scRNAseq analysis of mouse L4 whole dorsal root ganglions following sciatic nerve cush, dorsal root crush and spinal cord injury

After an injury in the adult mammalian central nervous system, lesioned axons fail to regenerate. This failure to regenerate contrasts with the remarkable potential of axons to grow following an injury in the peripheral nervous system. Peripheral sensory neurons with cell soma in dorsal root ganglia (DRG) switch to a regenerative state after nerve injury to enable axon regeneration and functional recovery. Decades of research have focused on the signaling pathways elicited by injury in sensory neurons and in Schwann cells that insulate axons as central mechanisms regulating nerve repair. However, neuronal microenvironment is far more complex and is composed of multiple cell types including endothelial, immune and glial cells. Whether the microenvironment surrounding neuronal soma contribute to the poor regenerative outcomes following central injuries remains largely unexplored. To answer this question, we performed a single cell transcriptional profiling of the DRG neuronal microenvironment response to peripheral and central injuries. In dissecting the roles of the microenvironment contribution, we have focused on a poorly studied glia population of Satellite Glial Cells (SGC) surrounding the neuronal cell soma. Upon a peripheral injury, SGC contribute to axon regeneration via Fatty acid synthase (Fasn)-PPARa signaling pathway. Our analysis reveals that in response to central injuries, SGC do not activate the PPAR signaling pathway. However, induction of this pathway with fenofibrate, an FDA- approved PPARa agonist used for dyslipidemia treatment, rescued axon regeneration following an injury to the central nerves. Collectively, our results uncovered a previously unappreciated role of the neuronal microenvironment differential response in central and peripheral injuries. We have applied the high-throughput single-cell mRNA sequencing technique, using the Chromium Single Cell Gene Expression Solution (10x Genomics) to mouse L4 DRG in naive and injured conditions (3 days post sciatic nerve crush injury, dorsal root crush or spinal cord injury), n=2 biological samples (NAI,DRC,SCI) n=1 (SNC)成年哺乳动物中枢神经系统损伤后，受损轴突无法实现再生。这一再生失败现象，与周围神经系统损伤后轴突所具备的显著生长潜能形成鲜明反差。胞体位于背根神经节（dorsal root ganglia, DRG）的外周感觉神经元，在神经损伤后会切换至再生状态，从而支持轴突再生与功能恢复。数十年来的研究均聚焦于感觉神经元及包裹轴突的施万细胞（Schwann cells）中由损伤触发的信号通路，将其作为调控神经修复的核心机制。然而，神经元微环境的构成远为复杂，包含内皮细胞、免疫细胞及神经胶质细胞等多种细胞类型。中枢神经系统损伤后，神经元胞体周围的微环境是否会导致较差的再生结局，目前仍未得到充分探索。为解答这一科学问题，我们对背根神经节神经元微环境在周围与中枢神经系统损伤后的响应开展了单细胞转录组分析。在解析微环境的调控贡献时，我们重点关注了此前研究较少的、包裹神经元胞体的卫星胶质细胞（Satellite Glial Cells, SGC）群体。研究发现，外周神经损伤后，卫星胶质细胞可通过脂肪酸合酶（Fatty acid synthase, Fasn）-过氧化物酶体增殖物激活受体α（PPARα）信号通路促进轴突再生。我们的分析结果显示，在应对中枢神经系统损伤时，卫星胶质细胞并不会激活该PPAR信号通路。不过，使用非诺贝特（fenofibrate）——一种经美国食品药品监督管理局（FDA）批准、用于治疗血脂异常的PPARα激动剂——诱导该通路激活后，可挽救中枢神经损伤后的轴突再生能力。综上，本研究揭示了神经元微环境在中枢与外周损伤中存在此前未被重视的差异化响应作用。我们采用10x Genomics公司的Chromium单细胞基因表达解决方案（Chromium Single Cell Gene Expression Solution, 10x Genomics），对未处理及损伤状态下的小鼠L4背根神经节进行了高通量单细胞mRNA测序。损伤模型包括坐骨神经压榨伤后3天、背根压榨伤或脊髓损伤，其中NAI、DRC、SCI组各设2份生物学重复，SNC组设1份生物学重复。