistent with bilayer defects caused by membrane instability. These data shed new insights into potential mechanisms of toxicity of oligomeric α-synuclein species.
在过去的十年中，神经元蛋白α-突触核蛋白的聚集如何导致帕金森病中的神经元毒性一直是密集研究的主题。最近，人们的注意力已经从淀粉样纤维转移到α-突触核蛋白聚集过程中的可溶性寡聚中间体。这些寡聚体被假设为具有细胞毒性，并通过在双层中形成孔状复合物来使细胞膜穿孔。尽管α-突触核蛋白寡聚物-膜相互作用的主题引起了广泛关注，但只有有限的证据支持α-突触核蛋白寡聚物形成孔。此外，现有数据相互矛盾。
方法/主要发现：在这里，我们使用多种体外双层系统和测定法详细研究了一种经过充分表征的α-突触核蛋白寡聚物物种破坏脂质双层的机制。发现寡聚α-突触核蛋白诱导的囊泡中的染料外排是快速的全有或全无过程。单个囊泡迅速失去其内容物，但囊泡整体形态保持不变。基于与葡聚糖偶联染料的新开发测定法表明，非平衡过程主导了囊泡的破坏。在寡聚物添加后，膜对溶质流入具有高度通透性，之后膜完整性部分恢复。膜的通透性可能与双层的固有不稳定性有关。通常用于测量α-突触核蛋白-脂质相互作用的带负电荷的脂质组成的囊泡通常由于蛋白质吸附而不稳定。
结论/意义：在添加α-突触核蛋白后，带负电荷的囊泡中的染料外排已被假设通过寡聚体膜孔的形成发生。然而，我们的结果表明，染料外排特征与由膜不稳定性引起的双层缺陷一致。这些数据为寡聚α-突触核蛋白物种的潜在毒性机制提供了新的见解。
