Partial FMRP expression is sufficient to normalize neuronal hyperactivity in Fragile X neurons
Partial FMRP expression is sufficient to normalize neuronal hyperactivity in Fragile X neurons
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DOI：
10.1111/ejn.14660
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发表时间：
2020-02-04
影响因子：
3.4
通讯作者：
Wallace, Owen
中科院分区：
文献类型：
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作者：
Graef, John D.;Wu, Hao;Wallace, Owen
脆性X综合征(FXS)是由脆性X智力低下基因FMR1的5‘端非编码区CGG重复扩增引起的最常见的遗传性智能障碍，导致表观遗传沉默和FMRP蛋白的缺失。FMR1的重新激活代表了一种针对FXS的遗传根本原因的有吸引力的治疗策略。然而，在FXS领域，很大程度上缺乏对FMR1重新激活才能挽救依赖FMRP的突变表型的理解。在这里，我们利用FXS患者来源的兴奋性神经元在体外建立FXS模型，并证实FMRP的缺乏会导致神经元的过度活动。我们进一步确定了FMRP的水平和纠正这种表型所需的FMRP阳性细胞的百分比，利用混合和镶嵌神经元培养系统以及CRISPR、反义和表达技术的组合来滴定FXS和WT神经元中的FMRP。我们的数据表明，在FXS iPSC来源的神经元中，恢复大于5%的总FMRP表达水平或大于20%的FMRP嵌合模式表达的神经元足以使FMRP依赖的、过度活跃的表型正常化。
Fragile X syndrome (FXS) is the most common genetic form of intellectual disability caused by a CGG repeat expansion in the 5 '-UTR of the Fragile X mental retardation gene FMR1, triggering epigenetic silencing and the subsequent absence of the protein, FMRP. Reactivation of FMR1 represents an attractive therapeutic strategy targeting the genetic root cause of FXS. However, largely missing in the FXS field is an understanding of how much FMR1 reactivation is required to rescue FMRP-dependent mutant phenotypes. Here, we utilize FXS patient-derived excitatory neurons to model FXS in vitro and confirm that the absence of FMRP leads to neuronal hyperactivity. We further determined the levels of FMRP and the percentage of FMRP-positive cells necessary to correct this phenotype utilizing a mixed and mosaic neuronal culture system and a combination of CRISPR, antisense and expression technologies to titrate FMRP in FXS and WT neurons. Our data demonstrate that restoration of greater than 5% of overall FMRP expression levels or greater than 20% FMRP-expressing neurons in a mosaic pattern is sufficient to normalize a FMRP-dependent, hyperactive phenotype in FXS iPSC-derived neurons.