iPSC-derived models of PACS1 syndrome reveal transcriptional and functional deficits in neuron activity

PACS1 syndrome is a neurodevelopmental disorder characterized by intellectual disability and distinct craniofacial abnormalities resulting from a de novo p.R203W variant in phosphofurin acidic cluster sorting protein 1 (PACS1). PACS1 is known to play roles in the endosomal pathway and nucleus, but how the p.R203W variant affects developing neurons is not fully understood. In this study we differentiated stem cells towards 2D and 3D neuronal models to investigate the impact of the PACS1 syndrome-causing variant on neurodevelopment. While few deleterious effects were detected in PACS1(+/R203W) neural precursors, mature PACS1(+/R203W) glutamatergic neurons in cortical organoids exhibited impaired expression of genes involved in synaptic signaling processes. The single-cell RNA sequencing data from dorsal cortical organoids after 40 and 88 days of differentiation can be found here. Subsequent characterization of neural activity using calcium imaging and multielectrode arrays revealed the p.R203W PACS1 variant leads to a prolonged neuronal network burst duration mediated by an increased inter-spike interval. These findings demonstrate the impact of the PACS1 p.R203W variant on developing human neural tissue and uncover putative electrophysiological underpinnings of disease. Five day 40 (1 Control 1, 1 Control 3, 1 CRISPR R203W, 1 Affected 1, and 2 combined Affected 2) and four day 88 (2 Control 2, 1 CRISPR R203W, and 1 Affected 1) cortical organoids across four independent differentiations were cultured and processed for single-cell RNA sequencing analysis.PACS1综合征是一种神经发育障碍，由磷酸弗林酸性簇分选蛋白1（phosphofurin acidic cluster sorting protein 1, PACS1）的新发p.R203W变异所致，以智力障碍与特征性颅面部畸形为核心表型。已知PACS1参与内体通路与细胞核的生理功能，但p.R203W变异如何调控发育中神经元的功能尚未完全明确。本研究通过将干细胞定向分化为二维（2D）与三维（3D）神经元模型，探究该PACS1综合征致病变异对神经发育的影响。尽管在PACS1(+/R203W)型神经前体细胞中仅检测到少量有害效应，但皮层类器官内的成熟PACS1(+/R203W)型谷氨酸能神经元，其参与突触信号传导过程的基因表达却呈现显著受损状态。分化40天与88天后的背侧皮层类器官的单细胞RNA测序（single-cell RNA sequencing）数据可于此处获取。后续通过钙成像与多电极阵列对神经活动进行表征后发现，p.R203W型PACS1变异可导致峰间期延长所介导的神经元网络爆发时长增加。本研究结果阐明了PACS1 p.R203W变异对人类发育神经组织的影响，并揭示了该疾病潜在的电生理学发病基础。本研究共开展4次独立分化实验，所培养并用于单细胞RNA测序分析的样本包括：5例分化40天的皮层类器官（1例对照1、1例对照3、1例CRISPR编辑R203W样本、1例受累1样本，以及2例合并受累2样本），以及4例分化88天的皮层类器官（2例对照2、1例CRISPR编辑R203W样本、1例受累1样本）。