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. Author manuscript; available in PMC: 2017 Oct 1.
Published in final edited form as: Acta Neuropathol. 2016 Jul 29;132(4):545–561. doi: 10.1007/s00401-016-1600-y

Fig. 2.

Fig. 2

The C. elegans calcineurin A homolog, tax-6, regulates TDP-43 phosphorylation in vivo. (a) Two different tax-6 loss of function alleles, p675 and ok2065, increase phosphorylation (pTDP) and total protein levels of WT TDP-43 transgenic C. elegans. (b) Bar graphs represent measurement of 3 independent replicate immunoblots. Significance was evaluated using one-way analysis of variance with Tukey’s multiple comparison test among TDP-43(WT), TDP-43(WT); tax-6(p675), and TDP-43(WT); tax-6(ok2065). p=0.001 comparing relative pTDP band intensities. p=0.005 comparing relative total TDP-43 band intensities. (c) tax-6 loss of function alleles increase pTDP and total TDP in A315T TDP-43 transgenic C. elegans. (d) Bar graphs represent measurement of 4 independent replicate immunoblots. Significance was evaluated using one-way analysis of variance with Tukey’s multiple comparison test among TDP-43(A315T), TDP-43(A315T); tax-6(p675), and TDP-43(A315T); tax-6(ok2065). p=0.008 comparing relative pTDP band intensities. p=0.0002 comparing total TDP-43 band intensities. (e) TDP-43(A315T) and TDP-43(A315T); tax-6(ok2065) were subjected to sequential protein extraction with detergents of increasing solubilizing strengths. Levels of sarkosyl-insoluble (SARK) TDP-43 and pTDP are increased with loss of tax-6. (f) tax-6(ok2065) increases levels of total TDP-43 in non-phosphorylatable TDP-43(M337V SS/AA); tax-6(ok2065). (g) Bar graphs represent measurement of 4 independent replicate immunoblots. Significance was evaluated using Student’s t-test between TDP-43(M337V SS/AA) and TDP-43(M337V SS/AA); tax-6(p675), p=0.0002. (h-i) Calcineurin loss of function significantly worsens TDP-43 tg motor dysfunction. Dispersal velocity of developmentally staged L4 larvae was measured by calculating the radial distance traveled from a designated central starting point over 30 minutes. (h) N=166 for TDP-43(WT), N=144 for TDP-43(WT); tax-6(p675), N=149 for TDP-43(WT); tax-6(ok2065). Significance was evaluated using one-way analysis of variance with Tukey’s multiple comparison test among TDP-43(WT), TDP-43(WT); tax-6(p675), and TDP-43(WT); tax-6(ok2065). p<0.0001 versus TDP-43(WT). (i) N=364 for TDP-43(A315T), N=177 for TDP-43(A315T); tax-6(p675), N=140 for TDP-43(A315T); tax-6(ok2065). Significance was evaluated using one-way analysis of variance with Tukey’s multiple comparison test among TDP-43(A315T), TDP-43(A315T); tax-6(p675), and TDP-43(A315T); tax-6(ok2065). p<0.0001 versus TDP-43(A315T). (j) TDP-43(M337V) with mutations of serines 409/410 to non-phosphorylatable alanines (SS/AA) do not exhibit exacerbated motor phenotypes with calcineurin loss of function. N=210 for TDP-43(M337V SS/AA), N=118 for TDP-43(M337V SS/AA); tax-6(ok2065). Significance was evaluated using Student’s t-test between TDP-43(M337V SS/AA) and TDP-43(M337V SS/AA); tax-6(ok2065). n.s. is not significant.