Translational profiling of retinal ganglion cell optic nerve regeneration in Xenopus laevis

Unlike adult mammals, adult frogs regrow and regenerate their optic nerve following a crush injury. Using Translational Ribosome Affinity Purification (TRAP), a method to isolate mRNAs actively undergoing translation in a target cell population, we have generated a transcriptional profile by RNA-Seq for retinal ganglion cells (RGC) during the period of recovery following an optic nerve injury. Based on bioinformatics analysis using the JGI 9.1 Xenopus laevis gene models, our results reveal a profound shift in the composition of actively translating mRNAs during the early stages of RGC regeneration: as factors involved in cell signaling are rapidly downregulated, and those involved in core metabolism are upregulated. We identified one highly upregulated gene in response to injury, uchl1, which coupled to downregulation of the synucleins (snca, scng), was previously implicated in neurodegenerative diseases. Our injury-screen in Xenopus identified a previously unknown gene, gng8, as being associated with the regenerative process. Our generated online database provides the Xenopus community a valuable resource for the identification of genes involved in the regeneration process to target for future functional studies. To investigate the changes in gene expression that occur as retinal ganglion cells (RGCs) recover and regrow following injury, we have used the TRAP method. With TRAP we are able to isolate the actively translating pool of mRNAs from a specific cell type, in this case RGCs. To do this, we created lines of transgenic frogs which express an eGFP-tagged variant of the large ribosomal protein rpl10a under the control of an RGC-specific promoter from the islet2b locus. In our experimental framework, we quantify gene expression changes in RGCs recovering from optic nerve crush by comparing mRNA levels in samples collected from the eye undergoing a surgical crush (right) to the contralateral eye (left). At discrete time points following optic nerve crush in the left eye, both eyes are rapidly dissected and the ribosome-associated RNAs purified from tissue extracts using eGFP antibodies conjugated to magnetic beads. To control for the effects of surgery on RGCs, gene expression was also quantified in animals that underwent sham surgeries with no optic nerve crush ("sham" samples). To control for the systemic effects of the surgical procedure per se, gene expression was also quantified in animals that did not undergo any surgery ("naive" sample). These mRNA pools were used to construct libraries for RNA-Seq using poly(A) selection and 2x multiplexing.

与成年哺乳动物不同，成年青蛙在视神经挤压损伤后可实现视神经的再生与修复。本研究采用翻译核糖体亲和纯化（Translational Ribosome Affinity Purification, TRAP）技术——一种可从目标细胞群体中分离正在活跃翻译的mRNA的方法——对视神经损伤后恢复阶段的视网膜神经节细胞（retinal ganglion cells, RGC）进行了RNA测序（RNA-Seq），并构建了其转录组表达图谱。基于JGI 9.1版非洲爪蟾（Xenopus laevis）基因模型的生物信息学分析结果显示，在视网膜神经节细胞再生早期，活跃翻译的mRNA组成发生了显著改变：参与细胞信号传导的因子快速下调，而参与核心代谢通路的因子则显著上调。我们鉴定出一个在损伤应答中高度上调的基因uchl1，该基因与突触核蛋白家族（snca、scng）的下调存在关联，而突触核蛋白此前已被证实与神经退行性疾病密切相关。本研究在非洲爪蟾的损伤筛选实验中发现了一个此前未被报道的基因gng8，其与视神经再生过程显著相关。我们构建的在线数据库可为非洲爪蟾研究领域的科研人员提供宝贵的资源，助力鉴定参与再生过程的靶基因，为后续功能验证研究提供方向。为探究视网膜神经节细胞（RGC）在损伤后恢复与再生过程中的基因表达变化，我们采用了TRAP技术。借助该技术，我们能够从特定细胞类型——即本研究中的视网膜神经节细胞——中分离得到活跃翻译的mRNA池。为此，我们构建了转基因青蛙品系，该品系在islet2b基因位点的视网膜神经节细胞特异性启动子调控下，表达带有增强型绿色荧光蛋白（enhanced green fluorescent protein, eGFP）标签的大亚基核糖体蛋白rpl10a。在本实验的研究框架中，我们通过比较接受手术挤压的右眼与对侧左眼的mRNA水平，量化分析视网膜神经节细胞在视神经挤压损伤后的基因表达变化。在视神经挤压术后的不同时间点，我们快速分离双侧眼球，并利用结合了磁珠的eGFP抗体从组织提取物中纯化核糖体结合RNA。为控制手术操作本身对视网膜神经节细胞的影响，我们还对接受假手术（未进行视神经挤压）的动物样本（"假手术"组）进行了基因表达定量分析。为排除手术操作带来的系统性干扰，我们也对未接受任何手术的动物样本（"未处理"组）进行了基因表达定量分析。这些mRNA池被用于构建RNA测序文库，构建过程采用了poly(A)选择和2×多重测序策略。