Deficit of mitogen-activated protein kinase phosphatase 1 (DUSP1) accelerates progressive hearing loss
- Spanish National Research Council-Autonomous University of Madrid (CSIC-UAM), Spain
- CIBER, ISCIII, Spain
- Hospital La Paz Institute for Health Research (IdiPAZ), Spain
- Autonomous University of Madrid, Spain
- Biomedicine Unit UCLM-CSIC, Spain
Figures
Figure 1 with 1 supplement
Expression of Dusp1 and related phosphatases in the mouse cochlea.
(A) Cochlear gene expression of Dusp1 in MF1 × 129 Sv mice from embryonic (E) to adult stages (measured in days (d) and months (m)). Expression levels were measured by RT-qPCR and calculated as 2–ΔΔC…
Figure 1—figure supplement 1
Expression of MAP kinase and MAP kinase phosphatases in the mouse cochlea.
(A-C) Cochlear expression of MKPs genes from the first (A), second (B) and third (C) classes in Dusp1+/+ (lighter color bars) and Dusp1–/– mice (darker color bars) of 2, 4–5 and 8–9 months of age. (D…
Figure 2 with 1 supplement
Comparative longitudinal hearing evaluation of Dusp1+/+ and Dusp1–/– mice.
(A) Representative ABR recordings showing the typical I–V waves obtained in response to click stimuli from Dusp1+/+ and Dusp1–/– mice of 2, 4–5, 8–9 and 12 months of age, showing the hearing …
Figure 2—figure supplement 1
ABR latencies and amplitudes of Dusp1+/+ and Dusp1–/– mice.
(A) Input–output function of wave I latency, the mean latency (± SEM) of ABR peak one is plotted against sound intensity (dB SPL) for mice of 2 months of age (Dusp1+/+, n = 23; Dusp1–/–, n = 24). (B)…
Figure 3 with 2 supplements
Comparative cochlear cytoarchitecture of Dusp1+/+ and Dusp1–/– mice.
(A) Representative microphotographs of hematoxylin-eosin-stained paraffin cochlear mid-modiolar sections of Dusp1+/+ and Dusp1–/– mice, showing the spiral ganglion and the organ of Corti from the …
Figure 3—figure supplement 1
Middle and inner ear morphology of Dusp1+/+ and Dusp1–/– mice.
(A–D) Representative images taken after soft tissue cleaning of 2-month-old Dusp1+/+ (n = 4) and Dusp1–/– mice (n = 4). Lateral (A), medial (B), rostral (C) and caudal (D) views. Co, cochlea; Lsc, …
Figure 3—figure supplement 2
Comparative cochlear cytoarchitecture of 2-month-old Dusp1+/+ and Dusp1–/– mice.
Representative microphotographs of hematoxylin-eosin-stained paraffin cochlear mid-modiolar sections of Dusp1+/+ and Dusp1–/– mice showing the spiral ganglion, the organ of Corti and the lateral …
Figure 4
Organ of Corti degeneration in Dusp1–/– mice.
(A) Representative confocal maximal projection images of the organ of Corti of the middle and basal turns of 5-month-old Dusp1+/+ (n = 4) and Dusp1–/– (n = 4) mice immunolabeled for hair cells …
Figure 5 with 2 supplements
Cochlear oxidative stress status of Dusp1+/+ and Dusp1–/– mice.
(A) Cochlear expression of redox regulation and apoptosis genes in Dusp1+/+ (lighter color bars) and Dusp1–/– mice (darker color bars) of 2, 4–5 and 8–9 months of age. Expression levels were …
Figure 5—figure supplement 1
3-Nitrotyrosine ( 3-NT) immunohistochemistry.
(A) Representative microphotographs of 3-NT levels detected by immunohistochemistry in the spiral ganglion of 2-month-old Dusp1+/+ and Dusp1–/–(n = 3 per genotype). (B) Quantification of the 3-NT …
Figure 5—figure supplement 2
Production of reactive oxygen species and DNA damage in MEF cells.
(A) Dusp1+/+ (white) and Dusp1–/– (gray) MEF cells were treated with H2O2 (200 µM) for 30 min. The results for non-treated control cells are also shown for comparison (NT). After harvesting the …
Figure 6 with 1 supplement
Exacerbated inflammatory response in Dusp1–/– mice.
(A) Cochlear expression of inflammatory response genes in Dusp1+/+ (lighter color bars) and Dusp1–/– mice (darker color bars) of 2, 4–5 and 8–9 months of age. Expression levels were calculated as 2–Δ…
Figure 6—figure supplement 1
TNF-α induces caspase 3 activation in Dusp1–/– MEF cells.
(A) MEFs cells from Dusp1+/+ or Dusp1–/– were treated or not with TNFα (10 ng/ml) and harvested 30 min, 1 hr, 2 hr and 4 hr after application of a stimulus. 20 μg of WCE were resolved in SDS-PAGE, …
Figure 7
Hearing loss following noise-exposure of Dusp1 and Mkp14 null mice.
(A) ABR thresholds (mean ± SEM) in response to click and tone burst stimuli before noise exposure (baseline) and 3 and 14 days post-noise (VSS, 107 dB, 30 min) exposure in Dusp1+/+ and Dusp1–/– mice …
Author response image 1
Comparative longitudinal hearing evaluation of Dusp1+/+, Dusp1+/- and Dusp1-/-mice.
