RNA-seq analysis of FACS-sorted control and Tsc2 cKO nociceptors with and without sciatic nerve crush

The goals of this study are to compare the transcriptomes of control heterozygous and Tuberous sclerosis 2 (Tsc2) homozygous nociceptors 3 days after sciatic nerve injury that have been enriched by fluorescence-associated cell sorting (FACS) with the aim of identifying differences in genes associated with axon regeneration. Nav1.8-Cre transgenic mice were used to delete Tsc2 as well as express Green fluorescent protein from the Rosa26 locus. Tsc2 deletion constitutively activates mTORC1 signaling cell autonomously. Dissociated dorsal root ganglia from adult mice were FACS-sorted for GFP and analyzed by RNA-seq. We find that the expression of a number of regeneration-associated transcription factors including cJun, Atf3, Sox11 and other are upregulated in Tsc2 mutant neurons in the absence of an injury. Additionally, the target genes of several other normally expressed regeneration-associated transcription factors are differentially expressed in Tsc2 mutant neurons, such as Creb1 and Klf’s. We conclude Tsc2 deletion and consequent mTORC1 activation establishes a pro-regenerative gene expression profile in nociceptors in the absence of an injury.

本研究旨在比较坐骨神经损伤3天后的对照杂合子与结节性硬化症2型（Tuberous sclerosis 2, Tsc2）纯合伤害感受器的转录组，上述样本已通过荧光激活细胞分选（fluorescence-associated cell sorting, FACS）完成富集，以期鉴定与轴突再生相关的基因表达差异。本研究采用Nav1.8-Cre转基因小鼠（Nav1.8-Cre transgenic mice），通过该工具既可敲除Tsc2基因，又可从Rosa26位点（Rosa26 locus）表达绿色荧光蛋白（Green fluorescent protein, GFP）。Tsc2基因敲除可通过细胞自主性方式持续激活mTORC1信号通路（mTORC1 signaling）。研究人员从成年小鼠体内解离背根神经节（dorsal root ganglia），针对GFP阳性细胞进行FACS分选，并通过RNA测序（RNA-seq）开展表达分析。研究结果显示，在未受损伤的Tsc2突变神经元中，包括c-Jun、Atf3、Sox11在内的多种再生相关转录因子（regeneration-associated transcription factors）的表达水平显著上调。此外，多种正常表达的再生相关转录因子的靶基因在Tsc2突变神经元中呈现差异表达，例如环腺苷酸应答元件结合蛋白1（Creb1）与Krüppel样因子（Klf）家族。综上，Tsc2基因敲除及其引发的mTORC1通路激活，可在未受损伤的伤害感受器中建立促再生基因表达谱（pro-regenerative gene expression profile）。