Supplementary Figure 6: Frontal cortical RNA-seq, tau staining and validation in line 3 mice.
From: TDP-43 gains function due to perturbed autoregulation in a Tardbp knock-in mouse model of ALS-FTD
(a) Quality control measures of sequencing reads from 20 frontal cortical libraries from 5 month old mice (coloured dots represent libraries for individual mice). A mean of 58.7m reads−per−mouse (range 42.8−76.3m) were obtained. (b) Filtering of frontal cortex DESeq2 alternative splice events that are significantly different between 5−month−old wild−type and TDP−43Q331K/Q331K mice. Non−expression hits reflect changes in splice junction usage exceeding a 1.5−fold change relative to expression of the gene from which they are derived. Log Reg hits include splice junctions whose usage changes relative to another junction with the same start or end position. (c) Immunostaining for tau in the cortices of 20−month−old mice. Neuronal cells have been stained with NeuN. Representative images shown. Scale bar, 25 μm. (d) Marbles buried by line ♯3 mice (n = 4 wild−type, 8 TDP−43Q331K/+, 5 TDP−43Q331K/Q331K mice). Pairwise comparisons: wild−type vs. TDP−43Q331K/+: P = 0.691 (ns); wild−type vs. TDP−43Q331K/Q331K: P = 0.020 (*); Kruskal−Wallis followed by Dunn’s test. Error bars represent median and interquartile range. (e) qPCR of expression and splicing changes in line ♯3 mice (n = 5 wild−type, 5 TDP−43Q331K/+, 5 TDP−43Q331K/Q331K mice). Pairwise comparisons: Tardbp expression: wild−type vs. TDP−43Q331K/+: P = 0.076 (ns); wild−type vs. TDP−43Q331K/Q331K: P = 0.0016 (**); TDP−43Q331K/+ vs. TDP−43Q331K/Q331K: P = 0.036 (*); 0N: wild−type vs. TDP−43Q331K/+: P = 0.072 (ns); wild−type vs. TDP−43Q331K/Q331K: P = 0.495 (ns); TDP−43Q331K/+ vs. TDP−43Q331K/Q331K: P = 0.03 (*); 2N/0N: wild−type vs. TDP−43Q331K/+: P = 0.877 (ns); wild−type vs. TDP−43Q331K/Q331K: P = 0.002 (**); TDP−43Q331K/+ vs. TDP−43Q331K/Q331K: P = 0.002 (**); P < 0.0001 (****); one−way ANOVA followed by Holm−Sidak post−hoc tests for pairwise comparisons. Error bars denote s.e.m. (f) Immunohistochemistry for parvalbumin in cortices of line ♯3 mice. Representative images shown. Scale bar, 250μm and quantification of parvalbumin−positive neurons (n = 4 mice per genotype). Comparison: P = 0.006 (**); unpaired t test. Error bars denote s.e.m. (g) Filtering of 5−month−old frontal cortex DESeq2 alternative splice events that are significantly different between MB+ and MB− TDP−43Q331K/Q331K mice. Refer to subfigure (b). (h) Hierarchical clustering of alternative splice events in 5−month−old frontal cortices comparing MB+ and MB− TDP−43Q331K/Q331K mice (n = 6 wild−type, 4 MB+ TDP−43Q331K/Q331K and 4 MB− TDP−43Q331K/Q331K mice); Comparison: DESeq2 MB+ vs MB−.
