Development of brain organoids from human induced pluripotent stem cells for studying the rare leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts

Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC) is a rare neurological disorder that belongs to astrocytopathies, a group of leukodystrophies distinguished by progressive cystic degeneration of myelin and mainly due to astrocyte dysfunctions. Disease features include cerebral oedema, the formation of subcortical cysts, myelin vacuolation, and astrocyte swelling, which together result in clinical manifestations such as macrocephaly, progressive cognitive and motor impairment featuring ataxia, spasticity, and epilepsy. The condition is primarily linked to mutations in the MLC1 gene, which encodes the MLC1 protein. In the human brain, this protein is almost exclusively localized at the astrocyte-astrocyte junctions and the perivascular astrocyte end-feet, where it is believed to play a crucial role in regulating cellular volume in response to physiological or pathological stimuli and consequently in controlling astrocyte activation. While the exact role of MLC1 and the mechanisms underlying MLC pathogenesis remain poorly understood, it is hypothesized that the resulting dysfunctional astrocytes may be incapable of properly supporting brain development. Indeed, astrocytes fulfil critical roles during neurodevelopment, including the regulation of neurogenesis, synaptogenesis, oligodendrocyte differentiation, myelination, and blood-brain barrier formation. In this study, we sought to investigate how MLC1 mutations impact early neurodevelopment by utilizing human brain organoids generated from human induced pluripotent stem cells (hiPSCs) derived from three healthy individuals and four MLC patients carrying mutations in the MLC1 gene. Western blotting, quantitative PCR, and immunostaining analyses confirmed that the organoids successfully developed the major cell populations of the central nervous system. This was evidenced by the expression of astrocyte markers (MLC1, SOX9, Vimentin, GFAP, Glutamine synthetase, EAAT1, EAAT2, Cx43, Kir4.1, and AQP4), neuronal markers (NEFH and MAP2), and oligodendrocyte markers (PDGFRA, CNPASE, OLIG2, and MBP). After 50 and 70 days of culture, organoids derived from MLC patients showed a reduced expression of some astrocyte-specific markers (MLC1, GFAP, Cx43, AQP4, Kir4.1, and SOX9) compared to controls, suggesting that the absence of functional MLC1 leads to defects in terminal differentiation and maturation of astrocytes. Furthermore, immunofluorescence staining of 50-day-old organoids revealed a downregulation of oligodendrocyte markers (PDGFRA and MBP). These findings point to a "cascade effect" where the primary dysfunction in astrocytes could negatively influence the differentiation of oligodendrocytes, which are essential for proper myelination. In conclusion, these hiPSC-derived brain organoids represent a sophisticated and powerful system for dissecting the pathophysiological mechanisms of MLC, marking a critical step toward the discovery of effective pharmacological treatments.