Table 1.
Candidate TDP-43 kinases identified by RNAi screening.
Figure 1.
The kinases TTBK1/2 phosphorylate TDP-43 in C. elegans and in vitro.
(A) Developmentally synchronized day 1 adult dkf-2(−/−);TDP-43, cdc-7(−/−);TDP-43, and H05L14.1(−/−);TDP-43 kinase mutants have decreased phosphorylated TDP-43 relative to TDP-43 transgenic animals alone. See S2 Figure for overexposure of immunoblots. Measurement of protein levels of three independent immunoblots is presented for phospho-TDP-43 (B) and total TDP-43 (C). Signal is normalized to the parental TDP-43 transgenic control strain, and graphs are plotted in arbitrary units of intensity. * P<0.05, Student's t-test relative to TDP-43 transgenic control. (D) Developmentally staged kinase mutant/TDP-43 transgenic L4 larvae exhibit significantly higher dispersal velocity relative to TDP-43 transgenic animals with intact kinase genes. Animals were measured for the linear distance traveled from a central reference point over time, N>70 for each genotype. *P<0.05 versus TDP-43. Non-transgenic animals disperse at an average velocity of 5.9 µm/second. (E) In vitro kinase assays testing the kinase activity of TTBK1, TTBK2, and PRKD2 against wild-type TDP-43 demonstrate purified TTBK1 and TTBK2 phosphorylate wild-type TDP-43, while PRKD2 does not. Immunoblots are probed with antibodies for phosphorylated (P-TDP-43) and total TDP-43. (F) In vitro kinase assays demonstrate purified TTBK1 and TTBK2 but not PRKD2 phosphorylate M337V mutant TDP-43. See S4 Figure for controls of kinase activity on known protein substrates.
Figure 2.
Tau tubulin kinase activation promotes TDP-43 phosphorylation and recruitment into cytoplasmic inclusions.
(A) Overexpression of TTBK1 and TTBK2 in HEK293 cells induces robust TDP-43 phosphorylation in the absence of other cellular stressors. Quantitative analysis of band intensities from three independent replicate transfections is shown for (B) TTBK1 and (C) TTBK2. Graphs are plotted in arbitrary units of intensity. *P = 0.004 and **P = 0.035 versus control transfection, Student's t-test. Differences in total TDP-43 are not statistically significant. (D, E) TTBK2 is expressed throughout the cytoplasm, and overlaps with phosphorylated TDP-43 in SHSY-5Y cells. Pearson coefficient of correlation for colocalization (D) = 0.9853, (E) = 0.9793.
Figure 3.
Reduced TTBK1 protects against TDP-43 phosphorylation.
(A) NSC-34 cells treated with siRNA targeting TTBK1 exhibit reduced TDP-43 phosphorylation following induction of P-TDP-43 with ethacrynic acid (EA). (B) Quantitative analysis of band intensities from three independent replicate siRNA experiments. Band intensities are graphed in arbitrary units. *P = 0.025 versus control+EA, Student's t-test.
Figure 4.
Upregulated Tau tubulin kinases are also co-expressed with phospho-TDP-43 pathology.
Representative photomicrographs depicting TTBK1 (A, C) and TTBK2 (B, D) immunoreactivity in cortical neurons in normal (A, B) and FTLD-TDP Type B (C, D) cases. The cellular distribution is both cytoplasmic and nuclear (insets), and immunoreactivity appears to be more widespread in FTLD cases relative to normal controls. Cortical layers I-VI are indicated (C). Quantification of immunostaining demonstrated a statistically significant increase in both TTBK1 (E) and TTBK2 (F) in FTLD cases compared to normal controls (**P = 0.003; ***P<0.0001). The distribution of phospho-TDP-43 immunoreactivity in the cortex of an FTLD case (G) overlaps with TTBK1 (C) and TTBK2 (D). Double label immunohistochemical experiments suggest co-localization of phospho-TDP-43 with TTBK1 (H) and TTBK2 (I) in an FTLD case. Scale bars: 100 µm A–D,G; 50 µm insets A–D; 25 µm H,I. See S5 Figure for controls for antibody specificity.
Figure 5.
Tau tubulin kinases are co-expressed with phospho-TDP-43 pathology in ALS cases.
TTBK1 (brown; A, C, E, F) and TTBK2 (brown; B, D, G, H) co-localize with phospho-TDP-43 (black) in spinal cord motor neurons (A, B), hippocampal dentate granule cells (C, D), cortical neurons (E, G), hippocampal CA3 pyramidal neurons (F) and subiculum (H). Scale bars = 50 µm.
Figure 6.
TTBK1/2 co-localize with phosphorylated TDP-43 in ALS spinal cord aggregates.
Double-label immunofluorescence of ALS spinal cord of (A) TTBK1 and (B) TTBK2 show significant co-localization of TTBK1/2 with phospho-TDP-43 within neuronal cytoplasmic inclusions. Significance was determined using Pearson coefficient of colocalization.