Impaired neuronal activity and differential gene expression in STXBP1 encephalopathy patient iPSC-derived GABAergic neurons
Impaired neuronal activity and differential gene expression in STXBP1 encephalopathy patient iPSC-derived GABAergic neurons
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DOI：
10.1093/hmg/ddab113
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发表时间：
2021-05-07
影响因子：
3.5
通讯作者：
Hirose, Shinichi
中科院分区：
文献类型：
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作者：
Ichise, Eisuke;Chiyonobu, Tomohiro;Hirose, Shinichi
合成素结合蛋白1(STXBP1；又称Munc18-1)由其编码，是控制突触小泡停靠和融合的分子机制的重要组成部分。STXBP1的从头开始致病变异会导致一系列复杂的神经障碍，即STXBP1脑病(STXBP1-E)，包括癫痫、神经发育障碍和神经退行性变。一些动物研究表明，GABA能功能障碍在STXBP1-E的发病机制中起重要作用。然而，这些患者的GABA能神经元的病理生理变化仍然知之甚少。在这里，我们通过瞬时表达转录因子ASCL1和DLX2，从STXBP1-E患者来源的诱导多能干细胞(IPSCs)中独家生成GABA能神经元。我们还产生了CRISPR/Cas9编辑的IPSC衍生的GABA能(IPSC GABA)等基因神经元作为对照。我们发现，患者来源的IPSC GABA神经元中的STXBP1蛋白水平与对照神经元相比略有下降(约20%)，尽管STXBP1 mRNA水平下降了50%。使用基于微电极阵列的分析，我们发现患者来源的iPSC GABA神经元表现出功能障碍的成熟，自发尖峰和猝发的数量减少。这些发现强化了这样的观点，即GABA能功能障碍是STXBP1-E发病的关键因素。此外，基因表达分析显示，先前与癫痫、神经发育和神经变性有关的基因在患者来源的iPSC GABA神经元中存在特定的失调，即KCNH1、KCNH5、CNN3、RASGRF1、SEMA3A、SIAH3和INPP5F。因此，我们的研究为理解STXBP1-E广泛神经病理特征背后的生物学过程提供了新的见解。
Syntaxin-binding protein 1 (STXBP1; also called MUNC18-1), encoded by STXBP1, is an essential component of the molecular machinery that controls synaptic vesicle docking and fusion. De novo pathogenic variants of STXBP1 cause a complex set of neurological disturbances, namely STXBP1 encephalopathy (STXBP1-E) that includes epilepsy, neurodevelopmental disorders and neurodegeneration. Several animal studies have suggested the contribution of GABAergic dysfunction in STXBP1-E pathogenesis. However, the pathophysiological changes in GABAergic neurons of these patients are still poorly understood. Here, we exclusively generated GABAergic neurons from STXBP1-E patient-derived induced pluripotent stem cells (iPSCs) by transient expression of the transcription factors ASCL1 and DLX2. We also generated CRISPR/Cas9-edited isogenic iPSC-derived GABAergic (iPSC GABA) neurons as controls. We demonstrated that the reduction in STXBP1 protein levels in patient-derived iPSC GABA neurons was slight (approximately 20%) compared to the control neurons, despite a 50% reduction in STXBP1 mRNA levels. Using a microelectrode array-based assay, we found that patient-derived iPSC GABA neurons exhibited dysfunctional maturation with reduced numbers of spontaneous spikes and bursts. These findings reinforce the idea that GABAergic dysfunction is a crucial contributor to STXBP1-E pathogenesis. Moreover, gene expression analysis revealed specific dysregulation of genes previously implicated in epilepsy, neurodevelopment and neurodegeneration in patient-derived iPSC GABA neurons, namely KCNH1, KCNH5, CNN3, RASGRF1, SEMA3A, SIAH3 and INPP5F. Thus, our study provides new insights for understanding the biological processes underlying the widespread neuropathological features of STXBP1-E.