Appendix 233 & models. Nevertheless, the models showed transcriptomic changes in 4H neurons, specifically downregulation of genes related to synaptic and cortical development and upregulation of genes related to morphogenesis and ribosomal genes. This reconfirmed a role for neuronal dysfunction in 4H and can be used to determine focus for future research. To further explore differences between leukodystrophies and mechanisms underlying 4H specific hypomyelination Chapter 4: Towards a 3D spheroid system for modelling leukodystrophies introduces a 3D brain spheroid platform derived from iPSCs to model 4H, GLD, and Canavan Disease (CD). Spheroids exhibited robust cellular diversity, including neurons, astrocytes, and oligodendrocytes, and formed compact myelin as shown by immunofluorescence and electron microscopy. Single-cell RNA sequencing identified 27 cell clusters. Interestingly, with a consistent underrepresentation of cycling radial glia and neural progenitors across leukodystrophies. We report differential expressed genes and significantly different gene sets that can guide future research directions. Although the spheroids encompass diverse cell types that mimic the in vivo brain, the model did not include microglia, while those cells are increasingly recognized as key players in brain development and disease. Therefore, Chapter 5: Human pluripotent stem cellderived microglia shape neuronal morphology and enhance network activity in vitro describes a co-culture system using hPSC-derived microglia and neurons. We showed that the microglia incorporate into the cultures where they reduced nuclear debris and altered neuronal morphology by decreasing axonal and dendritic segments and reducing synapse density. Despite the decrease in synapse density, neuronal network activity increased. Interestingly, addition of microglia from adrenoleukodystrophy (ALD) patients caused different axonal changes compared to the addition of 4H and control microglia. These findings demonstrate the importance of including microglia to more accurately model neuron–glia interactions in disease. To explore how patient-specific genetic variants affect POLR3 gene and protein expression during neuronal lineage differentiation in 4H leukodystrophy, we examined POLR3 expression levels, protein localization, and developmental dynamics across iPSCs, neural epithelial cells (NES) and neurons in Chapter 6: POLR3 gene and protein expression dynamics in 4H leukodystrophy using iPSC-derived neuronal lineages. We identified elevated POLR3 gene expression in NES. However, Pol III protein levels were notably reduced in 4H patient cells. Despite these protein-level alterations, overall Pol III-
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