Journal: The Journal of Neuroscience

Article Title: The Ste20 Family Kinases MAP4K4, MINK1, and TNIK Converge to Regulate Stress-Induced JNK Signaling in Neurons

doi: 10.1523/JNEUROSCI.0905-17.2017

Figure Lengend Snippet: Inhibitors targeting MAP4K4, TNIK, and MINK1 block DLK/JNK pathway activation in axons and induction of DLK and p-JNK in the soma after NGF withdrawal. A, Schematic illustration of experimental setup for isolation of distal axons (lower compartment, orange) from DRG cell bodies/proximal axons (inner compartment, blue) of DRG explants. Western blot analysis of DRG cell bodies/proximal axons (left, blue) and axons (orange, right), demonstrates localization of MAP4K4, MINK1, and TNIK in isolated axons as well as cell bodies. B–G, Immunoblot analysis of isolated DRG axons and cell bodies after NGF-withdrawal at 1, 3, and 6 h in the presence of DMSO or GNE-495. B–D, The presence of MAP4Ki GNE-495 reduced phosphorylation of c-Jun in explants (blue) (C) and JNK in explants and axons (red) (D) after NGF deprivation compared with DMSO control. Immunoblot intensities were normalized to TUJ1 and +NGF control (0 h of NGF withdrawal). p-c-Jun: n = 4–5/condition, p-JNK: n = 8–9/condition. E, Immunoblot analysis of DLK protein levels and p-c-Jun in isolated DRG axons and cell bodies at 1, 3, and 6 h in the presence of DMSO or GNE-495 (top). Illustration at the bottom shows how the activated fraction of DLK was calculated based on the Western blot banding pattern: molecular-weight-shifted DLKshift (red) divided by total DLKtotal (blue) normalized to actin immunoblot band intensity. The dashed line highlights the magnified region of full blot. F, Total DLK protein levels increase in cell bodies/proximal axons over time, but decrease in axons. GNE-495 block DLK induction in explants (n = 7–8/condition). G, Active fraction of DLK protein (DLKactive, see example illustration in E) is increased significantly in axons and cell bodies/proximal axons over time. GNE-495 treatment prevents DLK activation in both axons and explants (n = 7–8/condition). H, Immunofluorescence images demonstrating that both p-JNK (green) and DLK (red) protein is increased in the DRG cell bodies (DAPI: blue, TUJ-1: white) 3 h after NGF withdrawal. MAP4K inhibitors block the somal intensity of DLK and p-JNK. I, Quantification of somal p-JNK intensity relative to NGF p-JNK intensity in experiment H. J, Quantification of somal DLK intensity relative to NGF DLK intensity in experiment H (n = 40 cells/condition, obtained from 4 independent wells). One-way ANOVA followed by Bonferroni post hoc test was used for statistical comparison. Data are represented as mean ± SEM. Statistical significance level between treatment for all time points are displayed above graphs in C, D, F, and G; statistical significance level of individual time points are presented in the graphs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The half-maximal inhibitory concentration (IC 50 ) for GNE-495 of TNIK activity was determined using a TNIK kinase assay (V4158; Promega).

Techniques: Blocking Assay, Activation Assay, Isolation, Western Blot, Molecular Weight, Immunofluorescence

Journal: The Journal of Neuroscience

Article Title: The Ste20 Family Kinases MAP4K4, MINK1, and TNIK Converge to Regulate Stress-Induced JNK Signaling in Neurons

doi: 10.1523/JNEUROSCI.0905-17.2017

Figure Lengend Snippet: Small-molecule screen in primary DRG neurons identifies MAP4K4 inhibitors as regulators of JNK signaling. A, Representative immunofluorescence images showing c-Jun phosphorylation in embryonic DRG cultures after NGF withdrawal (2 h). Small-molecule inhibitors targeting MAP4K4 block p-c-Jun upregulation. Green: p-c-Jun; blue: DRAQ5 (nuclei); red: HuD (cytoplasm). Scale bar, 50 μm. IC50 (nm): GNE-495, 2.24; MAP4Ki_10, 1.05; MAP4Ki_29, 14.5; and MAP4Ki_26, 16.0. Concentrations of inhibitors: GNE-495, 200 nm; MAP4Ki_10, 833 nm; MAP4Ki_29, 6.67 μm; and MAP4Ki_26, 3.30 μm. DMSO was used as a control. B, Dose–response curves showing the ratio of p-c-Jun-positive nuclei after 2 h NGF withdrawal treated with varying concentrations of four different MAP4K4 inhibitors to that in cultures treated with vehicle alone (expressed as a percentage). Each concentration was tested in duplicate wells, with both values plotted. EC50 concentrations (μm) calculated from the mean IC50 values of two plate replicates are as follows: GNE-495, 0.0476; MAP4Ki_10, 0.0343; MAP4Ki_29, 1.605; and MAP4Ki_26, 1.027.

