Myeloid C3 causes neuronal injury prior to synapse loss [snMulti-ome-seq]. Myeloid C3 causes neuronal injury prior to synapse loss [snMulti-ome-seq]

Complement component C3 mediates pathology in several CNS neurodegenerative diseases, but the cellular and molecular mechanisms leading to neuronal injury remain unclear. Herein, we examined how C3 deletion affects glial profiles and anterior visual pathway pathology in an animal model of neuroinflammation. scRNA-seq from mouse brain and optic nerve revealed that C3 expression defined disease-associated glial subtypes which were characterized by increased mTOR activation, cell metabolism, and translation. Deletion of C3 restored these glia towards homeostatic profiles. Myeloid-derived C3 mediated injury in optic nerve axons and retinal ganglion cells (RGCs) at the peak of EAE. To elucidate the timing of pathology we examined retinas prior to symptom onset and found reductions in Brn3a, an RGC transcription factor involved in dendritic arborization and protection from apoptosis. Our study supports a direct role for C3 in activating the mTOR-ribosomal biogenesis axis in glia which subsequently mediate early neuro-axonal stress and later synapse loss. Overall design: Optic nerves from 4 pools of mice (2 C3 WT, 2 C3 KO, each pool containing optic nerves from 5 pooled mice) at peak of EAE were flash frozen then subjected to paired snRNAseq and snATACseq analyses via 10X Multi-Ome technology

补体成分C3（Complement component C3）在多种中枢神经系统（Central Nervous System, CNS）神经退行性疾病中介导病理进程，但导致神经元损伤的细胞与分子机制仍未阐明。本研究针对神经炎症动物模型，探讨了C3缺失对胶质细胞谱及视觉前通路病理变化的影响。我们对小鼠大脑与视神经开展单细胞RNA测序（single-cell RNA sequencing, scRNA-seq），结果显示C3的表达可界定一类疾病相关胶质细胞亚型，该亚型以mTOR（mammalian target of rapamycin）激活、细胞代谢增强及蛋白质翻译水平提升为典型特征。C3缺失可使这类胶质细胞向稳态表型恢复。在实验性自身免疫性脑脊髓炎（experimental autoimmune encephalomyelitis, EAE）发病高峰期，髓系来源的C3可介导视神经轴突与视网膜神经节细胞（retinal ganglion cells, RGCs）的损伤。为明确病理发生的时序，我们在小鼠出现症状前检测了视网膜组织，发现Brn3a——一种参与树突分支形成并抑制细胞凋亡的RGC转录因子——的表达水平显著降低。本研究证实，C3可直接激活胶质细胞中的mTOR-核糖体生物发生轴，进而介导早期神经轴突应激与后续的突触丢失。总体实验设计：于EAE发病高峰期，采集4组混合小鼠视神经样本（2组为C3野生型（wild type, WT），2组为C3敲除型（knockout, KO）；每组包含5只小鼠的视神经混合样本），经快速冷冻后，通过10X Multi-Ome技术开展配对单细胞核RNA测序（single-nucleus RNA sequencing, snRNA-seq）与单细胞核转座酶可及性染色质测序（single-nucleus assay for transposase-accessible chromatin sequencing, snATAC-seq）分析。