Table_1_Transcriptome and Proteome Analysis in LUHMES Cells Overexpressing Alpha-Synuclein.XLSX

LUHMES cells share many characteristics with human dopaminergic neurons in the substantia nigra, the cells, the demise of which is responsible for the motor symptoms in Parkinson's disease (PD). LUHMES cells can, therefore, be used bona fide as a model to study pathophysiological processes involved in PD. Previously, we showed that LUHMES cells degenerate after 6 days upon overexpression of wild-type alpha-synuclein. In the present study, we performed a transcriptome and proteome expression analysis in alpha-synuclein-overexpressing cells and GFP-expressing control cells in order to identify genes and proteins that are differentially regulated upon overexpression of alpha-synuclein. The analysis was performed 4 days after the initiation of alpha-synuclein or GFP overexpression, before the cells died, in order to identify processes that preceded cell death. After adjustments for multiple testing, we found 765 genes being differentially regulated (439 upregulated, 326 downregulated) and 122 proteins being differentially expressed (75 upregulated, 47 downregulated). In total, 21 genes and corresponding proteins were significantly differentially regulated in the same direction in both datasets, of these 13 were upregulated and 8 were downregulated. In total, 13 genes and 9 proteins were differentially regulated in our cell model, which had been previously associated with PD in recent genome-wide association studies (GWAS). In the gene ontology (GO) analysis of all upregulated genes, the top terms were “regulation of cell death,” “positive regulation of programmed cell death,” and “regulation of apoptotic signaling pathway,” showing a regulation of cell death-associated genes and proteins already 2 days before the cells started to die. In the GO analysis of the regulated proteins, among the strongest enriched GO terms were “vesicle,” “synapse,” and “lysosome.” In total, 33 differentially regulated proteins were associated with synapses, and 12 differentially regulated proteins were associated with the “lysosome”, suggesting that these intracellular mechanisms, which had been previously associated with PD, also play an important role in our cell model.

LUHMES细胞（LUHMES cells）与黑质内的人多巴胺能神经元具有诸多高度相似的特征，而这类神经元的丧失正是帕金森病（Parkinson's disease, PD）运动症状的核心致病机制。因此，LUHMES细胞可作为可靠的体外模型，用于探究帕金森病相关的病理生理过程。此前本团队已证实，过表达野生型α-突触核蛋白（alpha-synuclein）后，LUHMES细胞会在培养6天后发生退行性病变。本研究中，我们分别对α-突触核蛋白过表达细胞与绿色荧光蛋白（GFP）过表达的对照细胞开展转录组与蛋白质组表达谱分析，以期筛选出α-突触核蛋白过表达后发生差异调控的基因与蛋白质。为捕捉细胞死亡前的早期调控事件，我们在α-突触核蛋白或GFP过表达诱导4天后即开展了该分析，此时细胞尚未出现死亡。经多重检验校正后，我们共鉴定得到765个差异调控基因（其中439个上调、326个下调），以及122个差异表达蛋白质（其中75个上调、47个下调）。两类组学数据中共有21个基因及其对应蛋白质呈现出同向的显著差异调控，其中13个为上调表达，8个为下调表达。在本细胞模型中，共有13个基因与9个蛋白质曾在近期的全基因组关联研究（genome-wide association studies, GWAS）中被报道与帕金森病存在显著关联。对所有上调基因进行基因本体（Gene Ontology, GO）富集分析后，排名前列的核心条目包括“细胞死亡调控”“程序性细胞死亡正调控”以及“凋亡信号通路调控”，这表明在细胞开始死亡前2天，与细胞死亡相关的基因与蛋白质已发生调控变化。对差异调控蛋白质的GO富集分析显示，富集程度最高的条目涵盖“囊泡”“突触”与“溶酶体”。总计33个差异调控蛋白质与突触结构或功能相关，12个差异调控蛋白质与溶酶体相关，这提示此前已被证实与帕金森病相关的这些细胞内机制，在本细胞模型中同样扮演着关键角色。