Chapter 7 216 (Iulianella & Stanton-Turcotte, 2019). Other evidence of the involvement of Shh in 4H leukodystrophy, is found in the gene set enrichment analysis (GSEA) of spheroid single cell mRNA sequencing. Here, the gene set “Shh signalling” is downregulated in several neuron cell clusters (Chapter 4), including glutamatergic neurons and SST-expressing GABAergic neurons. While dysregulation of ARX was not consistent across all experiments, Shh signalling appears to be relevant as shown by independent experiments. Until now, we did not successfully interfere with the pathway to ameliorate 4H disease phenotypes. Hence, future research should investigate the therapeutic potential of targeting these pathways and assess their interactions. 1.3 Other molecular pathways Since reduced ARX expression pointed us to the possible involvement of the Shh pathway, it is tempting to try to relate every new finding to this hypothesis. However, other assays revealed significant genes, that could also be linked to other pathways. For example, while CHL1 contributes to Shh-related processes, it can also trigger PTCH1-, SMO-, RhoA- and ROCK-dependent signal transduction pathways in cerebellar development and promote neuronal survival (Katic et al., 2017). Besides hedgehog signalling, GSEA analysis of cortical neurons showed possible relevance of mTORC1 signalling (Chapter 2). Interestingly, this pathway is also often significant in cell clusters in the single cell data (Chapter 4). In this dataset, it is also intriguing that oxidative phosphorylation is highly significant in many clusters in all leukodystrophies (Chapter 4). The diversity of these findings do not point towards one disease mechanism involved in 4H. This highlights the need for integrative pathway analyses to identify converging nodes of dysfunction and potential points of therapeutic intervention. Expanding the focus to these broader molecular networks could help uncover novel targets beyond Shh signalling, addressing unresolved aspects of neuronal pathology in 4H. In summary, our research on different in vitro models of 4H provides a foundation for understanding the intrinsic neuronal defects in 4H leukodystrophy, highlighting specific vulnerabilities in interneurons, potential dysregulation of the Shh pathway, and the involvement of broader molecular networks. Future research should prioritize integrative approaches that investigate whether these pathways are causal in 4H pathology or a consequence. Identifying key drivers of 4H pathology will be critical for guiding the development of effective targeted therapies.
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