MAPK signaling promotes axonal degeneration by speeding the turnover of the axonal maintenance factor NMNAT2

  1. Lauren J Walker
  2. Daniel W Summers
  3. Yo Sasaki
  4. EJ Brace
  5. Jeffrey Milbrandt
  6. Aaron DiAntonio  Is a corresponding author
  1. Washington University Medical School, United States
  2. Hope Center for Neurological Disorders, United States
7 figures

Figures

MKK4/7 are necessary for NAD+ and ATP loss after axotomy but not in response to constitutively active SARM1.

MKK4/7 are necessary for NAD+ and ATP depletion in axons following axotomy but not following direct SARM1 activation. Axonal NAD+ (A) and ATP (B) levels were assayed at indicated timepoints after …

https://doi.org/10.7554/eLife.22540.002
Figure 2 with 1 supplement
MAPKs are required for injury-induced but not SARM1-TIR-induced axon degeneration.

(A) Depletion of MKK4/7 (sh4/7) protects axons for 24 hours after axotomy but not when axon degeneration is induced by dimerization of the SARM1-TIR domains with addition of the ligand AP20187 (n = 4…

https://doi.org/10.7554/eLife.22540.003
Figure 2—figure supplement 1
Dimerization of the SARM1-TIR domains with the ligand AP20187 results in MKK4 phosphorylation on Ser257/Thr261.

Lystate from DRGs was collected at the indicated timepoints after addition of AP20187.

https://doi.org/10.7554/eLife.22540.004
Figure 3 with 1 supplement
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). …

https://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 …

https://doi.org/10.7554/eLife.22540.006
Figure 4 with 1 supplement
MAPKs modulate dNMNAT in vivo in Drosophila larvae.

Immunostaining in third instar Drosophila larvae of HA-dNMNAT (green) expressed in nerves from the motorneuron driver OK6-Gal4, with HRP labeling (red) to counterstain the nerve. (A) Expression of …

https://doi.org/10.7554/eLife.22540.007
Figure 4—figure supplement 1
Depletion of JNK with RNAi or expression of JNK dominant negative (DN) protects Drosophila larval axons 24 hr after pinch injury.

(A) Representative images of injured axons. Usually two single axons within the nerve are labeled with GFP driven by m12-GAL4. Total nerve is labeled with HRP. (B) Quantification of axon …

https://doi.org/10.7554/eLife.22540.008
Figure 5 with 2 supplements
MKK4/7 promote turnover of axon survival factors.

(A) Immunostaining for NMNAT2-myc and SCG10 in distal axons four hours after axotomy. Fluorescence signal is diminished four hours after cut in control axons; however, with depletion of MKK4/7 …

https://doi.org/10.7554/eLife.22540.009
Figure 5—figure supplement 1
Pre-treatment with JNKi VIII (30 min) delays degradation of NMNAT2 and SCG10 after cycloheximide (CHX).

Western blot analysis from axon-only lysate from DRG cultures treated with CHX for the indicated timepoints to block protein synthesis to show turnover of endogenous NMNAT2 (A) and SCG10 (B). TUJ1 …

https://doi.org/10.7554/eLife.22540.010
Figure 5—figure supplement 2
Elevation of NMNAT2 and SCG10 does not delay turnover of survival factors within axons.

Representative western blots from axon-only lysate from DRG cultures treated with cycloheximide (CHX) for the indicated timepoints, showing turnover of endogenous NMNAT2 (A) and endogenous SCG10 (B).…

https://doi.org/10.7554/eLife.22540.011
Figure 6 with 1 supplement
MAPK signaling promotes axonal degeneration via regulation of survival factors.

MKK4/7 requires NMNAT2 to protect axons. Cas9 knock-in DRGs were cultured and treated with scrambled (gScrambled) or NMNAT2 (gNMNAT2) guide RNAs. (A) Knockdown of MKK4/7 (sh4/7) protects axons at 24 …

https://doi.org/10.7554/eLife.22540.012
Figure 6—figure supplement 1
MKK4/7 requires NMNAT2 to protect axons.

Cas9 knock-in DRGs were cultured and treated with scrambled (gScrambled) or Nmnat2 (gNMNAT2) guide RNAs (gRNA). (A) Uninjured Cas9 knock-in DRGs are intact when NMNAT2 is knocked out using guide RNA …

https://doi.org/10.7554/eLife.22540.013
Model of the axonal degeneration program.

In this model, MAPK signaling limits the levels of NMNAT2 and SCG10. NMNAT2 blocks activation of SARM1, and SCG10 may facilitate NMNAT2 function. Upon axon injury, delivery of the labile survival …

https://doi.org/10.7554/eLife.22540.014

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