DataSheet_1_RGC-32 Acts as a Hub to Regulate the Transcriptomic Changes Associated With Astrocyte Development and Reactive Astrocytosis.zip

Response Gene to Complement 32 (RGC-32) is an important mediator of the TGF-β signaling pathway, and an increasing amount of evidence implicates this protein in regulating astrocyte biology. We showed recently that spinal cord astrocytes in mice lacking RGC-32 display an immature phenotype reminiscent of progenitors and radial glia, with an overall elongated morphology, increased proliferative capacity, and increased expression of progenitor markers when compared to their wild-type (WT) counterparts that make them incapable of undergoing reactive changes during the acute phase of experimental autoimmune encephalomyelitis (EAE). Here, in order to decipher the molecular networks underlying RGC-32’s ability to regulate astrocytic maturation and reactivity, we performed next-generation sequencing of RNA from WT and RGC-32 knockout (KO) neonatal mouse brain astrocytes, either unstimulated or stimulated with the pleiotropic cytokine TGF-β. Pathway enrichment analysis showed that RGC-32 is critical for the TGF-β-induced up-regulation of transcripts encoding proteins involved in brain development and tissue remodeling, such as axonal guidance molecules, transcription factors, extracellular matrix (ECM)-related proteins, and proteoglycans. Our next-generation sequencing of RNA analysis also demonstrated that a lack of RGC-32 results in a significant induction of WD repeat and FYVE domain-containing protein 1 (Wdfy1) and stanniocalcin-1 (Stc1). Immunohistochemical analysis of spinal cords isolated from normal adult mice and mice with EAE at the peak of disease showed that RGC-32 is necessary for the in vivo expression of ephrin receptor type A7 in reactive astrocytes, and that the lack of RGC-32 results in a higher number of homeodomain-only protein homeobox (HOPX)+ and CD133+ radial glia cells. Collectively, these findings suggest that RGC-32 plays a major role in modulating the transcriptomic changes in astrocytes that ultimately lead to molecular programs involved in astrocytic differentiation and reactive changes during neuroinflammation.

补体32应答基因（Response Gene to Complement 32, RGC-32）是转化生长因子-β（TGF-β）信号通路的重要介导因子，越来越多的研究证据表明该蛋白在调控星形胶质细胞生物学功能中发挥关键作用。我们近期的研究发现，敲除RGC-32的小鼠脊髓星形胶质细胞呈现出类似神经祖细胞与放射状胶质细胞的未成熟表型：整体形态呈细长状，增殖能力增强，且相较于野生型（wild-type, WT）对照小鼠，其祖细胞标志物的表达水平显著上调，同时在实验性自身免疫性脑脊髓炎（experimental autoimmune encephalomyelitis, EAE）急性期无法发生反应性变化。为解析RGC-32调控星形胶质细胞成熟与反应性的分子网络机制，本研究对未受刺激或经多效性细胞因子TGF-β刺激的野生型与RGC-32基因敲除（knockout, KO）新生小鼠脑星形胶质细胞的RNA进行了下一代测序（next-generation sequencing, NGS）分析。通路富集分析结果显示，RGC-32对于TGF-β诱导的脑发育与组织重塑相关蛋白编码转录本的上调至关重要，这类蛋白包括轴突导向分子、转录因子、细胞外基质（extracellular matrix, ECM）相关蛋白以及蛋白聚糖。本次RNA下一代测序分析还证实，RGC-32缺失会显著诱导含WD重复基序与FYVE结构域蛋白1（WD repeat and FYVE domain-containing protein 1, Wdfy1）与斯钙素-1（stanniocalcin-1, Stc1）的表达。对正常成年小鼠及疾病高峰期实验性自身免疫性脑脊髓炎（EAE）模型小鼠的脊髓组织进行免疫组织化学分析后发现，RGC-32是反应性星形胶质细胞中 Eph 受体A7（ephrin receptor type A7）在体内表达的必要条件；而RGC-32缺失会导致同源盒结构域仅蛋白（homeodomain-only protein homeobox, HOPX）阳性与CD133阳性放射状胶质细胞的数量显著增多。综上，上述研究结果表明，RGC-32在调控星形胶质细胞的转录组变化中发挥核心作用，最终介导神经炎症过程中星形胶质细胞分化与反应性变化相关的分子程序。