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

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

MSUT2 is a determinant of susceptibility to tau neurotoxicity

Lesions containing abnormal aggregated tau protein are one of the diagnostic hallmarks of Alzheimer's disease (AD) and related tauopathy disorders. How aggregated tau leads to dementia remains enigmatic, although neuronal dysfunction and loss clearly contribute. We previously identified sut-2 as a gene required for tau neurotoxicity in a transgenic Caenorhabditis elegans model of tauopathy. Here, we further explore the role of sut-2 and show that overexpression of SUT-2 protein enhances tau-induced neuronal dysfunction, neurotoxicity and accumulation of insoluble tau. We also explore the relationship between sut-2 and its human homolog, mammalian SUT-2 (MSUT2) and find both proteins to be predominantly nuclear and localized to SC35-positive nuclear speckles. Using a cell culture model for the accumulation of pathological tau, we find that high tau levels lead to increased expression of MSUT2 protein. We analyzed MSUT2 protein in age-matched post-mortem brain samples from AD patients and observe a marked decrease in overall MSUT2 levels in the temporal lobe of AD patients. Analysis of post-mortem tissue from AD cases shows a clear reduction in neuronal MSUT2 levels in brain regions affected by tau pathology, but little change in regions lacking tau pathology. RNAi knockdown of MSUT2 in cultured human cells overexpressing tau causes a marked decrease in tau aggregation. Both cell culture and post-mortem tissue studies suggest that MSUT2 levels may influence neuronal vulnerability to tau toxicity and aggregation. Thus, neuroprotective strategies targeting MSUT2 may be of therapeutic interest for tauopathy disorders

Upregulated Tau tubulin kinases are also co-expressed with phospho-TDP-43 pathology.

<p>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 (**<i>P</i> = 0.003; ***<i>P</i><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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004803#pgen.1004803.s005" target="_blank">S5 Figure</a> for controls for antibody specificity.</p

Tau tubulin kinases are co-expressed with phospho-TDP-43 pathology in ALS cases.

<p>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.</p

The kinases TTBK1/2 phosphorylate TDP-43 in <i>C. elegans</i> and <i>in vitro</i>.

<p>(A) Developmentally synchronized day 1 adult <i>dkf-2(−/−)</i>;TDP-43, <i>cdc-7(−/−)</i>;TDP-43, and H05L14.1(−/−);TDP-43 kinase mutants have decreased phosphorylated TDP-43 relative to TDP-43 transgenic animals alone. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004803#pgen.1004803.s002" target="_blank">S2 Figure</a> 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. * <i>P</i><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. *<i>P</i><0.05 versus TDP-43. Non-transgenic animals disperse at an average velocity of 5.9 µm/second. (E) <i>In vitro</i> 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) <i>In vitro</i> kinase assays demonstrate purified TTBK1 and TTBK2 but not PRKD2 phosphorylate M337V mutant TDP-43. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004803#pgen.1004803.s004" target="_blank">S4 Figure</a> for controls of kinase activity on known protein substrates.</p

Tau tubulin kinase activation promotes TDP-43 phosphorylation and recruitment into cytoplasmic inclusions.

<p>(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. *<i>P</i> = 0.004 and **<i>P</i> = 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.</p