Biological Engineering Discipline, Indian Institute of Technology Gandhinagar, Palaj, Gujarat, India.
Center for Biomedical Engineering, Indian Institute of Technology Gandhinagar, Palaj, Gujarat, India.
Traffic. 2022 Jul;23(7):391-410. doi: 10.1111/tra.12859. Epub 2022 Jun 8.
Alpha-synuclein (α-Syn), an intrinsically disordered protein (IDP), is associated with neurodegenerative disorders, including Parkinson's disease (PD or other α-synucleinopathies. Recent investigations propose the transmission of α-Syn protein fibrils, in a prion-like manner, by entering proximal cells to seed further fibrillization in PD. Despite the recent advances, the mechanisms by which extracellular protein aggregates internalize into the cells remain poorly understood. Using a simple cell-based model of human neuroblastoma-derived differentiated neurons, we present the cellular internalization of α-Syn PFF to check cellular uptake and recycling kinetics along with the standard endocytic markers Transferrin (Tf) marking clathrin-mediated endocytosis (CME) and Galectin3 (Gal3) marking clathrin-independent endocytosis (CIE). Specific inhibition of endocytic pathways using chemical inhibitors reveals no significant involvement of CME, CIE and caveolae-mediated endocytosis (CvME). A substantial reduction in cellular uptake was observed after perturbation of actin polymerization and treatment with macropinosomes inhibitor. Our results show that α-Syn PFF mainly internalizes into the SH-SY5Y cells and differentiated neurons via the macropinocytosis pathway. The elucidation of the molecular and cellular mechanism involved in the α-Syn PFF internalization will help improve the understanding of α-synucleinopathies including PD, and further design specific inhibitors for the same.
α-突触核蛋白（α-Syn）是一种无序的蛋白（IDP），与神经退行性疾病有关，包括帕金森病（PD）或其他α-突触核蛋白病。最近的研究表明，α-Syn 蛋白纤维以类朊病毒的方式传递，通过进入邻近细胞，在 PD 中进一步引发纤维形成。尽管最近取得了进展，但细胞外蛋白聚集体如何进入细胞的机制仍知之甚少。我们使用一种简单的基于人神经母细胞瘤衍生的分化神经元的细胞模型，研究了 α-Syn PFF 的细胞内化，以检查细胞摄取和再循环动力学，以及转铁蛋白（Tf）标记网格蛋白介导的内吞作用（CME）和半乳糖凝集素 3（Gal3）标记网格蛋白非依赖内吞作用（CIE）的标准内吞标记物。使用化学抑制剂特异性抑制内吞途径，发现 CME、CIE 和小窝蛋白介导的内吞作用（CvME）没有明显参与。细胞内吞作用显著减少，这是在肌动蛋白聚合扰动和巨胞饮抑制剂处理后观察到的。我们的结果表明，α-Syn PFF 主要通过巨胞饮途径内化到 SH-SY5Y 细胞和分化神经元中。阐明 α-Syn PFF 内化所涉及的分子和细胞机制将有助于提高对包括 PD 在内的α-突触核蛋白病的理解，并进一步设计针对该疾病的特异性抑制剂。
