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Fig. 1 | Molecular Brain

Fig. 1

From: ALS-linked TDP-43M337V knock-in mice exhibit splicing deregulation without neurodegeneration

Fig. 1

Characterization of TDP-43M337V knock-in mice. a Schematic illustration of introducing TDP-43M337V mutation into an endogenous murine Tardbp exon 6 (left panel). The representative genotyping result is also shown (right panel). Nhe I restriction site is introduced in the mutant allele, resulting in no change of the amino acid at Nhe I site. b The expression level of Tardbp mRNA was not altered in the brains of 700-days-old TDP-43M337V/M337V (M337 V/M337 V) mice and wild-type (WT) littermates. c Alternation in splicing of genes regulated by TDP-43. The level of mRNA containing exons included by TDP-43 (Kcnip2 exon 2 and 3) was increased, while the levels of mRNA containing exons excluded by TDP-43 (Sort1 exon 17b and Sema3f exon 5) were reduced, suggesting a gain-of-function mechanism in TDP-43M337V/M337V mice. Relative expression levels of mRNA normalized to the WT control are plotted with SD (n = 3 each (b, c)) and were analyzed by unpaired t-tests. d Representative immunoblots of TDP-43 and β-actin in the brains and spinal cords of 700-days-old TDP-43M337V/M337V mice and WT littermates. Asterisk denotes a non-specific band. e and f Representative immunofluorescence images of the anterior horn in lumbar spinal cords of 700-days-old TDP-43M337V/M337V mice and WT littermates stained with anti-TDP-43 (3H8, green) and anti-ChAT (red) antibodies along with the merged images. TDP-43 was not mislocalized in motor neurons of TDP-43M337V/M337V mice (e). Low magnification images stained with anti-ChAT antibody (f). Scale bars: 20 μm (e), 100 μm (f). g Quantification of the numbers of ChAT-positive motor neurons per each anterior horn (AH) in the lumbar spinal cords of 700-days-old TDP-43M337V/M337V mice and WT littermates. For quantification, 20 AHs in three animals per each genotype were counted, and data are plotted as mean ± SD, and were analyzed by an unpaired t-test. h and i TDP-43M337V/M337V mice did not show any motor dysfunction phenotypes in the measurement of clasping score (h) and rotarod test (i). Data are plotted as mean ± SD, and were analyzed with two-way ANOVA. n = 15 for WT and 14 for M337 V/M337 V

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