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The tau tubulin kinases TTBK1/2 promote accumulation of pathological TDP-43.

Liachko NF, McMillan PJ, Strovas TJ, Loomis E, Greenup L, Murrell JR, Ghetti B, Raskind MA, Montine TJ, Bird TD, Leverenz JB, Kraemer BC - PLoS Genet. (2014)

Bottom Line: TTBK1/2 overexpression drives phosphorylation and relocalization of TDP-43 from the nucleus to cytoplasmic inclusions reminiscent of neuropathologic changes in disease states.Furthermore, protein levels of TTBK1 and TTBK2 are increased in frontal cortex of FTLD-TDP patients, and TTBK1 and TTBK2 co-localize with TDP-43 inclusions in ALS spinal cord.These kinases may represent attractive targets for therapeutic intervention for TDP-43 proteinopathies such as ALS and FTLD-TDP.

View Article: PubMed Central - PubMed

Affiliation: Geriatric Research Education and Clinical Center, Veterans Affairs Puget Sound Health Care System, Seattle, Washington, United States of America; Department of Medicine, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
Pathological aggregates of phosphorylated TDP-43 characterize amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP), two devastating groups of neurodegenerative disease. Kinase hyperactivity may be a consistent feature of ALS and FTLD-TDP, as phosphorylated TDP-43 is not observed in the absence of neurodegeneration. By examining changes in TDP-43 phosphorylation state, we have identified kinases controlling TDP-43 phosphorylation in a C. elegans model of ALS. In this kinome-wide survey, we identified homologs of the tau tubulin kinases 1 and 2 (TTBK1 and TTBK2), which were also identified in a prior screen for kinase modifiers of TDP-43 behavioral phenotypes. Using refined methodology, we demonstrate TTBK1 and TTBK2 directly phosphorylate TDP-43 in vitro and promote TDP-43 phosphorylation in mammalian cultured cells. TTBK1/2 overexpression drives phosphorylation and relocalization of TDP-43 from the nucleus to cytoplasmic inclusions reminiscent of neuropathologic changes in disease states. Furthermore, protein levels of TTBK1 and TTBK2 are increased in frontal cortex of FTLD-TDP patients, and TTBK1 and TTBK2 co-localize with TDP-43 inclusions in ALS spinal cord. These kinases may represent attractive targets for therapeutic intervention for TDP-43 proteinopathies such as ALS and FTLD-TDP.

No MeSH data available.


Related in: MedlinePlus

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.
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pgen-1004803-g001: 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.

Mentions: RNAi can inactivate multiple genes simultaneously depending on their sequence similarity, potentially confounding the identification of any single gene responsible for TDP-43 phosphorylation. To unambiguously determine the effects of single kinase gene loss of function on TDP-43 phosphorylation, we generated TDP-43 transgenic animals with viable deletion mutants eliminating the kinase active domain of each candidate gene of interest (Table 1). Each of these kinase mutants was tested for changes in the amount of phosphorylated TDP-43 by immunoblot. Three of the kinase loss of function mutations tested, cdc-7(−/−), H05L14.1(−/−), and dkf-2(−/−), dramatically reduce TDP-43 phosphorylation with only moderate or no changes in total levels of TDP-43, consistent with the results from the initial RNAi screen (Fig. 1A–C and S2 Figure). We observed a slight decrease in levels of a shorter 37 kDa isoform of TDP-43 (Fig. 1A), but the appearance of higher or lower molecular weight species, including multimers, post-translationally modified protein species, or translational variants, appears relatively unchanged (see S2 Figure for the full α-TDP-43 immunoblot), and after quantitation, only dkf-2(−/−) exhibited significant differences in total TDP-43 levels. cdc-7(−/−) has been previously characterized as a TDP-43 kinase [23], but we are including analysis of its mutant phenotypes in Fig. 1 for comparison with H05L14.1(−/−) and dkf-2(−/−).


