Quantitative Analysis of α-Synuclein Solubility in Living Cells Using Split GFP Complementation

Presently incurable, Parkinson's disease (PD) is the most common neurodegenerative movement disorder and affects 1% of the population over 60 years of age. The hallmarks of PD pathogenesis are the loss of dopaminergic neurons in the substantia nigra pars compacta, and the occurrence of proteinaceous cytoplasmic inclusions (Lewy bodies) in surviving neurons. Lewy bodies are mainly composed of the pre-synaptic protein alpha-synuclein (αsyn), an intrinsically unstructured, misfolding-prone protein with high propensity to aggregate. Quantifying the pool of soluble αsyn and monitoring αsyn aggregation in living cells is fundamental to study the molecular mechanisms of αsyn-induced cytotoxicity and develop therapeutic strategies to prevent αsyn aggregation. In this study, we report the use of a split GFP complementation assay to quantify αsyn solubility. Particularly, we investigated a series of naturally occurring and rationally designed αsyn variants and showed that this method can be used to study how αsyn sequence specificity affects its solubility. Furthermore, we demonstrated the utility of this assay to explore the influence of the cellular folding network on αsyn solubility. The results presented underscore the utility of the split GFP assay to quantify αsyn solubility in living cells.

帕金森病（Parkinson's disease, PD）目前尚无法治愈，是最为常见的神经退行性运动障碍疾病，在60岁以上人群中的患病率达1%。帕金森病发病机制的标志性特征为黑质致密部（substantia nigra pars compacta）多巴胺能神经元丢失，以及存活神经元内出现蛋白质类细胞质包涵体——路易小体（Lewy bodies）。路易小体主要由突触前蛋白α-突触核蛋白（alpha-synuclein, αsyn）构成，该蛋白本身固有无序、易发生错误折叠，且具有极强的聚集倾向。定量检测活细胞内可溶性αsyn的总量并监测其聚集情况，是研究αsyn诱导的细胞毒性分子机制、开发预防αsyn聚集治疗策略的核心基础。本研究采用分裂绿色荧光蛋白（split GFP）互补实验法对αsyn的可溶性进行定量检测。具体而言，我们对一系列天然存在及理性设计的αsyn突变体进行了研究，结果表明该方法可用于探究αsyn的序列特异性如何影响其可溶性。此外，我们还验证了该实验方法可用于探究细胞折叠网络对αsyn可溶性的调控作用。本研究结果进一步证实了分裂GFP互补实验法定量检测活细胞内αsyn可溶性的实用价值。