GGC重复扩增体现在NOTCH2NLC的5’非翻译区(UTR)中，与广泛的神经系统疾病有关，尤其是神经元核内内含体病(NIID)。研究发现，NOTCH2NLC中的GGC重复扩增可以诱导形成含有聚甘氨酸(polyG)的蛋白质，该蛋白质参与了神经内部核内包涵体的形成。然而，NOTCH2NLC GGC重复扩增引起的神经毒性机制还不清楚。在这里，我们使用NIID患者特异性的iPSC来源的3D脑器官(3DCOs)和细胞模型来研究NOTCH2NLC GGC重复扩增的病理生理机制。iPSC来源的3DCOs和细胞模型显示有聚甘氨酸含量的核内包涵体的沉积。NOTCH2NLC GGC重复扩增可能引起自噬流的升高、集成应激反应的增强和EIF2α磷酸化的激活。iPSC来源的神经元的大规模RNA测序和iPSC来源的3DCOs的单细胞RNA测序(scRNA-seq)显示，NOTCH2NLC GGC重复扩增可能与核糖体生物合成和翻译功能障碍有关。此外，NOTCH2NLC GGC重复扩增可能引起NPM1核质转位,增加核仁应激,损伤核糖体生物合成和诱导核糖体RNA(rRNA)的离子束固定，提示NIID细胞模型中膜无器官功能障碍。核糖体生物合成和磷酸化EIF2α的功能障碍以及由此导致G3BP1阳性应激颗粒的形成，可能会共同导致全细胞翻译抑制，最终导致细胞死亡。有趣的是，scRNA-seq显示在3DCOs发育过程中，NOTCH2NLC GGC重复扩增可能与不成熟神经元的比例显著降低有关。综上，我们的研究结果强调了患者特异性iPSC来源的3DCOs在研究聚甘氨酸疾病的机制中的价值，特别是人类特异性基因重复引起的疾病。© 作者(们)2023年。由牛津大学出版社代表Brain基金会保证。所有权利均受保护。请发送电子邮件至journals.permissions@oup.com以获取授权。

GGC repeat expansion in the 5' untranslated region (UTR) of NOTCH2NLC is associated with a broad spectrum of neurological disorders, especially neuronal intranuclear inclusion disease (NIID). Studies have found that GGC repeat expansion in NOTCH2NLC induces the formation of polyglycine (polyG)-containing protein, which is involved in the formation of neuronal intranuclear inclusions. However, the mechanism of neurotoxicity induced by NOTCH2NLC GGC repeats is unclear. Here, we used NIID patient-specific iPSC-derived 3D cerebral organoids (3DCOs) and cellular models to investigate the pathophysiological mechanisms of NOTCH2NLC GGC repeat expansion. IPSC-derived 3DCOs and cellular models showed the deposition of polyG-containing intranuclear inclusions. The NOTCH2NLC GGC repeats could induce the upregulation of autophagic flux, enhance integrated stress response, and activate EIF2α phosphorylation. Bulk RNA sequencing for iPSC-derived neurons and single-cell RNA sequencing (scRNA-seq) for iPSC-derived 3DCOs revealed that NOTCH2NLC GGC repeats may be associated with dysfunctions in ribosome biogenesis and translation. Moreover, NOTCH2NLC GGC repeats could induce the NPM1 nucleoplasm translocation, increase nucleolar stress, impair ribosome biogenesis, and induce ribosomal RNA (rRNA) sequestration, suggesting dysfunction of membraneless organelles in the NIID cellular model. Dysfunctions in ribosome biogenesis and phosphorylated EIF2α and the resulting increase in the formation of G3BP1-positive stress granules may together lead to whole-cell translational inhibition, which may eventually cause cell death. Interestingly, scRNA-seq revealed that NOTCH2NLC GGC repeats may be associated with a significantly decreased proportion of immature neurons while 3DCOs were developing. Together, our results underscore the value of patient-specific iPSC-derived 3DCOs in investigating the mechanisms of polyG diseases, especially those caused by repeats in human-specific genes.© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.