MRI of Patient with the p.Arg423His mutation.

<p>Cerebellar atrophy in a 48 year old female (H2591) with ataxia and the KCNC3^Arg423His mutation. Midsagittal T1-weighted MRI of the brain shows a small atrophic cerebellum with a normal appearing brainstem.</p

Clinical and mutation findings of p.Arg423His index patient and relatives.

<p>Nucleotide numbering for cDNA –based nomenclature uses 1+ as to the A of the ATG translation initiation codon in Genebank RefSeq NM_004977.2.</p><p>The initiation codon is codon 1. RefSeq NG_008134.1, NP_004968.2.</p

Mutation kinetics in <i>X. laevis</i> oocytes.

<p>p.Gly263Asp alters activation and deactivation kinetics in <i>X. laevis</i> oocytes. (<b>A</b>) p.Gly263Asp currents were evoked by stepping from −90 mV to voltages ranging from −90 mV to +70 mV in 10 mV increments. (<b>B</b>) Normalized isochronal tail current amplitudes have been plotted versus voltage for wild-type (▪, <i>n</i> = 84) and p.Gly263Asp (□, <i>n</i> = 8). (<b>C</b>) Activation time constant (τ_act) was plotted versus voltage. *Wild-type (▪, <i>n</i> = 36) and p.Gly263Asp (□, <i>n</i> = 8) values differed significantly , p<0.05. Inset: Wild-type (solid) and p.Gly263Asp (dotted) currents were evoked at 0 mV, scaled and overlaid. (<b>D</b>) Deactivation time constant (τ_deact) was plotted versus repolarization voltage. Wild-type (▪, <i>n</i> = 10) and p.Gly263Asp (□, <i>n</i> = 8) values differed significantly, p<0.05. Inset: Wild-type (solid) and p.Gly263Asp (dotted) tail currents were recorded at −60 mV, scaled and overlaid.</p

Sequence Variants Found in Index Cases.

<p>RefSeq NG_008134.1, NM_004977.2, NP_004968.2, % reflects allele frequency.</p><p>*Variant Found in patient with p.Arg423His and sister.</p><p>**Known SNP rs35578310.</p

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