Presenilin/γ-secretase-dependent EphA3 processing mediates axon elongation through non-muscle myosin IIA
- Universitat Autònoma de Barcelona, Spain
- Universitat de Lleida, Spain
- Monash University, Australia
Figures
Figure 1 with 1 supplement
PS1/γ-secretase is required for axon growth in the developing brain.
(A) Reduced axon length in embryonic Psen1-/- mouse brains. Confocal microscope images showing reduced axon length in the ventricular zone of Psen1-/- mouse brains (E15.5). Axons (green) are labeled …
Figure 1—figure supplement 1
PS1 colocalizes and interacts with EphA3 in axons.
(A) Expression of EphAs during neuronal polarization. Levels of Epha mRNAs measured by qRT-PCR at different neuronal polarization stages (2, 4 and 7 DIV). Levels of mRNA were normalized to Gapdh and …
Figure 2 with 1 supplement
PS1/γ-secretase mediates EphA3 processing in mammalian cells.
(A) Accumulation of EphA3 CTFs in Psen1-/- mouse brains. Western blot analysis of brain lysates from Psen1+/+ and Psen1-/- mouse embryos (E15.5). Values represent mean ± SEM (n = 4); Unpaired …
Figure 2—figure supplement 1
Overexpression of EphA3 mutants and Epha3 inactivation.
(A) Biochemical analyses showing expression of exogenous EphA3 ICD (C-19 antibody) and ΔICD (L-18 antibody) in HEK293 cells. (G) ShRNA-mediated Epha3 knock-down in neurons. Reduced EphA3 levels in Ep…
Figure 3 with 1 supplement
EphA3 ICD reverses axon defects in PS/γ-secretase- and EphA3-deficient neurons.
(A) EphA3 ICD reverses axon growth defects in Psen1-/- hippocampal neurons. Immunofluorescence staining (left) and quantitative analysis (right) of axon length (indicated by arrowheads) in …
Figure 3—figure supplement 1
Effect of EphA3 ICD on axon growth cone collapse.
EphA3 ICD regulates growth cone collapse in hippocampal neurons. Hippocampal Psen1+/+ and Psen1-/- neurons were transfected with vector (pWPI-GFP) or EphA3 ICD (green) at 2 DIV and fixed and stained …
Figure 4
PS1/γ-secretase regulates axon growth independently of EphA3-ephrin signaling.
(A) Inhibition of PS1/γ-secretase does not affect EphA3 phosphorylation. Biochemical analysis of total and phosphorylated (Tyr779) EphA3 and EphA3 CTFs in hippocampal cultured neurons (4 DIV) …
Figure 5
PS/γ-secretase-dependent EphA3 cleavage mediates axon elongation partially through RhoA signaling.
(A) ROCK inhibition reverses axon length deficits in Psen1-/- neurons. Immunofluorescence images of hippocampal neurons from Psen1+/+ and Psen1-/- embryos treated with vehicle or the RhoA inhibitor …
Figure 6 with 2 supplements
EphA3 ICD interacts with non-muscle myosin IIA and regulates its phosphorylation levels.
(A) Proteomic analysis of EphA3 interacting proteins. HEK293 cells overexpressing vector (Flag) or EphA3 ICD-Flag (ICD-Flag) were immunoprecipitated with anti-Flag antibody, and proteins were …
Figure 6—figure supplement 1
Reduced pNMIIA/F-actin colocalization in growth cones of Psen1-/- neurons.
Colocalization of pNMIIA and F-actin at growth cones of neurons from Psen1+/+ and Psen1-/- embryos. EphA3 ICD overexpression increases the pNMIIA spots that colocalize with F-actin at growth cones. …
Figure 6—figure supplement 2
NMIIA regulates axon retraction.
Overexpression of NMIIA WT and NMIIA S1943A (deficient for S1943 phosphorylation) cause axonal retraction in hippocampal neurons. Hippocampal neurons were transfected with NMIIA WT and NMIIA S1943A …
Figure 7
Molecular mechanisms of axon growth mediated by ephrin-dependent and -independent EphA3 signaling.
