Leptin signalling promotes axonal regeneration in the peripheral and central nervous system [LepvGFP RNA-seq]

Axonal growth is limited or absent following peripheral or central nervous system injury respectively, inhibiting repair. The identification of novel growth-promoting molecular mechanisms is therefore a priority. In the search for dietary-dependent mechanisms that control neuronal regenerative ability, we found, via RNA sequencing, that growth-promoting intermittent fasting (IF) induced leptin expression in sensory neurons of the dorsal root ganglia (DRG). Surprisingly, leptin signalling, whose canonical function is to control energy homeostasis, was critical for the IF-dependent regenerative phenotype. In fact, neuronal conditional deletion of the leptin receptor significantly impaired the regenerative response elicited by IF. Overexpression of leptin in vivo in DRG neurons enhanced axonal regeneration following peripheral sciatic nerve crush (SNC) and central spinal cord injury. Lastly, RNA sequencing following leptin overexpression in DRG neurons showed a significant increase in regenerative gene expression and transcription after SCI, indicating a role for leptin in inducing a euchromatic, transcriptionally active environment that facilitates nervous system repair after injury. AAV-GFP or AAV-Leptin-GFP was injected bilaterally into the sciatic nerve 4 weeks prior to a dorsal column axotomy. 24 hours post-injury, DRG were extracted and a neruonal enriched population processed for RNA-seq

周围神经系统与中枢神经系统损伤后，轴突生长分别受到限制或完全缺失，进而阻碍损伤修复。因此，发掘全新的促轴突生长分子机制成为当前研究的重点方向。在探究调控神经元再生能力的饮食依赖型分子机制过程中，我们通过RNA测序（RNA Sequencing）发现，具有促生长作用的间歇性禁食（Intermittent Fasting, IF）可诱导背根神经节（Dorsal Root Ganglia, DRG）感觉神经元表达瘦素（Leptin）。令人意外的是，瘦素信号通路的经典功能为调控能量稳态，但其却在依赖间歇性禁食的再生表型中发挥关键作用。事实上，神经元条件性敲除瘦素受体可显著削弱间歇性禁食诱导的再生应答反应。在背根神经节神经元体内过表达瘦素，可促进周围坐骨神经压榨伤（Sciatic Nerve Crush, SNC）与中枢脊髓损伤后的轴突再生。最后，对背根神经节神经元中瘦素过表达后的样本进行RNA测序发现，脊髓损伤（Spinal Cord Injury, SCI）后再生相关基因的表达与转录水平显著上调，这表明瘦素可诱导形成常染色质化、转录活跃的微环境，从而促进损伤后神经系统的修复。本实验于背柱轴突切断术前4周，将腺相关病毒（Adeno-Associated Virus, AAV）-GFP（绿色荧光蛋白，Green Fluorescent Protein, GFP）或AAV-Leptin-GFP双侧注射至坐骨神经。损伤24小时后，提取背根神经节并分离富集神经元群体以进行RNA测序（RNA-seq）。