Fig. 3: The TARDBP M337V mutation results in transcriptomic dysregulation unique to motor neurons.

a Schematic of the generation of the TARDBP M337V mutant iPSC line, differentiation into motor neurons and subsequent single-cell sorting and RNA-sequencing. b UMAP projections of all single cells in the study that passed quality control. The two UMAPs are colored by assigned cell type (left) or cell line (right). c UMAP projections of all neuronal cells in the study that passed quality control, again colored by assigned cell type (left) or cell line (right). d TDP-43 is mainly nuclear in the TARDBP M337V line. Confocal fluorescence microscopy with immunostaining of antibodies against TDP-43 and phospho-TDP-43 (S409/S410) in motor neurons (TUBB3 + , ISL1/2 + ) at day 28. Scale bars are 10 µm. e, f Violin plots for the quantitative immunofluorescence measurements of intracellular TDP-43 levels and phospho-TDP-43 levels in ISL1⁺ TUBB3+ motor neurons. The nucleocytoplasmic ratio for TDP-43 was calculated and shown in (e) and the cytoplasmic levels of phospho-TDP-43 (S409/S410) is shown in (f). The dots with black represent the average per independent differentiation (n = 3), while other dots represent individual well averages. The p-values were obtained from two-sided Wilcoxon rank sum test. g Violin plots of the TARDBP mRNA expression in the control and TARDBP M337V lines across cell types. The normalized expression is given as RPKM. Differential expression was performed within cell type, across lines. Error bars show mean ± SEM, * p  <  0.05, ** p  <  0.01, *** p  <  0.001, derived from two-sided DEA testing using DESeq2 corrected for an FDR of 0.05. The p-value for the V2a to motor neuron comparison in the TARDBP M337V line is 0.11. h Upset plots showing the overlap between DEGs in motor neurons, V2a interneurons and other interneurons between the TARDBP M337V mutant lines versus the control cell line. Each column represents a section of a Venn diagram of the cell types as indicated in the table below. The corresponding Venn diagram is shown in an insert. The red top and blue bottom columns represent upregulated and downregulated genes, respectively. Genes with an adjusted p-value of <0.05 (two-sided DEA with DESeq2) were considered differentially expressed. i Analysis of upstream regulators in the DEGs between TARDBP M337V and control motor neurons using the Ingenuity Pathway Analysis suite. The regulator TARDBP is highlighted with an activation z-score of -3.13, indicative of its inhibition in the TARDBP M337V cell line.