Towards a 3D spheroid system for modelling leukodystrophies 127 4 Disease specific differential gene expression in neuron lineage Lastly, we investigated the DEG in the neuronal lineage (Fig. 9 and 10). For GLD NPCs specifically, significant genes in both timepoints show to be mainly upregulated (Fig. 9A). Some genes that were differentially expressed in the oligo lineage are also downregulated in the neuron lineage, specifically DPP6 and CSMD1 (Fig. 9B-C). Similarly to NPC, also the inhibitory NP population shows mainly significant upregulated genes (Fig. 10A). MEG3 is upregulated in D100 and down regulated in D150 of SST and GABAergic neurons, and was upregulated in 4H oligo’s as well (Fig. 10B-C). MEG3 again is upregulated in 4H neuronal clusters as well (Fig. 9D-F). On the contrary, gene CHCHD2, observed to be downregulated in GLD oligo’s, is significantly upregulated in some 4H neuron clusters. For 4H, it also stands out that CHL1 is downregulated in all but the serotonergic cluster (Fig. 9D-F and Fig. 10D-F). In the inhibitory cluster it is interesting to note that SST, a gene related to subtypes of interneurons, is downregulated in 4H (Fig. 10D-F). Additionally, while 4H has shown quite consistent up and down regulation of significant genes, in the GABAergic cluster there seems to be less consistency (Fig. 10F). For CD, EBF1, EBF3 and GRIK2 are quite often upregulated (Fig. 9G-I and 10G-I). And while there is high consistency of down and upregulation of significant genes in 5HT neurons, there is quite some inconsistency again in the other neuronal clusters (Fig. 9G-I and 10G-I). Interestingly, in the NPC lineage there seems to be mainly upregulation of gene expression for GLD, while there is mainly down regulation in 4H and an inconsistent pattern in CD. This are potentially interesting clusters for future investigations (Fig. 9A, D, G). To conclude, there are many differential expressed genes. Future investigation should explore their potential involvement in disease processes in the different leukodystrophies diseases.
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