Provincial and Ministerial Co-founded Collaborative Innovation Center of Rehabilitation Technology, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 350122, China.
National-Local Joint Engineering Research Center of Rehabilitation Medicine Technology, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 350122, China.
Exp Neurol. 2023 Dec;370:114571. doi: 10.1016/j.expneurol.2023.114571. Epub 2023 Oct 15.
Astrocytes have been demonstrated to undergo conversion into functional neurons, presenting a promising approach for stroke treatment. However, the development of small molecules capable of effectively inducing this cellular reprogramming remains a critical challenge.
Initially, we introduced a glial cell marker gene, GFaABC1D, as the promoter within an adeno-associated virus vector overexpressing miR-124 into the motor cortex of an ischemia-reperfusion model in rats. Additionally, we administered NeuroD1 as a positive control. Lentiviral vectors overexpressing miR-124 were constructed and transfected into primary rat astrocytes. We assessed the cellular distribution of GFAP, DCX, and NeuN on days 7, 14, and 28, respectively.
In rats with ischemic stroke, miR-124-transduced glial cells exhibited positive staining for the immature neuron marker doublecortin (DCX) and the mature neuron marker NeuN after 4 weeks. In contrast, NeuroD1-overexpressing model rats only expressed NeuN, and the positive percentage was higher in co-transfection with miR-124 and NeuroD1. Overexpression of miR-124 effectively ameliorated neurological deficits and motor functional impairment in the model rats. In primary rat astrocytes transduced with miR-124, DCX was not observed after 7 days of transfection, but it appeared at 14 days, with the percentage further increasing to 44.6% at 28 days. Simultaneously, 15.1% of miR-124-transduced cells exhibited NeuN positivity, which was not detected at 7 and 14 days. In vitro, double fluorescence assays revealed that miR-124 targeted Dll4, and in vivo experiments confirmed that miR-124 inhibited the expression of Notch1 and DLL4.
The overexpression of miR-124 in astrocytes demonstrates significant potential for improving neurological deficits following ischemic stroke by inhibiting DLL4 expression, and it may facilitate astrocyte-to-neuronal transformation.
星形胶质细胞已被证明可转化为功能性神经元，为中风治疗提供了一种很有前途的方法。然而，开发能够有效诱导这种细胞重编程的小分子仍然是一个关键挑战。
最初，我们将一个胶质细胞标记基因，GFaABC1D，作为启动子引入腺相关病毒载体中，在大鼠缺血再灌注模型的运动皮层中过表达 miR-124。此外，我们还使用 NeuroD1 作为阳性对照。构建了过表达 miR-124 的慢病毒载体，并转染原代大鼠星形胶质细胞。分别在第 7、14 和 28 天评估 GFAP、DCX 和 NeuN 的细胞分布。
在缺血性中风大鼠中，miR-124 转导的胶质细胞在 4 周后表现出不成熟神经元标记物双皮质素（DCX）和成熟神经元标记物 NeuN 的阳性染色。相比之下，NeuroD1 过表达模型大鼠仅表达 NeuN，并且与 miR-124 和 NeuroD1 共转染时阳性百分比更高。miR-124 的过表达有效改善了模型大鼠的神经功能缺损和运动功能障碍。在转导 miR-124 的原代大鼠星形胶质细胞中，转染后 7 天未观察到 DCX，但在 14 天出现，28 天进一步增加到 44.6%。同时，15.1%的 miR-124 转导细胞呈 NeuN 阳性，7 天和 14 天未检测到。在体外，双荧光实验表明 miR-124 靶向 Dll4，体内实验证实 miR-124 抑制 Notch1 和 DLL4 的表达。
星形胶质细胞中 miR-124 的过表达通过抑制 DLL4 的表达显示出改善缺血性中风后神经功能缺损的巨大潜力，并且可能促进星形胶质细胞向神经元的转化。
