NEDD9将星形胶质细胞转分化为神经元

Injury to central nervous system triggers the pronounced reactivity of astrocytes, leading to the glial scar formation and rendering the lesion unfavorable to nerve regeneration. Recent studies have shown that astrocytes, the most plentiful cells in the central nervous system, hold great promise, especially upon transdifferentiation into neurons or neural stem/progenitor cells (NSCs) that can be used to replace the lost cells or ameliorate the lesion conditions, for nerve repair after injury. In this study, based on our intriguing preliminary results that overexpression of NEDD9 (neural precursor cell expressed, developmentally down-regulated 9) can transdifferentiate astrocytes into neurons, we will analyze in detail the effects of NEDD9 overexpression on the conversion of astrocytes into neurons and/or NPCs in relation to the specificity of the produced neurons and the expression of the neurogenic transcription factors, and further delineate the signaling molecules that participate in the transdifferentiation of astrocytes into neurons by NEDD9. Moreover, by using a spinal cord injury model, we will evaluate the contribution of the reactive astrocytes that have migrated to the lesion sites and overexpressed NEDD9 by lentivirus infection to the structural and functional recovery of the injured spinal cord, shedding light on the new therapeutic strategy for nerve repair by using astrocytes as a powerful and plentiful cell source.

中枢神经系统的损伤引起大量星形胶质细胞向损伤部位的聚集，如何利用或改善损伤区域星形胶质细胞促进损伤修复近年来成为研究热点。我们的初步工作首次发现高表达NEDD9可以将星形胶质细胞转分化为神经元，本项目将利用慢病毒Dox诱导表达系统阐明高表达NEDD9对星形胶质细胞的转分化作用以及参与NEDD9转分化作用的相关信号分子，并进一步回答以下问题:(1)NEDD9持续高表达和高表达不同时间后撤除Dox变为低表达后转分化作用是否相同？(2)转分化过程是否经过神经干/祖或前体细胞阶段？(3)NEDD9是否能将星形胶质细胞转分化为特定神经元？(4)体内是否具有同样的转分化作用？在此基础上，利用脊髓损伤模型通过病毒感染高表达NEDD9探讨将损伤区域的星形胶质细胞转分化为神经元后对神经损伤的修复作用，为利用星形胶质细胞转分化治疗神经损伤提供理论指导和可能的治疗方案。

中枢神经系统的损伤引起大量星形胶质细胞向损伤部位的聚集，如何利用或改善损伤区域星形胶质细胞促进损伤修复近年来成为研究热点。我们通过形态跟踪、神经元标志物表达、GFAP启动子报告基因的失活以及Tuj1启动子报告基因的激活研究发现高表达NEDD9可以将星形胶质细胞转分化为神经元。qRT-PCR检测发现转分化4周时DCX和Tuj1的表达分别上调6.15和1.63倍，细胞呈现GFAP阴性、Tuj1阳性、成熟神经元标志物MAP2、NeuN和GAP43阳性。进一步转录组测序分析及谷氨酸能、GABA能和多巴胺能神经元特异性标志物 vGlut1、GAD67和TH的表达检测显示，星形胶质细胞高表达NEDD9 30天后获得的神经元可能为多巴胺能神经元。另外，细胞增殖、Nestin启动子报告基因的活性变化、成球能力以及多向分化潜能研究证实NEDD9诱导的转分化过程经过神经干/组细胞阶段。RNA测序分析、qRT-PCR检测以及信号特异抑制剂处理实验结果表明，多种神经分化相关的转录因子及Wnt/β-catenin、MAPK和Notch信号参与NEDD9诱导的星形胶质细胞向神经元的转分化。在此基础上，利用脊髓损伤模型通过病毒感染高表达NEDD9能够将损伤区域的星形胶质细胞转分化为神经元，促进损伤组织修复，为利用星形胶质细胞转分化治疗神经损伤提供理论指导和可能的治疗方案。