University of Cambridge, Department of Oncology, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 0XZ, UK John van Geest Centre for Brain Repair, University of Cambridge, Forvie Site, Robinson Way, Cambridge CB2 0PY, UK.
University of Cambridge, Department of Oncology, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 0XZ, UK.
Development. 2014 Jun;141(11):2216-24. doi: 10.1242/dev.106377. Epub 2014 May 12.
Generation of neurons from patient fibroblasts using a combination of developmentally defined transcription factors has great potential in disease modelling, as well as ultimately for use in regeneration and repair. However, generation of physiologically mature neurons in vitro remains problematic. Here we demonstrate the cell-cycle-dependent phosphorylation of a key reprogramming transcription factor, Ascl1, on multiple serine-proline sites. This multisite phosphorylation is a crucial regulator of the ability of Ascl1 to drive neuronal differentiation and maturation in vivo in the developing embryo; a phosphomutant form of Ascl1 shows substantially enhanced neuronal induction activity in Xenopus embryos. Mechanistically, we see that this un(der)phosphorylated Ascl1 is resistant to inhibition by both cyclin-dependent kinase activity and Notch signalling, both of which normally limit its neurogenic potential. Ascl1 is a central component of reprogramming transcription factor cocktails to generate neurons from human fibroblasts; the use of phosphomutant Ascl1 in place of the wild-type protein significantly promotes neuronal maturity after human fibroblast reprogramming in vitro. These results demonstrate that cell-cycle-dependent post-translational modification of proneural proteins directly regulates neuronal differentiation in vivo during development, and that this regulatory mechanism can be harnessed to promote maturation of neurons obtained by transdifferentiation of human cells in vitro.
使用发育定义的转录因子从患者成纤维细胞中生成神经元在疾病建模方面具有巨大的潜力，最终也可用于再生和修复。然而，在体外生成生理成熟的神经元仍然存在问题。在这里，我们证明了关键重编程转录因子 Ascl1 在多个丝氨酸-脯氨酸位点上的细胞周期依赖性磷酸化。这种多位点磷酸化是 Ascl1 在体内驱动胚胎发育中神经元分化和成熟的关键调节剂；Ascl1 的磷酸突变形式在非洲爪蟾胚胎中显示出显著增强的神经元诱导活性。从机制上讲，我们发现这种未磷酸化的 Ascl1 对细胞周期依赖性激酶活性和 Notch 信号的抑制均具有抗性，而这两种活性通常会限制其神经生成潜力。Ascl1 是从人成纤维细胞生成神经元的重编程转录因子鸡尾酒的核心成分；在体外人成纤维细胞重编程中，使用磷酸突变体 Ascl1 替代野生型蛋白可显著促进神经元成熟。这些结果表明，在发育过程中，原神经蛋白的细胞周期依赖性翻译后修饰直接调节体内神经元分化，并且可以利用这种调节机制来促进体外人细胞转分化获得的神经元的成熟。
