Appendix 234 transcribed transcript levels, were unchanged in 4H cells. Notably, patient-specific genetic backgrounds were found to have a significant impact on POLR3A expression. These results underscore the necessity of considering individual genetic backgrounds and specific developmental cell states when investigating the pathology of 4H leukodystrophy. The last chapter, the discussion, integrates findings across model systems to show that 4H leukodystrophy involves neuron-intrinsic dysfunction, particularly interneurons. While the Shh pathway may contribute to pathology, other molecular processes—including mTORC1 signalling and oxidative phosphorylation—also emerge as candidates for 4H but also GLD and CD. The thesis underscores the strengths and limitations of mono-culture, co-culture, and spheroid systems, emphasizing the need for strategic model selection and study design. Finally, the work advocates for precision medicine approaches that combine patientderived iPSCs, transcriptomics, and functional assays to identify new therapeutic targets and screening platforms. In conclusion this thesis advances the understanding of 4H leukodystrophy by demonstrating alterations in neurons - particularly interneurons – in 4H. It introduces and validates a range of iPSC-derived in vitro models, including spheroids and microglia cocultures, as powerful tools for dissecting leukodystrophy mechanisms, while emphasizing on careful selection of suitable in vitro models. By revealing candidate pathological pathways this work lays a foundation for future therapeutic development in 4H and related disorders.
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