Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Int J Mol Sci. 2020 Jul 22;21(15):5193. doi: 10.3390/ijms21155193.
Genetic and biochemical evidence attributes neuronal loss in Parkinson's disease (PD) and related brain diseases to dyshomeostasis of the 14 kDa protein α-synuclein (αS). There is no consensus on how αS exerts toxicity. Explanations range from disturbed vesicle biology to proteotoxicity caused by fibrillar aggregates. To probe these mechanisms further, robust cellular toxicity models are needed, but their availability is limited. We previously reported that a shift from dynamic multimers to monomers is an early event in αS dyshomeostasis, as caused by familial PD (fPD)-linked mutants such as E46K. Excess monomers accumulate in round, lipid-rich inclusions. Engineered αS '3K' (E35K+E46K+E61K) amplifies E46K, causing a PD-like, L-DOPA-responsive motor phenotype in transgenic mice. Here, we present a cellular model of αS neurotoxicity after transducing human neuroblastoma cells to express yellow fluorescent protein (YFP)-tagged αS 3K in a doxycycline-dependent manner. αS-3K::YFP induction causes pronounced growth defects that accord with cell death. We tested candidate compounds for their ability to restore growth, and stearoyl-CoA desaturase (SCD) inhibitors emerged as a molecule class with growth-restoring capacity, but the therapeutic window varied among compounds. The SCD inhibitor MF-438 fully restored growth while exerting no apparent cytotoxicity. Our αS bioassay will be useful for elucidating compound mechanisms, for pharmacokinetic studies, and for compound/genetic screens.
遗传和生化证据表明，帕金森病（PD）和相关脑部疾病中的神经元丧失归因于 14kDa 蛋白α-突触核蛋白（αS）的内稳态失调。目前对于αS如何产生毒性尚无共识。解释范围从囊泡生物学的紊乱到纤维状聚集物引起的蛋白毒性。为了进一步探究这些机制，需要有强大的细胞毒性模型，但它们的可用性有限。我们之前曾报道过，从动态多聚体向单体的转变是由家族性 PD（fPD）相关突变如 E46K 引起的αS 内稳态失调的早期事件。过量的单体在富含脂质的圆形包涵体中积累。工程化的αS“3K”（E35K+E46K+E61K）放大了 E46K 的作用，导致转基因小鼠出现类似 PD 的 L-DOPA 反应性运动表型。在这里，我们通过转导人神经母细胞瘤细胞以在强力霉素依赖性方式表达黄色荧光蛋白（YFP）标记的αS 3K 来建立αS 神经毒性的细胞模型。αS-3K::YFP 的诱导引起明显的生长缺陷，与细胞死亡相符。我们测试了候选化合物恢复生长的能力，发现硬脂酰辅酶 A 去饱和酶（SCD）抑制剂是一类具有生长恢复能力的分子，但化合物之间的治疗窗口不同。SCD 抑制剂 MF-438 完全恢复了生长，而没有明显的细胞毒性。我们的αS 生物测定将有助于阐明化合物的作用机制、进行药代动力学研究以及进行化合物/遗传筛选。
