GAS5抑制促DRG神经元轴突再生的作用及机制

The peripheral nervous system (PNS) has an intrinsic capacity to regenerate on its own. Deciphering the molecular mechanisms underlying nerve regeneration following mouse sciatic nerve transection contributes to identifying the triggers and regulators of nerve regeneration. Dorsal root ganglia (DRG) neurons spontaneously undergo robust neurite growth after axotomy. In preliminary experiments with the sciatic nerve injury model, we (1) systematically screened lncRNA profiling in DRGs neuron during nerve regeneration in vivo, in combination with lncRNA profiling in DRGs neuron at different stages of development. (2) performed functional investigation by lentivirus RNAi in vitro, and noted that GAS5 inhibition markedly promoted neurite outgrowth. In this project, we are going to search for the downstream factor that could be regulated by GAS5, and to explore the molecular mechanisms of GAS5 promoting regenerative outgrowth of DRG neurons by targeting Vimentin. Bioinformatics analysis revealed that GAS5 may regulate peripheral nerve regeneration through ceRNA. Additionally, retrograde Labeling of DRG neuron subtypes, which can regenerate axon, were determined by application of fluorescence gold. Moreover, we plan to investigate the impact of GAS5 inhibition in GAS5 conditional knockout mouse model by injection with AAV2/8 to determine whether the exposure to Cre or Vimentin could enhance axon growth. DRGs are also injected with AAV 2/8 virus that overexpresses GAS5 alone/in combination with Vimentin.In additionally, we are going to observe the role of GAS5 inhibition in optic nerve crush and repair.We believe that the oncoming findings may aid to developing a new route to stimulate intrinsic neurite growth in peripheral and central nerve regeneration.

神经元内在再生能力的激活是神经损伤后轴突再生的关键因素，我们通过损伤后能再生的坐骨神经来寻找调控神经再生的关键分子。利用坐骨神经夹伤模型，通过系统性分析DRG神经元在轴突再生不同时间点lncRNA表达谱的变化，结合DRG神经元在发育的不同阶段lncRNA表达谱的变化，经功能验证，得到关键差异lncRNA GAS5。进一步经RNA蛋白相互作用实验，发现其能与可溶性Vimentin绑定。本课题将通过构建GAS5条件性敲除小鼠，进一步阐明GAS5通过靶向Vimentin参与神经再生具体过程，以及可能的ceRNA调控机制；结合荧光金逆标，确定轴突能够再生的神经元亚型；DRGs注射过表达GAS5单独/联合过表达Vimentin的AAV2/8型病毒、在体观察轴突再生和神经修复的效果；并通过在体实验，研究GAS5抑制对视神经损伤修复的影响。探讨GAS5作为外周和中枢神经修复新靶点的应用价值。

轴突损伤后，外周神经系统(PNS)神经元具有较强的再生能力，但哺乳动物中枢神经系统(CNS)神经元普遍缺乏这种再生能力。越来越多的证据强调长链非编码RNA (LncRNA)在发育和疾病中的关键作用，但LncRNA在中枢神经系统和PNS中触发再生能力的作用尚未得到很好的研究。随着发育的过程，外周神经的再生能力也是不断减弱。在本研究中，我们发现lncRNA Gas5是轴突再生的抑制因子。我们利用RT2Profiler PCR阵列分析了坐骨神经挤压伤后不同时间点(0h、3h、9h、1d、4d、7d)和不同发育阶段(E14、E18、P1、P8、P28) DRGs的基因表达。我们分别在坐骨神经损伤和发育过程中分析了96个lncRNA (图1A和1B)。进一步对从坐骨神经损伤和发育模型中检测到的所有lncRNA进行聚类分析的系列检验，分别在两个模型中鉴定出2个具有显著lncRNA表达谱的聚类。其中，Gas5在DRG神经元发育过程中呈年龄依赖性上调，坐骨神经损伤后呈下调。在体外实验中，抑制Gas5的表达可促进小鼠和大鼠DRG神经元神经突的生长。Gas5过表达抑制小鼠DRG神经元轴突生长。在体内，Gas5基因敲除(Gas5-/-)小鼠坐骨神经损伤后神经再生能力增强。RNA pull-down分析表明，Gas5可以与可溶性Vimentin相互作用，这对周围神经的发育和再生至关重要。Vimentin的下调逆转了Gas5沉默调控的轴突促再生，说明Gas5的功能依赖于Vimentin。此外，抑制Gas5表达还能促进视神经再生，这表明Gas5沉默在中枢神经系统中具有潜在的促再生能力。我们的研究首次在体内提供了lncRNA在调节中枢轴突再生中的作用的直接证据，Gas5可能是PNS和CNS轴突再生的新治疗靶点。