Introduction 21 1 Less invasive treatment options such as drug therapies that target disrupted pathways could potentially slow or halt disease progression. Identifying such pathways and screening for compounds that alter the pathways requires realistic in vitro models that accurately replicate 4H pathology—models that are currently lacking. By establishing such models, this thesis aims to close this gap, facilitating both investigating of therapeutic targets as well as development of models that might be used in future therapeutic screening. This does not only apply to 4H, but might also be used for other leukodystrophies. THESIS OUTLINE In this thesis we aim 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. To address this question and also to unravel patient-specific genetics contributing to broad clinical presentations, we used iPSC lines from patients with either POLR3A or POLR3B variants, that are exonic or intronic and which are relatively common in the 4H patient population (Figure 4). To investigate if our findings are unique to 4H leukodystrophy or could be translated to other leukodystrophies, some studies also include iPSCs of other leukodsytrophies. Here we provide the research questions, approaches and main results of the following experimental chapters: Chapter 2: First we explored what genes are dysregulated in brain specific cell types in 4H patient cultures using patient iPSC technology in combination with RNAseq analysis on iPSC-derived cerebellar cells. Downregulation of ARX, a gene involved in early brain and interneuron development gave first suggestions to further explore interneuron dysfunction in 4H leukodystrophy. In cortical neuron cultures containing both glutamatergic and GABAergic neuron populations, we confirmed the downregulation of ARX in 4H and a reduction in GABAergic synapses. Functional analysis by MEA further pointed towards alterations in the inhibitory neuron population. Initial analysis points to no obvious intrinsic changes in the oligodendrocyte population. Since ARX is an important regulator of SHH gradients, we investigated whether targeting the SHH pathway could improve 4H associated GABAergic phenotypes, but no improvements were observed in our assays. In conclusion, we identified cortical interneuron development to be affected in 4H leukodystrophy.
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