Proposed model of ligand-dependent and -independent EphA3 signaling regulating axon growth in neurons. Left: Ephrin-A5 induces EphA3 phosphorylation (Tyr779), CrkII binding and activation of …
Author response image 1
Detection of EphA3 ICD in mouse brains in vivo.
PS/γ-secretase-mediated EphA3 processing in mouse brain. In vitro γ-secretase activity assay in brain lysates of embryonic mouse brains (E17.5). Biochemical analysis shows generation of EphA3 ICD …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (H. sapiens) | HEK293 (Human embryonic kidney293 cells) | American Type Culture Collection | ||
| Cell line (M. musculus) | PS1/PS2-/- MEF (mouse embryonic fibroblast cells) | B. De Strooper (Katholieke Universiteit Leuven, Belgium) | ||
| Strain (M. musculus) | Psen1+/+; Psen1-/-; Psen2-/- | J. Shen (Brigham and Women Hospital, USA) | MGI: 1202717 | PMID: 9160754 |
| Antibody | anti-EphA3 C19 (rabbit polyclonal) | Santa Cruz Biotechnology | Cat. #: sc-919 RRID: AB_2099221 | WB (1:1000) |
| Antibody | anti-EphA3 H80 | Santa Cruz Biotechnology | Cat. #: sc-25456 RRID: AB_2099214 | ICC (1:200) IHC (1:200) |
| Antibody | anti-EphA3 L18 (rabbit polyclonal) | Santa Cruz Biotechnology | Cat. #: sc-920 RRID: AB_2099218 | WB (1:1000) |
| Antibody | anti-EphA3 (mouse monoclonal) | ThermoFisher Scientific | Cat. #: 5E11F2 RRID: AB_10104885 | WB (1:1000) IP (5 μg) |
| Antibody | anti-CrkII | BD Biosciences | Cat. #: 610035 RRID: AB_397451 | WB (1:5000) IP (5 μl) |
| Antibody | anti-Neurofilament; SMI312 (mouse monoclonal) | Covance | Cat. #: SMI-312R RRID: AB_2314906 | ICC (1:1000) |
| Antibody | anti-tau; TNT-1 (mouse monoclonal) | Merck Millipore | Cat. #: MAB3420 RRID: AB_94855 | IHC (1:400) |
| Antibody | anti-Nestin; rat-401 (mouse monoclonal) | Abcam | Cat. #: ab11306 RRID: AB_1640723 | IHC (1:200) |
| Antibody | anti-NeuN (mouse monoclonal) | Merck Millipore | Cat. #: MAB377 RRID: AB_2298772 | IHC (1:1000) |
| Antibody | anti-MAP2 (mouse monoclonal) | Sigma-Aldrich | Cat. #: M9942 RRID: AB_477256 | ICC (1:300) IHC (1:800) |
| Antibody | anti-Doublecortin (rabbit polyclonal) | Abcam | Cat. #: ab18723 RRID: AB_732011 | IHC (1:1000) |
| Antibody | PS1; APS11 (mouse monoclonal) | Abcam | Cat. #: ab15456 RRID: AB_301867 | ICC (1:200) IHC (1:200) |
| Antibody | PS1 loop (rabbit polyclonal) | Merck Millipore | Cat. #: AB5308 RRID: AB_91785 | WB (1:4000) |
| Antibody | APP C-terminal Saeko (rabbit polyclonal; serum) | M. Shoji (Gunma Univ School Medicine, Japan) | - | WB (1:7500) |
| Antibody | ß-actin [AC-15] (mouse monoclonal) | Sigma-Aldrich | Cat. #: A1978 RRID: AB_476692 | WB (1:60000) |
| Antibody | anti-Flag [FG4R] (mouse monoclonal) | Abcam | Cat. #: ab127420 RRID: AB_11157374 | IP (10 μl) |
| Antibody | anti-HA (mouse monoclonal) | Cell Signaling Technology | Cat. #: 2362 | WB (1:1000) |
| Antibody | AlexaFluor 594-phalloidin | ThermoFisher Scientific | Cat. #: A12381 RRID: AB_2315633 | ICC (1:50) |
