Lu Rongrong, Xu Ying, Li Hao, Xiong Man, Zhou Wenhao, Feng Weijun, Zhao Rui
Department of Neurosurgery, National Children's Medical Center (Shanghai), Children's Hospital of Fudan University, No.399 Wan Yuan Avenue, Minhang District, Shanghai, 201102, China.
Institute of Pediatrics, Shanghai Key Laboratory of Medical Epigenetics, International Co-Laboratory of Medical Epigenetics and Metabolism, Institutes of Biomedical Sciences, Children's Hospital of Fudan University, Shanghai Medical College, Fudan University, Shanghai, 200032, China.
J Mol Neurosci. 2024 Dec 27;75(1):3. doi: 10.1007/s12031-024-02304-5.
Focal cortical dysplasia (FCD) II is a cortical malformation characterized by cortical architectural abnormalities, dysmorphic neurons, with or without balloon cells. Here, we systematically explored the pathophysiological role of the GATOR1 subunit NPRL3 variants including a novel mutation from iPSCs derived from one FCD II patient. Three FCD II children aged 0.5-7 years who underwent cerebral lesion resection in our hospital from March 2019 to October 2019 were included in this study. We generated patient-derived iPSCs and performed whole-exome sequencing to accurately identify somatic cells with mutations. The effect of the newly identified NPRL3 mutation found in one of our FCD II patients was evaluated using the personalized cortical organoid model and the NPRL3 knockout HEK293T cells. Whole-exome sequencing of iPSCs derived from FCD II patients revealed a novel NPRL3 C.767G > C (p.R256P) heterozygous mutation. Cortical organoids generated from iPSCs of FCD II patients were larger than control iPSCs, with increased number of p-S6 cells and NeuN neurons. In NPRL3 knockout HEK293T cells, overexpression of NPRL3 together with NPRL2 protein is necessary to reduce p-S6 level upon amino acid starvation. The reduced binding between NPRL3 and NPRL2 protein leads to downregulation of the relative total protein amount of both proteins in the cell. Our study describes a novel cortical organoid model generated from iPSCs of the FCD patients to investigate the underlying mechanism of NPRL3-related epilepsy. The mutation of NPRL3 impaired the function of NPRL3 protein via affecting the binding with NPRL2 protein, which resulted in unstable protein monomer.
局灶性皮质发育不良（FCD）II型是一种皮质畸形，其特征为皮质结构异常、神经元异型，伴有或不伴有气球样细胞。在此，我们系统地探究了GATOR1亚基NPRL3变体的病理生理作用，包括来自一名FCD II型患者的诱导多能干细胞（iPSC）中的一种新突变。本研究纳入了2019年3月至2019年10月在我院接受脑病变切除术的3名年龄在0.5至7岁的FCD II型儿童。我们生成了患者来源的iPSC，并进行全外显子测序以准确鉴定有突变的体细胞。使用个性化皮质类器官模型和NPRL3基因敲除的HEK293T细胞评估了在我们的一名FCD II型患者中发现的新鉴定的NPRL3突变的影响。对来自FCD II型患者的iPSC进行全外显子测序，发现了一种新的NPRL3基因C.767G＞C（p.R256P）杂合突变。FCD II型患者的iPSC生成的皮质类器官比对照iPSC更大，p-S6细胞和NeuN神经元数量增加。在NPRL3基因敲除的HEK293T细胞中，在氨基酸饥饿时，NPRL3与NPRL2蛋白一起过表达对于降低p-S6水平是必要的。NPRL3与NPRL2蛋白之间结合减少导致细胞中这两种蛋白的相对总蛋白量下调。我们的研究描述了一种由FCD患者的iPSC生成的新型皮质类器官模型，用于研究NPRL3相关癫痫的潜在机制。NPRL3突变通过影响与NPRL2蛋白的结合而损害了NPRL3蛋白的功能，导致蛋白单体不稳定。
