Corticospinal neurons (CSN) centrally degenerate in amyotrophic lateral sclerosis (ALS), along with spinal motor neurons, and loss of voluntary motor function in spinal cord injury (SCI) results from damage to CSN axons. For functional regeneration of specifically affected neuronal circuitry in vivo, or for optimally informative disease modeling and/or therapeutic screening in vitro, it is important to reproduce the type or subtype of neurons involved. No such appropriate in vitro models exist with which to investigate CSN selective vulnerability and degeneration in ALS, or to investigate routes to regeneration of CSN circuitry for ALS or SCI, critically limiting the relevance of much research. Here, we identify that the HMG-domain transcription factor Sox6 is expressed by a subset of NG2+ endogenous cortical progenitors in postnatal and adult cortex, and that Sox6 suppresses a latent neurogenic program by repressing inappropriate proneural Neurog2 expression by progenitors. We FACS-purify these genetically accessible progenitors from postnatal mouse cortex and establish a pure culture system to investigate their potential for directed differentiation into CSN. We then employ a multi-component construct with complementary and differentiation-sharpening transcriptional controls (activating Neurog2, Fezf2, while antagonizing Olig2 with VP16:Olig2). We generate corticospinal-like neurons from SOX6+/NG2+ cortical progenitors, and find that these neurons differentiate with remarkable fidelity compared with corticospinal neurons in vivo. They possess appropriate morphological, molecular, transcriptomic, and electrophysiological characteristics, without characteristics of the alternate intracortical or other neuronal subtypes. We identify that these critical specifics of differentiation are not reproduced by commonly employed Neurog2-driven differentiation. Neurons induced by Neurog2 instead exhibit aberrant multi-axon morphology and express molecular hallmarks of alternate cortical projection subtypes, often in mixed form. Together, this developmentally-based directed differentiation from genetically accessible cortical progenitors sets a precedent and foundation for in vitro mechanistic and therapeutic disease modeling, and toward regenerative neuronal repopulation and circuit repair.

中文翻译：

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功能性皮质脊髓样神经元与内源性 SOX6+/NG2+ 皮质祖细胞的定向分化

肌萎缩侧索硬化症 (ALS) 中的皮质脊髓神经元 (CSN) 以及脊髓运动神经元集中退化，脊髓损伤 (SCI) 中的自主运动功能丧失是由于 CSN 轴突损伤造成的。对于体内受特定影响的神经元回路的功能再生，或对于体外最佳信息疾病建模和/或治疗筛选，重现所涉及神经元的类型或亚型非常重要。不存在这样合适的体外模型来研究 ALS 中 CSN 的选择性脆弱性和退化，或研究 ALS 或 SCI 中 CSN 回路的再生途径，这严重限制了许多研究的相关性。在这里，我们确定 HMG 结构域转录因子 Sox6 由出生后和成人皮质中的 NG2+ 内源性皮质祖细胞亚群表达，并且 Sox6 通过抑制祖细胞不适当的原神经性 Neurog2 表达来抑制潜在的神经源性程序。我们从出生后小鼠皮质中流式细胞仪纯化这些可遗传的祖细胞，并建立纯培养系统来研究它们定向分化为 CSN 的潜力。然后，我们采用具有互补和分化锐化转录控制的多组分构建体（激活 Neurog2、Fezf2，同时用 VP16:Olig2 拮抗 Olig2）。我们从 SOX6+/NG2+ 皮质祖细胞生成皮质脊髓样神经元，并发现与体内皮质脊髓神经元相比，这些神经元的分化具有显着的保真度。它们具有适当的形态学、分子、转录组和电生理学特征，但没有替代皮质内或其他神经元亚型的特征。我们发现常用的 Neurog2 驱动的分化无法重现这些分化的关键细节。相反，由 Neurog2 诱导的神经元表现出异常的多轴突形态，并表达交替皮质投射亚型的分子特征，通常以混合形式。总之，这种基于发育的定向分化来自可遗传的皮质祖细胞，为体外机械和治疗疾病模型以及再生神经元重新填充和回路修复奠定了先例和基础。