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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

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.
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pgen-1004803-g002: 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.

Mentions: TTBK1/2 kinase hyperactivity may contribute to the pathological phosphorylated TDP-43 observed in both FTLD-TDP and ALS. To test whether increased cellular levels of TTBK1/2 activity suffice to drive TDP-43 phosphorylation, we transfected full-length TTBK1 and TTBK2 cDNAs into HEK293 cells. HEK293 cells have some neuronal characteristics and may be derived from a subpopulation of neuronal precursor cells in the embryonic kidney [35]. This cell line is especially useful for biochemical assays requiring high efficiency transfection rates. In the absence of other cellular stresses, we observed robust induction of TDP-43 phosphorylation by immunoblot following transfection with both TTBK1 and TTBK2 (Fig. 2A–C). Likewise, we utilized SH-SY5Y cells, a human neuroblastoma-derived cell line, to determine the location of phosphorylated TDP-43 produced by TTBK2 transfection. The phospho-TDP-43 produced by TTBK2 overexpression is localized throughout the cytoplasm overlapping with TTBK2 (Fig. 2D, E). Further, TTBK2 and phospho-TDP-43 appear concentrated in apparent aggregates, producing a pattern of TDP-43 and TTBK1/2 expression reminiscent of the neuronal cytoplasmic inclusion pathology observed in FTLD-TDP and ALS. SH-SY5Y cells are relatively recalcitrant to transfection; we observed less than 5% transfection efficiency with TTBK2. However, all the cells with strong TTBK2::GFP expression also had inclusions of phosphorylated TDP-43. We observed a similar pattern of TTBK2 transfection overlapping with large phospho-TDP-43 positive aggregates in HEK293 cells (S5 Figure).


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4256087&req=5

pgen-1004803-g002: 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.
Mentions: TTBK1/2 kinase hyperactivity may contribute to the pathological phosphorylated TDP-43 observed in both FTLD-TDP and ALS. To test whether increased cellular levels of TTBK1/2 activity suffice to drive TDP-43 phosphorylation, we transfected full-length TTBK1 and TTBK2 cDNAs into HEK293 cells. HEK293 cells have some neuronal characteristics and may be derived from a subpopulation of neuronal precursor cells in the embryonic kidney [35]. This cell line is especially useful for biochemical assays requiring high efficiency transfection rates. In the absence of other cellular stresses, we observed robust induction of TDP-43 phosphorylation by immunoblot following transfection with both TTBK1 and TTBK2 (Fig. 2A–C). Likewise, we utilized SH-SY5Y cells, a human neuroblastoma-derived cell line, to determine the location of phosphorylated TDP-43 produced by TTBK2 transfection. The phospho-TDP-43 produced by TTBK2 overexpression is localized throughout the cytoplasm overlapping with TTBK2 (Fig. 2D, E). Further, TTBK2 and phospho-TDP-43 appear concentrated in apparent aggregates, producing a pattern of TDP-43 and TTBK1/2 expression reminiscent of the neuronal cytoplasmic inclusion pathology observed in FTLD-TDP and ALS. SH-SY5Y cells are relatively recalcitrant to transfection; we observed less than 5% transfection efficiency with TTBK2. However, all the cells with strong TTBK2::GFP expression also had inclusions of phosphorylated TDP-43. We observed a similar pattern of TTBK2 transfection overlapping with large phospho-TDP-43 positive aggregates in HEK293 cells (S5 Figure).

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