Institute of Neuroscience, Key Laboratory of Molecular Neurobiology of Ministry of Education and the Collaborative Innovation Center for Brain Science, Naval Medical University, Shanghai 200433, China.
Theranostics. 2022 May 27;12(10):4548-4563. doi: 10.7150/thno.71378. eCollection 2022.
Direct reprogramming of astrocytes into neurons opens up a new avenue for neuroregenerative medicine. However, the poor understanding of the molecular mechanisms underpinning the latent neurogenic program in astrocytes has largely restricted this strategy towards safe and effective clinical therapies. Immunocytochemistry, immunohistochemistry, western blotting, qRT-PCR, gene knockdown and fate-mapping are performed to analyze the role of NOTCH1 signaling in regulation of the latent neurogenic program in reactive astrocytes after spinal cord injury. Western blotting analysis highlights that NOTCH1 is a key signaling mediating Ascl1- and Neurog2-driven astrocyte-to-neuron conversion. Inhibition of NOTCH1 signaling in cultured astrocytes by shRNA or DAPT (a NOTCH1 inhibitor) is sufficient to reprogram them into neurons by upregulating the expression of pro-neural transcription factors, including NeuroD1, NeuroD2, Pax6, Lmx1a and Lhx6. In the spinal cord of adult mouse, the expression of Notch1 is detected in resident astrocytes, which was significantly increased after spinal cord injury (SCI). Genetical knockdown of NOTCH1 signaling alone successfully triggers endogenous reactive astrocytes reprogramming into neurons in the injured adult spinal cord. Importantly, pharmacologically blocking NOTCH1 signaling with small molecule DAPT alone can also induce astrocyte-to-neuron conversion after SCI. We identify NOTCH1 as a key common signaling pathway in reactive astrocyte that provides a barrier for cell fate conversion. This proof-of-principle study will significantly expand our molecular understanding of astroglial-lineage reprogramming and overcoming the NOTCH1 gatekeeper with small molecules may provide a transgene-free approach for chemical neuronal reprogramming with potential clinical application in neuroregeneration.
星形胶质细胞直接重编程为神经元为神经再生医学开辟了新途径。然而，对星形胶质细胞中潜在神经发生程序的分子机制缺乏了解，在很大程度上限制了这一策略向安全有效的临床治疗的发展。免疫细胞化学、免疫组织化学、western blot、qRT-PCR、基因敲低和命运图谱用于分析 NOTCH1 信号在脊髓损伤后反应性星形胶质细胞中潜在神经发生程序的调节中的作用。Western blot 分析强调 NOTCH1 是一种关键信号，介导 Ascl1 和 Neurog2 驱动的星形胶质细胞向神经元的转化。通过 shRNA 或 DAPT（一种 NOTCH1 抑制剂）抑制培养的星形胶质细胞中的 NOTCH1 信号足以通过上调包括 NeuroD1、NeuroD2、Pax6、Lmx1a 和 Lhx6 在内的神经营养转录因子的表达将其重编程为神经元。在成年小鼠的脊髓中，检测到 Notch1 在常驻星形胶质细胞中表达，脊髓损伤（SCI）后其表达显著增加。单独遗传敲低 NOTCH1 信号成功触发内源性反应性星形胶质细胞在损伤的成年脊髓中重编程为神经元。重要的是，单独使用小分子 DAPT 阻断 NOTCH1 信号也可以在 SCI 后诱导星形胶质细胞向神经元转化。我们确定 NOTCH1 是反应性星形胶质细胞中关键的共同信号通路，为细胞命运转化提供了障碍。这项原理验证研究将极大地扩展我们对星形胶质细胞谱系重编程的分子理解，并且用小分子克服 NOTCH1 守门员可能为无转基因的化学神经元重编程提供潜在的临床应用于神经再生。