The tau tubulin kinases TTBK1/2 promote accumulation of pathological TDP-43.

Liachko NF, McMillan PJ, Strovas TJ, Loomis E, Greenup L, Murrell JR, Ghetti B, Raskind MA, Montine TJ, Bird TD, Leverenz JB, Kraemer BC - PLoS Genet. (2014)

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.
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pgen-1004803-g001: 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.
Mentions: RNAi can inactivate multiple genes simultaneously depending on their sequence similarity, potentially confounding the identification of any single gene responsible for TDP-43 phosphorylation. To unambiguously determine the effects of single kinase gene loss of function on TDP-43 phosphorylation, we generated TDP-43 transgenic animals with viable deletion mutants eliminating the kinase active domain of each candidate gene of interest (Table 1). Each of these kinase mutants was tested for changes in the amount of phosphorylated TDP-43 by immunoblot. Three of the kinase loss of function mutations tested, cdc-7(−/−), H05L14.1(−/−), and dkf-2(−/−), dramatically reduce TDP-43 phosphorylation with only moderate or no changes in total levels of TDP-43, consistent with the results from the initial RNAi screen (Fig. 1A–C and S2 Figure). We observed a slight decrease in levels of a shorter 37 kDa isoform of TDP-43 (Fig. 1A), but the appearance of higher or lower molecular weight species, including multimers, post-translationally modified protein species, or translational variants, appears relatively unchanged (see S2 Figure for the full α-TDP-43 immunoblot), and after quantitation, only dkf-2(−/−) exhibited significant differences in total TDP-43 levels. cdc-7(−/−) has been previously characterized as a TDP-43 kinase [23], but we are including analysis of its mutant phenotypes in Fig. 1 for comparison with H05L14.1(−/−) and dkf-2(−/−).

Bottom Line: TTBK1/2 overexpression drives phosphorylation and relocalization of TDP-43 from the nucleus to cytoplasmic inclusions reminiscent of neuropathologic changes in disease states.Furthermore, protein levels of TTBK1 and TTBK2 are increased in frontal cortex of FTLD-TDP patients, and TTBK1 and TTBK2 co-localize with TDP-43 inclusions in ALS spinal cord.These kinases may represent attractive targets for therapeutic intervention for TDP-43 proteinopathies such as ALS and FTLD-TDP.

View Article: PubMed Central - PubMed

Affiliation: Geriatric Research Education and Clinical Center, Veterans Affairs Puget Sound Health Care System, Seattle, Washington, United States of America; Department of Medicine, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
Pathological aggregates of phosphorylated TDP-43 characterize amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP), two devastating groups of neurodegenerative disease. Kinase hyperactivity may be a consistent feature of ALS and FTLD-TDP, as phosphorylated TDP-43 is not observed in the absence of neurodegeneration. By examining changes in TDP-43 phosphorylation state, we have identified kinases controlling TDP-43 phosphorylation in a C. elegans model of ALS. In this kinome-wide survey, we identified homologs of the tau tubulin kinases 1 and 2 (TTBK1 and TTBK2), which were also identified in a prior screen for kinase modifiers of TDP-43 behavioral phenotypes. Using refined methodology, we demonstrate TTBK1 and TTBK2 directly phosphorylate TDP-43 in vitro and promote TDP-43 phosphorylation in mammalian cultured cells. TTBK1/2 overexpression drives phosphorylation and relocalization of TDP-43 from the nucleus to cytoplasmic inclusions reminiscent of neuropathologic changes in disease states. Furthermore, protein levels of TTBK1 and TTBK2 are increased in frontal cortex of FTLD-TDP patients, and TTBK1 and TTBK2 co-localize with TDP-43 inclusions in ALS spinal cord. These kinases may represent attractive targets for therapeutic intervention for TDP-43 proteinopathies such as ALS and FTLD-TDP.

No MeSH data available.


Related in: MedlinePlus