Investigating neuron intrinsic defects in 4H and Globoid Leukodystrophy 93 3 Significance of Transcriptomic Findings in 4H Neurons Transcriptomic analysis of 4H neurons revealed many significant differentially expressed genes between 4H and control neurons. Among the most significant upregulated genes was EFCC1, associated with calcium ion binding and osteogenesis imperfecta. So far, no apparent links with Pol III translation or leukodystrophies can be made. On the contrary, genes related to neural tube defects such as TRIM4, IRX1, 2 and 3 were differentially expressed and could be more easily linked to neurodevelopmental defects (Zhang et al., 2019). The gene RELN, an early marker for NDNF interneurons was differentially expressed, which is inline with previously established interneuron abnormalities in 4H (Dooves et al., 2023). GSEA analysis showed significant differences in many biological processes related to neuron signalling and development, reinforcing the presence of a neuronal phenotype in 4H neurons. From the cellular component gene sets many were significantly different between 4H and control. While these differences did not point to singular specific defect, it is interesting to note that they are mainly downregulated, potentially indicating an overall decline in neuronal functioning. This is however not captured by our morphological or axonal transport assessments, leading to the hypothesis that the transcriptional changes might precede measurable phenotypes in these parameters. Hence, this emphasizes the need for further research on more mature neurons to capture later-emerging defects. Although not adjusted for significance, hallmark gene set analysis revealed possible relevance of the upregulation of Notch and MTORC1 signalling. Those pathways are known to stimulate Pol III transcription and ribosome synthesis (Lee et al., 2012; Orellana et al., 2022; Wei et al., 2009). Similarly, upregulation of E2F target gene set was observed. E2F is thought to regulate the production of Pol III-dependent products through interaction with RB (Sizer et al., 2024). Together with the KEGG gene set analysis which shows upregulation of ribosome-related genes, these results suggest potential alterations in translation and Pol III transcription pathways. Lastly, hallmark gene set analysis shows the possible relevance of hedgehog signalling, previously linked to 4H pathology via ARX. Although, the targeting of the Shh pathway could not be confirmed as therapeutic target (Dooves et al., 2023), its recurrence in this analysis is intriguing. However, it is important to interpret this finding with caution, as this gene set consists of a small number of genes and a small percentage contributes to the observation.
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