ForeFront Dementia and Movement Disorders Laboratory, Brain and Mind Centre, Central Clinical School, Faculty of Medicine and Health, University of Sydney, Camperdown, New South Wales 2050, Australia.
School of Medical Sciences, Faculty of Medicine, University of New South Wales, Kensington, New South Wales 2033, Australia.
J Biol Chem. 2019 Sep 27;294(39):14241-14256. doi: 10.1074/jbc.RA119.008733. Epub 2019 Aug 2.
There is much interest in delineating the mechanisms by which the α-synuclein protein accumulates in brains of individuals with Parkinson's disease (PD). Preclinical studies with rodent and primate models have indicated that fibrillar forms of α-synuclein can initiate the propagation of endogenous α-synuclein pathology. However, the underlying mechanisms by which α-synuclein fibrils seed pathology remain unclear. To investigate this further, we have used exogenous fibrillar α-synuclein to seed endogenous α-synuclein pathology in human neuronal cell lines, including primary human neurons differentiated from induced pluripotent stem cells. Fluorescence microscopy and immunoblot analyses were used to monitor levels of α-synuclein and key autophagy/lysosomal proteins over time in the exogenous α-synuclein fibril-treated neurons. We observed that temporal changes in the accumulation of cytoplasmic α-synuclein inclusions were associated with changes in the key autophagy/lysosomal markers. Of note, chloroquine-mediated blockade of autophagy increased accumulation of α-synuclein inclusions, and rapamycin-induced activation of autophagy, or use of 5'-AMP-activated protein kinase (AMPK) agonists, promoted the clearance of fibril-mediated α-synuclein pathology. These results suggest a key role for autophagy in clearing fibrillar α-synuclein pathologies in human neuronal cells. We propose that our findings may help inform the development of human neural cell models for screening of potential therapeutic compounds for PD or for providing insight into the mechanisms of α-synuclein propagation. Our results further add to existing evidence that AMPK activation may be a therapeutic option for managing PD.
人们对于阐明帕金森病（PD）患者大脑中α-突触核蛋白积累的机制非常感兴趣。啮齿动物和灵长类动物模型的临床前研究表明，纤维状α-突触核蛋白可以引发内源性α-突触核蛋白病理学的传播。然而，α-突触核蛋白纤维引发病理学的潜在机制仍不清楚。为了进一步研究这一点，我们使用外源性纤维状α-突触核蛋白在包括源自诱导多能干细胞的原代人神经元在内的人神经元细胞系中引发内源性α-突触核蛋白病理学。荧光显微镜和免疫印迹分析用于监测外源性α-突触核蛋白纤维处理的神经元中α-突触核蛋白和关键自噬/溶酶体蛋白随时间的变化。我们观察到细胞质α-突触核蛋白包涵体的积累随时间的变化与关键自噬/溶酶体标志物的变化相关。值得注意的是，氯喹介导的自噬阻断增加了α-突触核蛋白包涵体的积累，雷帕霉素诱导的自噬激活或使用 5'-AMP 激活的蛋白激酶（AMPK）激动剂促进了纤维介导的α-突触核蛋白病理学的清除。这些结果表明自噬在清除人神经元细胞中纤维状α-突触核蛋白病理学中起着关键作用。我们提出，我们的发现可能有助于为 PD 潜在治疗化合物的筛选或提供对α-突触核蛋白传播机制的深入了解提供信息。我们的结果进一步增加了现有的证据，即 AMPK 激活可能是治疗 PD 的一种选择。
