Table_4_ZEB2 haploinsufficient Mowat-Wilson syndrome induced pluripotent stem cells show disrupted GABAergic transcriptional regulation and function.XLSX

Mowat-Wilson syndrome (MWS) is a severe neurodevelopmental disorder caused by heterozygous variants in the gene encoding transcription factor ZEB2. Affected individuals present with structural brain abnormalities, speech delay and epilepsy. In mice, conditional loss of Zeb2 causes hippocampal degeneration, altered migration and differentiation of GABAergic interneurons, a heterogeneous population of mainly inhibitory neurons of importance for maintaining normal excitability. To get insights into GABAergic development and function in MWS we investigated ZEB2 haploinsufficient induced pluripotent stem cells (iPSC) of MWS subjects together with iPSC of healthy donors. Analysis of RNA-sequencing data at two time points of GABAergic development revealed an attenuated interneuronal identity in MWS subject derived iPSC with enrichment of differentially expressed genes required for transcriptional regulation, cell fate transition and forebrain patterning. The ZEB2 haploinsufficient neural stem cells (NSCs) showed downregulation of genes required for ventral telencephalon specification, such as FOXG1, accompanied by an impaired migratory capacity. Further differentiation into GABAergic interneuronal cells uncovered upregulation of transcription factors promoting pallial and excitatory neurons whereas cortical markers were downregulated. The differentially expressed genes formed a neural protein-protein network with extensive connections to well-established epilepsy genes. Analysis of electrophysiological properties in ZEB2 haploinsufficient GABAergic cells revealed overt perturbations manifested as impaired firing of repeated action potentials. Our iPSC model of ZEB2 haploinsufficient GABAergic development thus uncovers a dysregulated gene network leading to immature interneurons with mixed identity and altered electrophysiological properties, suggesting mechanisms contributing to the neuropathogenesis and seizures in MWS.

莫瓦特-威尔逊综合征（Mowat-Wilson syndrome, MWS）是一种由编码转录因子ZEB2的基因发生杂合变异所导致的重型神经发育障碍。受累个体会出现脑结构异常、言语发育迟缓以及癫痫症状。在小鼠模型中，Zeb2的条件性敲除可引发海马体退行性变、γ-氨基丁酸能中间神经元（GABAergic interneurons）的迁移与分化异常——这类异质性群体主要为抑制性神经元，对维持正常神经兴奋性至关重要。为深入探究MWS中γ-氨基丁酸能神经元的发育与功能机制，本研究对MWS患者的ZEB2单倍体剂量不足诱导多能干细胞（induced pluripotent stem cells, iPSC）以及健康供体的诱导多能干细胞开展了对比分析。在γ-氨基丁酸能神经元发育的两个时间节点对RNA测序（RNA-sequencing）数据进行分析后发现，MWS患者来源的诱导多能干细胞中，中间神经元的成熟特征显著减弱，且富集了与转录调控、细胞命运转换以及前脑模式化相关的差异表达基因。ZEB2单倍体剂量不足的神经干细胞（neural stem cells, NSCs）表现出腹侧端脑特化相关基因的表达下调，例如FOXG1基因，同时其迁移能力受损。进一步向γ-氨基丁酸能中间神经元分化后，研究观察到促进背侧端脑与兴奋性神经元发育的转录因子表达上调，而皮层标志物的表达则出现下调。上述差异表达基因构成了一个神经源性蛋白质-蛋白质相互作用网络，且与已被证实的癫痫相关基因存在广泛关联。对ZEB2单倍体剂量不足的γ-氨基丁酸能神经元的电生理特性分析显示，其存在明显的功能异常，具体表现为重复动作电位发放能力受损。综上，本研究通过构建ZEB2单倍体剂量不足的γ-氨基丁酸能神经元发育模型，揭示了一条失调的基因网络，该网络会导致中间神经元发育不成熟且身份属性混杂，并伴随电生理特性改变，为阐释MWS的神经发病机制与癫痫发作的潜在致病机制提供了关键依据。