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FMR1 loss in a human stem cell model reveals early changes to intrinsic membrane excitability
Developmental Biology ( IF 2.5 ) Pub Date : 2020-09-22 , DOI: 10.1016/j.ydbio.2020.09.012
Sara G. Susco , Mario A. Arias-García , Violeta G. López-Huerta , Amanda Beccard , Anne M. Bara , Jessica Moffitt , Justin Korn , Zhanyan Fu , Lindy E. Barrett

Fragile X mental retardation 1 (FMR1) encodes the RNA binding protein FMRP. Loss of FMRP drives Fragile X syndrome (FXS), the leading inherited cause of intellectual disability and a leading monogenic cause of autism. While cortical hyperexcitability is a hallmark of FXS, the reported phenotypes and underlying mechanisms, including alterations in synaptic transmission and ion channel properties, are heterogeneous and at times contradictory. Here, we report the generation of new isogenic FMR1y/+ and FMR1y/- human pluripotent stem cell (hPSC) lines using CRISPR-Cas9 to facilitate the study of how complete FMRP loss, independent of genetic background, drives molecular and cellular alterations relevant for FXS. After differentiating these stem cell tools into excitatory neurons, we systematically assessed the impact of FMRP loss on intrinsic membrane and synaptic properties over time. Using whole-cell patch clamp analyses, we found that FMR1y/- neurons overall showed an increased intrinsic membrane excitability compared to age-matched FMR1y/+ controls, with no discernable alternations in synaptic transmission. Surprisingly, longitudinal analyses of cell intrinsic defects revealed that a majority of significant changes emerged early following in vitro differentiation and some were not stable over time. Collectively, this study provides a new isogenic hPSC model which can be further leveraged by the scientific community to investigate basic mechanisms of FMR1 gene function relevant for FXS. Moreover, our results suggest that precocious changes in the intrinsic membrane properties during early developmental could be a critical cellular pathology ultimately contributing to cortical hyperexcitability in FXS.



中文翻译:

人类干细胞模型中的FMR1丢失揭示了内在膜兴奋性的早期变化

脆性X智力低下1(FMR1)编码RNA结合蛋白FMRP。FMRP的丧失导致易碎X综合征(FXS),这是智力残疾的主要遗传病因,也是自闭症的主要单基因病因。尽管皮质兴奋性亢进是FXS的标志,但所报告的表型和潜在机制(包括突触传递和离子通道特性的改变)是异质的,有时是矛盾的。在这里,我们报告了新的等基因FMR1 y / +FMR1 y /-的生成人类多能干细胞(hPSC)系使用CRISPR-Cas9来促进研究完整的FMRP丢失如何独立于遗传背景如何驱动与FXS相关的分子和细胞改变。在将这些干细胞工具分化为兴奋性神经元后,我们系统地评估了FMRP损失对内在膜和突触特性的影响。使用全细胞膜片钳分析,我们发现与年龄匹配的FMR1 y / +对照相比,FMR1 y /-神经元总体上显示出内在的膜兴奋性增加,在突触传递中没有明显的交替。出乎意料的是,对细胞内在缺陷的纵向分析表明,大多数显着变化发生在体外早期分化和一些不稳定。总的来说,这项研究提供了一种新的等基因hPSC模型,科学界可以进一步利用该模型来研究与FXS相关的FMR1基因功能的基本机制。此外,我们的研究结果表明,早期发育过程中固有膜特性的过早变化可能是最终导致FXS皮质超兴奋性的关键细胞病理学。

更新日期:2020-09-30
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