Department of Physics.
Department of Chemistry, University of Alberta, Edmonton, Canada.
Biophys J. 2021 Mar 2;120(5):877-885. doi: 10.1016/j.bpj.2020.12.031. Epub 2021 Jan 28.
Aggregates of misfolded α-synuclein are a distinctive feature of Parkinson's disease. Small oligomers of α-synuclein are thought to be an important neurotoxic agent, and α-synuclein aggregates exhibit prion-like behavior, propagating misfolding between cells. α-Synuclein is internalized by both passive diffusion and active uptake mechanisms, but how uptake varies with the size of the oligomer is less clear. We explored how α-synuclein internalization into live SH-SY5Y cells varied with oligomer size by comparing the uptake of fluorescently labeled monomers to that of engineered tandem dimers and tetramers. We found that these α-synuclein constructs were internalized primarily through endocytosis. Oligomer size had little effect on their internalization pathway, whether they were added individually or together. Measurements of co-localization of the α-synuclein constructs with fluorescent markers for early endosomes and lysosomes showed that most of the α-synuclein entered endocytic compartments, in which they were probably degraded. Treatment of the cells with the Pitstop inhibitor suggested that most of the oligomers were internalized by the clathrin-mediated pathway.
聚集的错误折叠的α-突触核蛋白是帕金森病的一个显著特征。小的α-突触核蛋白寡聚物被认为是一种重要的神经毒性剂，α-突触核蛋白聚集表现出类朊病毒样行为，在细胞之间传播错误折叠。α-突触核蛋白通过被动扩散和主动摄取机制被内化，但摄取随寡聚物大小的变化如何尚不清楚。我们通过比较荧光标记的单体和工程串联二聚体和四聚体的摄取，研究了α-突触核蛋白进入活 SH-SY5Y 细胞的内化如何随寡聚物大小而变化。我们发现这些α-突触核蛋白构建体主要通过内吞作用内化。寡聚物大小对其内化途径几乎没有影响，无论是单独添加还是一起添加。α-突触核蛋白构建体与早期内体和溶酶体荧光标记物的共定位测量表明，大多数α-突触核蛋白进入内吞小泡，在其中可能被降解。用 Pitstop 抑制剂处理细胞表明，大多数寡聚物通过网格蛋白介导的途径被内化。
