Single-cell profiles of retinal neurons differing in resilience to injury reveal neuroprotective genes - Atlas of adult mouse retinal ganglion cells

Neuronal types in the central nervous system differ dramatically in their resilience to injury or insults. Here we studied selective resilience in mouse retinal ganglion cells (RGCs) following optic nerve crush (ONC), which severs their axons and leads to death of ~80% of RGCs in 2 weeks. To identify expression programs associated with differential resilience, we first used single-cell RNA-seq (scRNA-seq) to generate a comprehensive molecular atlas of 45 RGC types in adult retina. We tracked their survival after ONC, characterized transcriptomic, morphological, and physiological changes that preceded degeneration, and identified genes selectively expressed by each type. Finally, loss- and gain-of-function assays in vivo showed that manipulating some of these genes improved neuronal survival and axon regeneration following ONC. This study provides a systematic framework for parsing type-specific responses to injury, and demonstrates that these responses can be used to reveal molecular targets for intervention. In this experiment, we FACS sorted for retinal ganglion cells using a combination of a transgenic reporter (VGLUT2) and a surface protein CD90 from dissociated cells from retinas of adult (postnatal day 56) mice中枢神经系统内的神经元亚型对损伤或应激刺激的耐受性差异显著。本研究针对视神经钳夹伤（optic nerve crush, ONC）后小鼠视网膜神经节细胞（RGCs）的选择性耐受特性展开探究：该造模方式会切断RGCs的轴突，并在两周内导致约80%的RGCs死亡。为筛选与差异化耐受相关的基因表达程序，研究团队首先借助单细胞RNA测序（single-cell RNA-seq, scRNA-seq），构建了成年小鼠视网膜内45种RGC亚型的完整分子图谱。本研究追踪了ONC造模后RGCs的存活情况，对变性前的转录组、形态学及生理学变化进行了系统表征，并鉴定出各RGC亚型选择性表达的基因。最后，体内功能缺失与功能获得性实验证实，靶向调控部分上述基因可改善ONC造模后神经元的存活状态与轴突再生能力。本研究为解析神经元亚型特异性损伤应答提供了系统性框架，并证实可通过此类应答筛选干预性分子靶点。本实验中，研究团队从出生后第56天的成年小鼠视网膜解离细胞中，通过联合使用转基因报告基因（VGLUT2）与表面蛋白CD90的标记方案，借助荧光激活细胞分选（fluorescence-activated cell sorting, FACS）分离得到RGCs。