Figure 3. MKK4/7 regulates the levels of axon survival factors NMNAT2 and SCG10.

Loss of MKK4/7 (sh4/7) increases levels of endogenous NMNAT2 and SCG10 within cultured DRG neurons (A) and axons (B) as observed via western blot as compared to shLuciferase (ctrl) controls (n = 3). Vertical line in westerns blots denotes where lanes were removed; however, samples were run on the same gel and images are from the same exposure. We confirm that MKK4/7 functions through the canonical JNK pathway, as timecourse treatment with JNK inhibitor (JNKi) VIII (10 uM, 0–12 hr treatment; n = 4) increases endogenous NMNAT2 and SCG10 within neurons within two hours. (C) MKK4/7 regulate levels of exogenously-expressed NMNAT2-myc and endogenous SCG10 (D) as observed by immunostaining within uninjured DRG axons, indicating the modulation is post-transcriptional. The anti-Tuj1 antibody stains tubulin and labels all axons. Scale bar: 25 µm, n = 4 (E) Western blot from embryonic DRG neurons reveals that knockdown of MKK4/7 leads to an increase in the levels of endogenous NMNAT2 and SCG10 in both wildtype (WT) and SARM1 knockout (SARM1 KO) neurons. (F) Quantification of endogenous NMNAT2 and SCG10 in WT and SARM1 knockout neurons with depletion of MKK4/7 by shRNA. Data were normalized to protein levels with shLuciferase controls (not significant, n = 3). Also refer to Figure 3—figure supplement 1.

DOI: http://dx.doi.org/10.7554/eLife.22540.005

Figure 3—figure supplement 1. Depletion of MAPKs elevates survival factors and protects axons.

(A) Validation of specificity of shMKK4-2 and shMKK7 used in this study. All samples were run on the same gel. Vertical line indicates lanes cropped out of blot. (B) Western blot from embryonic DRGs derived from a Cas9 knock-in mouse demonstrating that depletion of MKK4 using two independent shRNAs or pooled guide RNAs (gRNA) can deplete MKK4 and modulate expression of NMNAT2 and SCG10. The shRNA used in this study to knockdown MKK4 was shMKK4-2. (C) Depletion of MKK4/7 protects axons 24 hr after injury compared to shLuciferase (ctrl) controls. Values represent the Degeneration Index, with higher DI values representing axon degeneration. Values are presented as mean ± SEM. ****p≤0.0001, **p≤0.01, n = 3–6. (D) Western blot from axon-only lysate demonstrating MKK4/7 regulate exogenously-expressed NMNAT2-myc. The NMNAT2-myc construct is expressed from the ubiquitin C promoter instead of the endogenous NMNAT2 promoter, suggesting that MAPK-dependent regulation of NMNAT2 is post-transcriptional. Samples were blotted with an anti-myc antibody (n = 3).

DOI: http://dx.doi.org/10.7554/eLife.22540.006