亚历山大病 (AxD) 是一种罕见且严重的神经退行性疾病，由胶质纤维酸性蛋白 (GFAP) 突变引起。虽然确切的疾病机制仍不清楚，但先前的研究表明突变型 GFAP 影响许多细胞过程，包括细胞骨架稳定性、机械传感、代谢和蛋白酶体功能。虽然大多数研究主要集中在表达 GFAP 的星形胶质细胞上，但放射状胶质细胞和神经祖细胞也表达 GFAP，这引发了关于 GFAP 突变对中枢神经系统 (CNS) 发育影响的问题。在这项研究中，我们观察到星形胶质细胞和神经元的共培养物以及神经类器官中星形胶质细胞和神经元的分化受损，这两种细胞器均由 AxD 患者衍生的具有 GFAPR239C 突变的诱导多能干 (iPS) 细胞产生。利用单细胞 RNA 测序 (scRNA-seq)，我们鉴定了突变 GFAP 培养物和校正的同基因对照之间的不同细胞群和转录组差异。这些发现得到了免疫细胞化学和蛋白质组学结果的支持。在共培养中，GFAPR239C 突变导致未成熟细胞丰度增加，而在无引导的神经类器官和皮质类器官中，我们观察到谱系定向改变和星形胶质细胞丰度减少。基因表达分析显示，AxD 培养物中的应激敏感性增加、细胞骨架异常、细胞外基质和细胞间通讯模式改变，并且应激后细胞死亡率更高。总体而言，我们的结果表明 AxD 患者来源的 iPS 细胞模型中的细胞分化发生了改变，为 AxD 研究开辟了新途径。© 2024 作者。 GLIA 由 Wiley periodicals LLC 出版。

Alexander disease (AxD) is a rare and severe neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP). While the exact disease mechanism remains unknown, previous studies suggest that mutant GFAP influences many cellular processes, including cytoskeleton stability, mechanosensing, metabolism, and proteasome function. While most studies have primarily focused on GFAP-expressing astrocytes, GFAP is also expressed by radial glia and neural progenitor cells, prompting questions about the impact of GFAP mutations on central nervous system (CNS) development. In this study, we observed impaired differentiation of astrocytes and neurons in co-cultures of astrocytes and neurons, as well as in neural organoids, both generated from AxD patient-derived induced pluripotent stem (iPS) cells with a GFAPR239C mutation. Leveraging single-cell RNA sequencing (scRNA-seq), we identified distinct cell populations and transcriptomic differences between the mutant GFAP cultures and a corrected isogenic control. These findings were supported by results obtained with immunocytochemistry and proteomics. In co-cultures, the GFAPR239C mutation resulted in an increased abundance of immature cells, while in unguided neural organoids and cortical organoids, we observed altered lineage commitment and reduced abundance of astrocytes. Gene expression analysis revealed increased stress susceptibility, cytoskeletal abnormalities, and altered extracellular matrix and cell-cell communication patterns in the AxD cultures, which also exhibited higher cell death after stress. Overall, our results point to altered cell differentiation in AxD patient-derived iPS-cell models, opening new avenues for AxD research.© 2024 The Author(s). GLIA published by Wiley Periodicals LLC.