Single-cell profiles of retinal neurons differing in resilience to injury reveal neuroprotective genes - Time course of transcriptomic changes in single retinal ganglion cells following optic nerve crush

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 these experiments, we FACS sorted for retinal ganglion cells using a combination of a transgenic reporter (VGLUT2) and a surface protein (CD90) from dissociated retinas of adult (6-8 wk old) mice, including uninjured controls, and at 6 time points following optic nerve crush (12h, 1d, 2d, 4d, 1w, 2w).

中枢神经系统内的神经元类型对损伤或侵害的耐受性差异显著。本研究探讨了视神经钳夹术（optic nerve crush, ONC）后小鼠视网膜神经节细胞（retinal ganglion cells, RGCs）的选择性耐受特性：该术式会切断其轴突，并在两周内导致约80%的RGC死亡。为鉴定与差异性耐受相关的基因表达程序，我们首先通过单细胞RNA测序（single-cell RNA-seq, scRNA-seq）构建了成年小鼠视网膜中45种RGC类型的全面分子图谱。我们追踪了这些细胞在ONC术后的存活情况，对变性发生前的转录组、形态学及生理学变化进行了表征，并鉴定出每种RGC类型特异性表达的基因。最后，体内的功能丧失与功能获得实验证实，对其中部分基因进行调控可改善ONC术后神经元的存活水平与轴突再生能力。本研究为解析神经元类型特异性损伤应答提供了系统性框架，并证明可通过这类应答挖掘干预性分子靶点。在本实验中，我们从6-8周龄成年小鼠的解离视网膜中，联合使用转基因报告基因（VGLUT2）与表面蛋白标记物（CD90），通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）分离视网膜神经节细胞，设置未损伤对照组，并在ONC术后的6个时间点（12h、1d、2d、4d、1w、2w）进行样本采集。