基于单细胞转录组的基因富集方法分析星形胶质细胞与阿尔茨海默病的关系

Study of the Relationship Between Astrocytes and Alzheimer’s Disease by Gene Set Enrichment Analysis Based on Single-cell RNA Sequence

目的:通过生物信息学方法分析阿尔茨海默病(Alzheimer disease,AD)中与星形胶质细胞相关的糖代谢通路,为揭示AD患者的星形胶质细胞在大脑中的糖代谢过程提供理论基础。方法:首先根据细胞特异性表达基因将AD患者和健康人脑组织单细胞转录组学测序结果进行降维分析,再根据星形胶质细胞不同亚型的基因表达特征进行细胞分群,对星形胶质细胞差异表达基因进行基因注释(Gene Ontology.GO)、信号通路分析(Kyoto Encyclopedia of Genes and Genomes,KEGG)以及基因集富集分析(Gene Set Enrichment Analysis,GSEA),采用转录调控网络分析与AD的星形胶质细胞相关的转录辅助因子。结果:所有细胞降维分析结果显示AD患者脑内星形胶质细胞和兴奋性神经元数量显著减少;星形胶质细胞降维分析结果显示其可以被进一步分为6个亚群,其中在AD患者中减少的星形胶质细胞主要为RASGEF1B^(+)SLC26A3^(+)亚群和NRGN^(+)CALM1^(+)亚群;GO分析结果显示AD患者与健康对照星形胶质细胞差异表达基因主要与轴突发生、神经元的迁移、胶质细胞分化、体内锌离子稳态、突触传递的正调控、血管运输有关。KEGG结果显示,上述差异基因主要与PI3K-Akt信号通路、AMPK信号通路、钙信号通路有关。GSEA分析结果显示,AD患者差异基因在糖酵解/糖异生通路中得到富集,其中丙酮酸激酶PKM、PFKL、ACSS1、乳酸脱氢酶LDHB在AD患者星形胶质细胞中下调。转录调控网络分析结果显示,星形胶质细胞中差异表达转录辅助因子有5个,其中PKM、SOX2、SOX9在AD患者星形胶质细胞中下调。SREBF1和BCL6在AD患者星形胶质细胞中上调。结论:AD患者脑内兴奋性神经元和星形胶质细胞数量降低,以及星形胶质细胞糖酵解相关基因下调。结合星形胶质细胞作为神经元的主要乳酸供应细胞,其数量减少和糖酵解能力减低提示星形胶质细胞供能不足可能是AD发生的机制之一。

Objective:To analyze the glucose metabolism pathway associated with astrocytes in Alzheimer’s disease(AD)by the bioinformatics method,and to provide a theoretical basis for revealing the glycolysis metabolism of astrocytes in the brains of AD patients.Methods:Firstly,the single-cell transcriptome data from AD patients and healthy persons were analyzed with t-distributed stochastic neighbor embedding(t-SNE);secondly,the genes differently expressed in astrocytes from AD patients and healthy persons were analyzed by Gene Ontology(GO)analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis and Gene Set Enrichment Analysis(GSEA);lastly,the transcription cofactors related to AD were analyzed by the transcriptional regulatory network.Results:The t-SNE analysis indicated that the ratio of excitatory neurons and astrocytes was decreased in the brains of AD patients.In addition,the decreased astrocytes were characterized with RASGEF1B^(+)SLC26A3^(+)or NRGN^(+)CALM1^(+);GO analysis showed that differentially expressed genes were mainly related to axongenesis,neuronal migration,glial cell differentiation,zinc homeostasis,positive regulation of synaptic transmission and vascular transport.KEGG analysis showed that the differentially expressed genes were mainly related to PI3K-Akt signaling pathway,AMPK signaling pathway and calcium signaling pathway.GSEA analysis results showed that the differentially regulated genes in AD patients were enriched in glycolysis and gluconeogenesis pathway.In addition,PKM,PFKL,ACSS1 and LDHB were down-regulated in astrocytes from AD patients.Transcriptional regulatory network analysis showed that PKM,SOX2 and SOX9 were downregulated in astrocytes from AD patients.SREBF1 and BCL6 were up-regulated in astrocytes of AD patients.Conclusion:The reduction of excitatory neurons and astrocytes in the brains of AD patients,as well as the downregulation of the glycolysis pathway in astrocytes from the brains of AD patients,suggest that the impaired glycolysis in astrocytes could be one of the mechanisms underlying the development of AD.