Investigating neuron intrinsic defects in 4H and Globoid Leukodystrophy 89 3 are also associated with embryonic morphogenesis and regionalization in general. In the biological process pathways there are also many non-significant but possible relevant mechanisms. Here we see the same division of downregulated neuron-related biological processes and upregulated morphogenesis and embryo development pathways (Supplementary Fig. 4). GO analysis cellular components identified significant downregulation in genes related to neuronal compartments such as the dendritic tree, somatodendritic compartment, glutamatergic synapse and neuron-to-neuron synapse. Also the non-significant but possibly interesting findings (P < 0.025) mainly involve neuron compartments (Fig. 3C). We did not find any significant upregulated pathways, however for the ribosomal subunits a large proportion of genes in this gene set (58% of 171) contribute to the findings, which suggests there might still be an increase in protein synthesis machinery. For molecular functions, only upregulation of DNA binding transcription repressor activity reached adjusted significance, and only GPCR activity was within our <0.25 cutoff (Fig. 3D). Hallmark analysis identified trends in pathways related to Notch signalling, Hedgehog signalling and E2F targets, though none reached statistical significance after adjustment (Fig. 3E). KEGG analysis highlighted ribosomal pathways with high gene set enrichment (72% of 86 genes), though again not significant after adjusting for multiple testing. Notch signalling reappeared in this analysis, potentially indicating disrupted developmental signalling relevant to 4H pathology. Summarized, bulk RNA sequencing of 4H and GLD neurons in neuron-mono culture conditions identified clear transcriptional differences in 4H. In 4H, we observed downregulation of neuronal processes related to synaptic signalling and cortical development, alongside upregulation of developmental pathways and ribosomal components. Pathway analysis in 4H further indicated potential dysregulation in Notch signalling and protein synthesis, underscoring molecular distinctions specific to 4H.
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