Extended Data Fig. 2: Identification and prioritization of TDP-43-dependent KCNQ2 mis-splicing.

(a) Verification of mis-spliced KCNQ2 in day 40 human iPSC-MNs. Representative image of n > 3 biological replicates; marker in base pairs. (b) KCNQ2^∆E5 abundance increases with TDP-43 knockdown level. Comparison of KCNQ2^∆E5 and TDP-43 level in reported datasets^9,11. (c-e) Upregulation of KCNQ2^∆E5 and decrease of KCNQ2^WT in Dox-inducible TDP-43 aggregation model in SH-SY5Y cells. (c) Detection of KCNQ2^∆E5 upon Dox induction of TDP-43 containing 12 Q/N domain repeats; marker in base pairs. (d) Percent Spliced Index (PSI) of KCNQ2 (exon 5 inclusion or skipping) in control versus aggregation-prone TDP-43 cells. Points represent n = 3 independent biological replicates; values are mean +/- SEM; p-values are the result of unpaired two-tailed t-test. (e) Immunofluorescence of TDP-43 aggregates in SH-SY5Y 12Q/N cells; NT=not treated control. (f) Conservation of genomic locus of KCNQ2 exons 4-6 between human and mouse. Mouse conservation is reflected on a black to white color scale where black is highly conserved and white is not conserved. Human KCNQ2 exonic sequences are conserved, while intronic sequences are poorly conserved in mice. The 1-kb long UG repeat found upstream of human exon 5 is not present in the mouse genome. (g-h) Splicing of murine Kcnq2 is not regulated by TDP-43; data from Polymenidou et al.⁴⁵. (g) TDP-43 depletion in mouse brains treated with Tardbp targeting ASOs. (h) Sashimi plot depicting HISAT2-mapped sequencing coverage and junction spanning reads of syntenic mouse Kcnq2 locus. There is no exon skipping following TDP-43 reduction (TDP-43 antisense oligonucleotide; ASO). (i) Comparison of KCNQ2^∆E5 abundance in human and mouse stem-cell derived neurons with TARDBP knockdown. qRT-PCR-based relative expression of KCNQ2^∆E5 and TARDBP. Circles represent relative expression of KCNQ2^∆E5 or TARDBP from n = 3 independent biological replicates of TARDBP knockdowns in MNs. Data are presented as mean +/- SEM; p-values are the result of unpaired t-test. (j) Top: Sashimi plot depicting HISAT2-mapped sequencing coverage and junction spanning reads of the entire mouse Kcnq2 gene. Bottom: gene models. TDP-43 depletion did not induce alternative splicing in this gene; Data from Polymenidou et al.⁴⁵.

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