Gaining insight into the pathology of 4H leukodystrophy using patient-specific iPSC-based models

4H leukodystrophy is a rare genetic disorder characterized by hypomyelination, hypodontia, and hypogonadotropic hypogonadism. Today treatments for 4H are solely symptomatic rather than curative. In this thesis we aimed to gain insight into how molecular and cellular mechanisms contribute to the pathology of 4H leukodystrophy using patient-specific iPSC-based models with the goal to facilitate development of new therapeutic strategies. In Chapter 2 we explored differential expression of genes in 4H cerebellar cells using patient iPSC technology in combination with RNAseq analysis. This revealed downregulation of ARX, a transcription factor required for interneuron development. Focused investigation of cortical neuron co-cultures showed showed a reduction in GABAergic synapses and elevated network activity in 4H neurons. Interestingly, myelination appeared normal. Expression of the parvalbumin-related gene ERBB4 was increased, suggesting disrupted development of specific interneuron subtypes. Taken together, we concluded that cortical interneuron development is affected in 4H leukodystrophy. In Chapter 3 we further explored neuronal phenotypes in 4H leukodystrophy. Additionally, we aimed to explore whether neuronal changes are 4H-specific. Hence control, 4H and GLD neurons were compared. Surprisingly, the in vitro cultures with GLD neurons did not show accumulation of psychosine, a hallmark of the disease. This highlights the heterogeneity of leukodystrophies and the necessity of refining disease 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 introduces a 3D brain spheroid platform to model 4H, GLD, and Canavan Disease. Spheroids exhibited robust cellular diversity and formed compact myelin. 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. Therefore, Chapter 5 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. Interestingly, addition of microglia from adrenoleukodystrophy patients caused different axonal changes compared to the addition of 4H and control microglia. These findings demonstrate the importance of including microglia to 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. 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-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, integrates findings across chapters and model systems. And concludes that 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, 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.