Astrocyte dysfunction in autosomal dominant leukodystrophy: morphofunctional insights from in vitro and ex vivo systems

Autosomal Dominant Leukodystrophy (ADLD) is a rare, fatal neurological disorder primarily caused by LMNB1 gene alterations, leading to progressive central nervous system demyelination. Despite knowledge of its genetic basis, the absence of reliable human models has limited mechanistic insight and biomarker discovery. The present study aimed to dissect ADLD pathophysiology across multiple biological systems, using complementary cellular, organoid, and ex vivo approaches. Human primary astrocytes overexpressing LMNB1 were generated to investigate morphofunctional alterations. These cells exhibited NF-κB and NFAT4 nuclear translocation, increased IL-9 secretion, and ultrastructural nuclear anomalies, indicating astrocyte activation and nuclear vulnerability. When co-cultured with oligodendrocyte precursor cells (OPC) on aligned microfiber scaffolds, LMNB1-astrocytes impaired OPC survival and impaired myelination, demonstrating the critical role of astrocytic health in supporting oligodendrocyte function. Patient-derived human induced pluripotent stem cell (hiPSC) models provided complementary insights. In 2D cultures, ADLD astrocytes displayed reduced nuclear circularity and increased aspect ratio, while neurons remained preserved. Similar nuclear alterations were confirmed in astrocytes within 3D cerebral organoids, reinforcing astrocytic susceptibility across experimental systems. Calcium live imaging revealed reduced spontaneous calcium transient amplitude and increased firing frequency in ADLD astrocytes, further supporting functional impairment, whereas neurons did not show significant differences. Extending to ex vivo validation, cerebrospinal fluid (CSF) analyses identified elevated IGFBP-2, TIMP-2, and reduced IGFBP-6, alongside metabolic alterations detected by Nuclear Magnetic Resonance (NMR) metabolomics. Increases in lactate, alanine and pyruvate pointed to disruptions of the lactate–alanine shuttle and astrocyte–neuron metabolic coupling. Together, these findings establish astrocytic dysfunction as a central driver of ADLD across nuclear, functional, and metabolic domains. By linking in vitro and ex vivo observations through cellular, organoid, and CSF analyses, the study provides a translational framework for mechanistic insight, biomarker discovery, and the advancement of precision medicine–oriented therapeutic strategies.