| Antibody | GFP (chicken polyclonal) | Abcam | Cat. #: 13970 RRID: AB_371416 | ICC (1:1000) |
| Recombinant DNA reagent | EphA3-GFP | P. W. Janes (Monash University, Australia) | ||
| Recombinant DNA reagent | EphA3-HA | This study | ||
| Recombinant DNA reagent | EphA3-∆ICD | P. W. Janes (Monash University, Australia) | ||
| Recombinant DNA reagent | EphA3-∆LBD | P. W. Janes (Monash University, Australia) | ||
| Recombinant DNA reagent | EphA3-∆PDZ | P. W. Janes (Monash University, Australia) | ||
| Recombinant DNA reagent | EphA3-ICD | This study | ||
| Recombinant DNA reagent | EphA3-ICD-Flag | This study | ||
| Recombinant DNA reagent | EphA3-ICD-pCAGIG | This study | ||
| Recombinant DNA reagent | RhoA T19N | X. R. Bustelo (Centro Investigación del Cáncer, CSIC, Spain) | ||
| Recombinant DNA reagent | RhoA Q63L | P. Crespo (IBBTEC, Santander, Spain) | ||
| Recombinant DNA reagent | NMIIA WT | A. Bresnick (Albert Einstein College Medicine, NY, USA) | ||
| Recombinant DNA reagent | NMIIA S1943A | A. Bresnick (Albert Einstein College of Medicine, NY, USA) | ||
| Sequenced-based reagent | Epha3 ShRNA | This study | ||
| Sequenced-based reagent | RT-qPCR primers | This study | See Materials and methods | |
| Peptide, recombinant protein | ephrin-A5 Fc chimera | R and D Systems | Cat. #: 374-EA | |
| Commercial assay or kit | RhoA G-LISA Activation Assay Kit | Cytoskeleton Inc | Cat. #: BK124 | |
| Commercial assay or kit | Rnease Mini kit | Qiagen | Cat. #: 74106 | |
| Chemical compound, drug | DAPT | Sigma-Aldrich | Cat. #: D5942 | 5 μM |
| Chemical compound, drug | L685,458 | Tocris | Cat. #: 2627 | 5 μM |
| Chemical compound, drug | GM6001 | Enzo | Cat. #: BML-EI300-0001 | 25 μM |
| Chemical compound, drug | 1,10-PNT | Sigma | Cat. #: 131377 | 83 μM |
| Chemical compound, drug | MMP9/13 | Calbiochem | Cat. #: 444252 | 10 μM |
| Chemical compound, drug | Y27632; ROCK-1 inhibitor | Calbiochem | Cat. #: 688000 | 10 μM |
| Software, algorithm | GraphPad Prism | GraphPad Prism (https://www.graphpad.com/) | RRID:SCR_015807 | Version 6 |
| Software, algorithm | ImageJ | ImageJ (https://imagej.nih.gov/ij/) | RRID:SCR_003070 | |
| Software, algorithm | Imaris | Imaris (https://imaris.oxinst.com/) | RRID:SCR_007370 | Version 8 |
Additional files
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Supplementary file 1
Proteomic analysis of the PS/γ-secretase-mediated EphA3 cleavage site.
Mass spectrometry spectra of the trypsin-digested band (~47–49 kDa) (top spectra) and specific mass spectrometry spectra obtained for peptide VLIGR showing the mass/charge (m/z) values (bottom spectra). Detected signals corresponding with theoretical ions are labeled with red asterisks.
- https://doi.org/10.7554/eLife.43646.014
-
Supplementary file 2
Identified peptides from NMIIA protein.
- https://doi.org/10.7554/eLife.43646.015
-
Transparent reporting form
- https://doi.org/10.7554/eLife.43646.016
