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.

Centro de Investigaciones Biológicas Margarita Salas Madrid Spain

Department of Cellular Neurophysiology Institute of Experimental Medicine of the Czech Academy of Sciences Prague Czechia

Department of Translational Neuroscience University Medical Centre Utrecht Brain Centre Utrecht University Utrecht The Netherlands

Division of Metabolism University Children's Hospital Zurich University of Zurich Zurich Switzerland

Faculty of Science Charles University Prague Czechia

Florey Institute of Neuroscience and Mental Health Parkville Victoria Australia

Glial and Neuronal Biology Lab Department of Experimental Medical Science Faculty of Medicine Lund Stem Cell Center Lund University Lund Sweden

Institute of Biomedicine University of Gothenburg Gothenburg Sweden

ITINERARE Innovative therapies in rare diseases University Research Priority Program University of Zurich Zurich Switzerland

Laboratory of Astrocyte Biology and CNS Regeneration Center for Brain Repair Department of Clinical Neuroscience Institute of Neuroscience and Physiology Sahlgrenska Academy at the University of Gothenburg Gothenburg Sweden

Laboratory of Gene Expression Institute of Biotechnology of the Czech Academy of Sciences Vestec Czechia

Oncode Institute and Molecular Cancer Research Center for Molecular Medicine University Medical Center Utrecht Utrecht University Utrecht The Netherlands

Stem Cells Aging and Neurodegeneration Lab Department of Experimental Medical Science Faculty of Medicine Lund Stem Cell Center Lund University Lund Sweden

University of Newcastle Newcastle New South Wales Australia

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