Chapter 4 132 sequencing work-up. For this, it might be useful to use bulk mRNA sequencing and search for differential expression of genes that are indicative for the underrepresented clusters. Interestingly, the underrepresentation of radial glia is a common feature in these LDs. This is an interesting lead for future research as shared pathology among leukodystrophies could mean that future therapeutic strategies developed for one leukodystrophy might be repurposed for others, potentially broadening the clinical applicability of future treatments. Disease-specific differential gene expression In addition to common mechanisms, the spheroid models were used to study disease specific mechanisms. A minimal requirement for the spheroids to serve as a disease model for LDs is the expression of disease genes, which was indeed confirmed. Specifically, 4H leukodystrophy patients have mutations in subunits of POLR3. As expected, the POLR3 subunits where broadly expressed in all cell types. Further DEG analysis revealed a number of interesting changes in 4H 3D spheroids. For example, the lncRNA Maternally Expressed Gene 3 (MEG3) shows upregulation in the different neuron cellular clusters of 4H cells. Previous studies showed that although MEG3 is expressed throughout the brain, it shows an especially high expression in the cerebellum and pituitary gland (Zhang et al., 2003). Interestingly, 4H patients often show cerebellar atrophy and hormonal issues associated with pituitary gland dysfunction (Wolf et al., 2014). Previous studies showed that upregulation of MEG3 improves cognitive impairment, alleviates neuronal damage, and inhibits activation of astrocytes in hippocampus tissues in Alzheimer's disease through inactivating the PI3K/Akt signalling pathway (Yi et al., 2019; Zhang et al., 2020). It is possible that the observed upregulation of MEG3 is an effect of neuronal stress, which is in line with the observed upregulation of neuroprotective peptide Y (NPY) in certain 4H neuronal clusters (Zhang et al., 2021). Disease-specific gene set enrichment analysis We have performed extensive, gene set enrichment analysis (GSEA) which revealed ample pathway dysregulations. Those findings provide a base for validation of existing diseasespecific hypothesis or the formation of new hypothesis to determine the direction of future research. However, the pathway findings warrant further investigation before publication as they are sensitive to bias. To exemplify, initially we discovered the pathway “translation” often to be differentially expressed in 4H compared to control. This aligned well with our hypothesis that POLR3A variants cause changes in translation, however, “translation” was also often significantly different in the other leukodystrophies. Although, “translation”
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