Chapter 3 88 Exploring Transcriptional Changes Using PCA To further explore neuronal changes at the molecular levels, we performed bulk RNA sequencing on neuron-mono cultures at day 25. Principal component analysis (PCA) revealed clear separation between control and 4H neurons (Supplementary Fig. 3A-C), while GLD samples did not clearly distinguish from controls and scattered throughout the plots (Supplementary Fig. 3A-B). The limited separation of GLD samples from other controls, made the differential gene expression analysis and gene set enrichment analysis unreliable. We tried to improve contrast by removing KO X19 from the analysis, as this seemed to be the outlier. However, this did not resolve the issue (Supplementary Fig. 3B). Hence, we proceed to discuss only the analysis for 4H compared to control (Supplementary Fig. 3C). In 4H neurons, we identified 575 differentially expressed genes (DEGs) compared to controls (Fig. 3A). Among these, 304 genes were downregulated, including 136 with log2 fold change less than -1, and 271 genes were upregulated, with 246 showing log2 fold change greater than 1. Notably, the gene TRIM4, a gene related to neural tube defects and associated with GO-terms for axons, was most significant downregulated (Zhang et al., 2019). Other interesting downregulated genes are SLC17A7 (Vglut1) which is synapse related, RELN which is linked to interneurons and GO-terms for neuronal migration (Yu et al., 2021) and LHX2 which is important for neural development (Chou & Tole, 2019). Conversely, EFCC1, which is related to GO-terms for calcium binding, is most significant upregulated. Also IRX1, IRX2, IRX3, NKX1-2, and NKX2-2 were significantly upregulated indicating potential dysregulation in pathways associated with neuronal tube patterning and neurogenesis (Bosse et al., 1997). Pathway analysis reveal 4H neuronal phenotypes To get a completer and more unbiased overview we performed Gene Set Enrichment Analysis (GSEA). Significant discoveries have an adjusted P-value of < 0.05. However, as this dataset was intended to be exploratory and to assist the formation of new hypothesis and directions for future research, we investigated all findings with an adjusted P < 0.25. Gene ontology (GO) analysis of DEGs revealed downregulation in several neuron-related biological processes, including synaptic signalling, regulation of transsynaptic signalling, pallium and cerebral cortex development and regionalization (Fig. 3B). Specifically, 5 out of 7 genes associated with cerebral cortex regionalization contribute to the finding, strongly suggesting impaired cortical development. Surprisingly, upregulated pathways were often kidney related, which is not meaningful considering our investigation of neuron-mono cultures. However, some genes associated with kidney development such as IRX1, 2 and 3,
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