Article Snippet: The half-maximal inhibitory concentration (IC 50 ) for GNE-495 of TNIK activity was determined using a TNIK kinase assay (V4158; Promega).

Techniques: Immunofluorescence, Blocking Assay, Concentration Assay

Journal: The Journal of Neuroscience

Article Title: The Ste20 Family Kinases MAP4K4, MINK1, and TNIK Converge to Regulate Stress-Induced JNK Signaling in Neurons

doi: 10.1523/JNEUROSCI.0905-17.2017

Figure Lengend Snippet: Inhibitors targeting MAP4K4 reduce DLK/JNK pathway activation after NGF deprivation. A, Western blot analysis of NGF-deprived DRG neuron lysates demonstrate phosphorylation of c-Jun and activation of DLK as measured by increased DLK abundance and a shift in molecular weight after 3 h. Treatment with inhibitors targeting MAP4K4, GNE-495 (800 nm), MAP4Ki_10 (1 μm), and MAP4Ki_29 (10 μm), block p-c-Jun and DLK activation. B, Treatment with GNE-495 (800 nm) blocks phosphorylation of JNK (p-JNK) and DLK (shift in molecular weight) after NGF deprivation. Lambda protein phosphatase (λpp) treatment of DRG samples confirms that the increased molecular weight of DLK is due to phosphorylation. C, Phosphorylation of DLK on T43 detected by an antibody targeting phosphorylated DLK-T43 (p-DLKT43) is induced by NGF withdrawal. Treatment with GNE-495 treatment suppresses p-DLKT43 and downstream p-c-Jun. D, Analysis of DLK protein stability after 4 and 8 h of NGF withdrawal using the protein synthesis inhibitor cycloheximide (5 μm). E, GNE-495 treatment attenuates the increase in DLK protein stability in response to stress, as demonstrated by NGF deprivation 4 and 8 h in the presence of cycloheximide. Relative DLK protein levels were normalized to cycloheximide-treated DRG control lysates in the presence of NGF for each time point. DLK 4 h: p = 0.03, 8 h: p = 0.004. n = 8–10/condition from six independent experiments. Student's t test was used for statistical analysis. Data are represented as mean ± SEM, *p < 0.05, **p < 0.01.

Article Snippet: The half-maximal inhibitory concentration (IC 50 ) for GNE-495 of TNIK activity was determined using a TNIK kinase assay (V4158; Promega).

Techniques: Activation Assay, Western Blot, Molecular Weight, Blocking Assay

Journal: The Journal of Neuroscience

Article Title: The Ste20 Family Kinases MAP4K4, MINK1, and TNIK Converge to Regulate Stress-Induced JNK Signaling in Neurons

doi: 10.1523/JNEUROSCI.0905-17.2017

Figure Lengend Snippet: Inhibitors targeting MAP4K4 protects from axon degeneration and apoptosis after NGF withdrawal. A, Four different inhibitors targeting MAP4K4 were able to block axon (β-tubulin) degeneration in DRG explants 20 h after NGF withdrawal. B, Quantification of axon degeneration in A using a five-point blinded scoring system (see Materials and Methods), demonstrating significant protection from axon fragmentation with inhibitors targeting MAP4K4: GNE-495, MAP4Ki_10, MAP4Ki_29, and MAP4Ki_26. n = 6/condition from 4–6 explants. C, Treatment with GNE-495 blocks caspase-3 (red) activation in dissociated primary DRG neurons labeled with tubulin (green) 8 h after NGF withdrawal to a similar degree as DLK inhibition via treatment with GNE-3511. n = 6/condition obtained from 4–6 explants. D, Quantification of activated caspase-3 in C calculated as percentage of caspase-3 positive, β-tubulin-colabeled, neurons. n = 4–8 wells per condition from 2 independent experiments (average of 2 fields of view per well, 20×). Scale bars, 100 μm. One-way ANOVA followed by Bonferroni post hoc test was used for statistical comparison. Data are represented as mean ± SEM, ****p < 0.0001.

Article Snippet: The half-maximal inhibitory concentration (IC 50 ) for GNE-495 of TNIK activity was determined using a TNIK kinase assay (V4158; Promega).

Techniques: Blocking Assay, Activation Assay, Labeling, Inhibition