Genome-wide analysis of the transcriptional profile of the CNS during EAE in B6 mice by scRNA-seq [scRNA-seq]. Genome-wide analysis of the transcriptional profile of the CNS during EAE in B6 mice by scRNA-seq [scRNA-seq]

We identify cellular heterogeneity during EAE in B6 mice. Multiple sclerosis (MS) is an autoimmune neurologic disease leading to demyelination and neurologic dysfunction controlled by both genetic and environmental factors. In addition to CNS-infiltrating immune cells, CNS-resident cells, such as astrocytes, are thought to play an important role in MS pathogenesis. However, a comprehensive understanding of the extent to which gene expression is disrupted in astrocytes is not known. Here, we implement single-cell RNA sequencing, in vivo genetic perturbations, cell-specific RNA profiling by Ribotag, as well as single-cell RNA sequencing of human MS patient samples to identify a transcriptional regulatory network in astrocytes that controls the pathogenesis of EAE and potentially, MS. We defined an astrocyte subpopulation characterized by expression of the small Maf protein, MAFG, which represses NRF2-driven antioxidant mechanisms and promotes EAE pathogenesis. Mechanistically, MAFG suppresses NRF2-dependent antioxidant genetic programs by cooperating with its cofactor, MAT2a, to promote DNA methylation in the context of CNS inflammation, which in turn increases pathogenic signaling processes in astrocytes. MAFG/MAT2a astrocytes are controlled by GM-CSF signaling, which drives EAE pathogenesis and MAFG expression. MAFG is activated in astrocytes derived from MS patients, which are characterized by DNA methylation programs, pro-inflammatory signaling processes including GM-CSF signaling, and repressed NRF2 activation. Together, these data create a transcriptional and epigenetic framework to analyze CNS inflammation in MS and may provide new therapeutic targets. Overall design: Naïve mice (n=6), CFA-only mice (n=3), EAE priming phase (n=6), EAE peak phase (n=6), EAE remission phase (n=6)

本研究旨在解析C57BL/6（B6）小鼠体内实验性自身免疫性脑脊髓炎（Experimental Autoimmune Encephalomyelitis, EAE）进程中的细胞异质性。多发性硬化（Multiple Sclerosis, MS）是一类受遗传与环境因素共同调控的自身免疫性神经系统疾病，可引发脱髓鞘病变与神经系统功能障碍。除中枢神经系统（Central Nervous System, CNS）浸润的免疫细胞外，星形胶质细胞等CNS驻留细胞被认为在MS发病机制中发挥关键作用，但目前学界尚未全面阐明星形胶质细胞内基因表达紊乱的具体程度与相关机制。本研究采用单细胞RNA测序（single-cell RNA sequencing）、在体遗传扰动技术、基于Ribotag的细胞特异性RNA谱分析，以及人类MS患者样本的单细胞RNA测序，旨在鉴定星形胶质细胞中的转录调控网络，该网络可调控EAE乃至潜在的MS发病进程。本研究鉴定出一类以小Maf蛋白MAFG表达为特征的星形胶质细胞亚群，该亚群可抑制核因子E2相关因子2（Nuclear Factor Erythroid 2-related Factor 2, NRF2）介导的抗氧化防御机制，并促进EAE发病。机制层面研究显示，MAFG可与其辅因子MAT2a协同作用，在中枢神经系统炎症微环境中促进DNA甲基化，进而抑制NRF2依赖的抗氧化基因程序，最终增强星形胶质细胞的致病性信号通路。MAFG/MAT2a阳性星形胶质细胞受粒细胞-巨噬细胞集落刺激因子（Granulocyte-Macrophage Colony-Stimulating Factor, GM-CSF）信号通路调控，该通路可驱动EAE发病并上调MAFG的表达水平。在MS患者来源的星形胶质细胞中，MAFG同样被激活，此类细胞的特征包括DNA甲基化程序异常、包括GM-CSF信号通路在内的促炎信号通路活化，以及NRF2激活受到抑制。综上，本研究构建了一套可用于分析MS患者中枢神经系统炎症的转录与表观遗传研究框架，或可为MS治疗提供全新的潜在靶点。实验整体设计：未免疫小鼠（n=6）、仅弗氏完全佐剂（Complete Freund's Adjuvant, CFA）处理小鼠（n=3）、EAE致敏期小鼠（n=6）、EAE高峰期小鼠（n=6）以及EAE缓解期小鼠（n=6）