Identification of transcription factor combinations that promote axonal sprouting in the injured spinal cord and regeneration-associated gene expression

Axon regeneration after injury to the central nervous system is limited by an inhibitory environment but also because injured neurons fail to initiate expression of regeneration associated genes (RAGs), thought to be required for robust long-distance growth. The potential of strong RAG expression to promote CNS regeneration is exemplified by the conditioning lesion model, whereby peripheral nerve injury promotes regeneration of centrally projecting branches of the injured neurons. RAG expression could potentially be induced by delivery of the right combination of transcription factors (TF), but the specific TFs required remain to be identified. We here begin with a focused bioinformatics approach, analyzing binding site motifs in the promoters of the RAG program to identify nine candidate growth-promoting TFs. In vitro screening then identified two combinations, KLF7/MEF2 and ATF3/KLF7/MEF2, that had potent neurite-growth promoting activity, the latter being the more powerful. We expressed TFs in vivo in L4/L5 dorsal root ganglia to test whether these combinations would promote regeneration and activate the RAG program, mimicking the conditioning lesion effect. KLF7 and MEF2 were also tested individually for comparison. KLF7/MEF2, but not ATF3/KLF7/MEF2 or the individual TFs, promoted axonal sprouting into the lesion site and improved functional recovery. We also generated gene expression profiles of laser dissected dorsal root ganglion neurons specifically expressing these TF combinations, and of DRG neurons that had been axotomized. Unexpectedly, the MEF2-VP16 construct used had little transcriptional activity in vivo, suggesting additional steps may be required to achieve full MEF2 activity. All combinations except MEF2 alone induced RAG expression mirroring that induced by axotomy to significant extents, while ATF3/KLF7/MEF2, KLF7 and ATF3, but not KLF7/MEF2 also induced apoptosis-related genes which may hinder regeneration. In conclusion, the combination KLF7/MEF2 partially mimics the conditioning lesion effect and promotes functional improvement. All samples are laser-dissected large diameter neurons from rat dorsal root ganglion, following either: adeno-associated viral vector delivery expressing transcription factors with fluorophores, or only fluorophore (control group); or sciatic nerve injury; or from naïve DRG (control group for nerve injury ). The TFs over-expressed in each group were: ATF3, KLF7, MEF2, KLF7/MEF2, ATF3/KLF7/MEF2, no TF (survival time 4 weeks); sciatic nerve injury survival times were 1 day and 7 days. All groups were performed with n=4.

中枢神经系统（central nervous system, CNS）损伤后轴突再生不仅受到抑制性微环境的限制，还因受损神经元无法启动再生相关基因（regeneration associated genes, RAGs）的表达——而后者被认为是实现高效长距离轴突生长的必要条件。强RAG表达促进中枢神经系统再生的潜力，可通过条件性损伤模型得到印证：外周神经损伤可促使受损神经元的中枢投射分支实现再生。理论上，通过递送合适的转录因子（transcription factors, TFs）组合即可诱导RAG表达，但目前仍未明确所需的特异性转录因子。本研究首先采用聚焦式生物信息学方法，分析RAG基因程序启动子区域的结合基序，筛选出9种潜在的促生长转录因子候选物。后续体外筛选实验发现两种组合具有显著的神经突起生长促进活性：KLF7/MEF2与ATF3/KLF7/MEF2，其中后者的促生长能力更强。为验证这两种组合是否可模拟条件性损伤效应，促进中枢神经系统再生并激活RAG基因程序，我们在L4/L5背根神经节（dorsal root ganglia, DRG）内活体过表达上述转录因子组合，同时单独过表达KLF7与MEF2作为对照。实验结果显示，KLF7/MEF2组合（而非ATF3/KLF7/MEF2组合或单个转录因子）可促进轴突发芽侵入损伤部位，并改善功能恢复。我们还对特异性表达上述转录因子组合的激光显微切割背根神经节神经元，以及轴突切断后的背根神经节神经元进行了基因表达谱分析。意外的是，所使用的MEF2-VP16融合蛋白在活体中几乎无转录活性，提示要完全激活MEF2的功能可能需要额外的调控步骤。除单独使用MEF2外，所有转录因子组合均能在显著程度上诱导RAG表达，其效果与轴突切断诱导的RAG表达相似；而ATF3/KLF7/MEF2组合、KLF7与ATF3（而非KLF7/MEF2组合）还会诱导凋亡相关基因的表达，这可能会对再生过程造成阻碍。综上，KLF7/MEF2组合可部分模拟条件性损伤的效应，并促进功能恢复。本研究的所有样本均取自大鼠背根神经节的大直径神经元，经激光显微切割获取，处理方式分为以下四类：① 经腺相关病毒（adeno-associated viral vector, AAV）载体递送，表达携带荧光标签的转录因子；② 仅递送荧光标签（对照组）；③ 坐骨神经损伤模型；④ 未经过任何处理的正常背根神经节（作为坐骨神经损伤模型的对照组）。各组过表达的转录因子分别为：ATF3、KLF7、MEF2、KLF7/MEF2、ATF3/KLF7/MEF2，以及无转录因子过表达组（存活时间为4周）；坐骨神经损伤模型的存活时间分别为1天与7天。所有实验组的样本量均为n=4。