Evolution of ABR thresholds (mean ± SEM) in response to click and tone bursts stimuli in Dusp1+/+(white circles), Dusp1+/- (grey circles) and Dusp1-/-(black circles) mice of 2 (Dusp1+/+, n=10; Dusp1+…
Author response image 2
Monthly evolution of auditory thresholds.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | Dusp1-/- | Dorfman et al., 1996 | RRID:MGI:4940296 | R Perona lab (Institute of Biomedical Research 'Alberto Sols', Madrid, Spain) |
| Genetic reagent (M. musculus) | Mapk14+/KI; Mapk14KI/KI | Ventura et al., 2007 | RRID:MGI:3716853 | AR Nebreda lab (Institute for Research in Biomedicine, Barcelona, Spain) |
| Cell line (M. musculus) | Dusp1–/– MEF | Dorfman et al., 1996 | RRID:MGI:4940296 | Primary culture of mouse embryonic fibroblasts, maintained in I Sánchez-Pérez's lab |
| Antibody | Anti-myelin P0 (chicken polyclonal) | Neuromics | Neuromics Cat# CH23009; RRID:AB_2737144 | IHC (1:150) |
| Antibody | Anti-nitrotyrosine (rabbit polyclonal) | Merck-Millipore | Millipore Cat# AB5411; RRID:AB_177459 | IHC (1:100) |
| Antibody | Anti-IBA1 (goat polyclonal) | Abcam | Abcam Cat# ab5076; RRID:AB_2224402 | IHC (1:100) |
| Antibody | Anti-MyoVIIA (rabbit polyclonal) | Proteus | Proteus Biosciences Cat# 25–6790; RRID:AB_2314838 | IHC (1:250) |
| Antibody | Anti-neurofilament (mouse monoclonal) | Merck-Millipore | Millipore Cat# CBL212; RRID:AB_93408 | IHC (1:100) |
| Antibody | Alexa Fluor 647 Phalloidin | Thermo Fisher Scientific | Thermo Fisher Scientific Cat# A22287; RRID:AB_2620155 | IHC (1:1000) |
| Antibody | Anti-P-p38 (rabbit polyclonal) | Cell Signaling | Cell Signaling Technology Cat# 9211, RRID:AB_331641 | WB (1:1000; 1:2000) |
| Antibody | Anti-P-JNK (rabbit polyclonal) | Cell Signaling | Cell Signaling Technology Cat# 4668; RRID:AB_823588 | WB (1:1000) |
| Antibody | Anti-P-ERK (rabbit polyclonal) | Cell Signaling | Cell Signaling Technology Cat# 9101, RRID:AB_331646 | WB (1:1000) |
| Antibody | Anti-MKP1 (C-19) (rabbit polyclonal) | Santa Cruz Biotechnology | Santa Cruz Biotechnology Cat# sc-370; RRID:AB_631385 | WB (1:1000) |
| Antibody | Anti-γ-H2AX Ser139 (rabbit polyclonal) | Cell Signaling | Cell Signaling Technology Cat# 2577; RRID:AB_2118010 | IHC (1:200); WB (1:1000) |
| Antibody | Anti-P22phox (rabbit polyclonal) | Santa Cruz Biotechnology | Santa Cruz Biotechnology Cat# sc-20781; RRID:AB_2090309 | WB (1:250) |
| Antibody | Anti-MnSOD (rabbit polyclonal) | Merck-Millipore | Millipore Cat# 06–984; RRID:AB_310325 | WB (1:1000) |
| Antibody | Anti-BAX (NT) (rabbit polyclonal) | Merck-Millipore | Millipore Cat# ABC11; RRID:AB_10561771 | WB (1:1000) |
| Antibody | Anti-PI3K (rabbit polyclonal) | Not commercially available | WB (1:10,000) From AM Valverde's lab (Institute of Biomedical Research 'Alberto Sols', Madrid, Spain) | |
| Antibody | Anti-p38 (C-20) (rabbit polyclonal) | Santa Cruz Biotechnology | Santa Cruz Biotechnology Cat# sc-535; RRID:AB_632138 | WB (1:1000) |
| Antibody | Anti-caspasa3 (rabbit polyclonal) | Cell Signaling | Cell Signaling Technology Cat# 9662; RRID:AB_331439 | WB (1:1000) |
| Antibody | Anti-β-actin (mouse monoclonal) | Sigma-Aldrich | Sigma-Aldrich Cat# A5441; RRID:AB_4767441 | WB (1:10000) |
| Antibody | Anti-β-tubulin (mouse monoclonal) | Sigma-Aldrich | Sigma-Aldrich Cat# T9026; RRID:AB_47759 | WB (1:1000) |
| Commercial assay or kit | Dead-End Fluorometric TUNEL System | Promega | Promega Cat# G3250 | |
| Chemical compound, drug | TNFα | Sigma-Aldrich | Sigma-Aldrich Cat# T6674 | (10 ng/ml) |
| Software, algorithm | BioSigRP TM | Tucker Davis Technologies (TDT) | RRID:SCR_014590 | |
| Software, algorithm | Fiji | Fiji (https://fiji.sc/) | RRID:SCR_002285 | |
| Software, algorithm | SPSS | IBM | RRID:SCR_002865 | |
| Software, algorithm | Cell Profiler | Cell Profiler (https://cellprofiler.org/) | RRID:SCR_007358 |
Additional files
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Supplementary file 1
Monthly ABR threshold (dB SPL).
- https://doi.org/10.7554/eLife.39159.016
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Supplementary file 2
Taqman essays for RT-qPCR.
- https://doi.org/10.7554/eLife.39159.017
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Supplementary file 3
Primers for RT-qPCR.
Primers for MKPs were designed using Primer Express 3.0 software and the mouse gene sequences available on the Ensembl genome database with references: NM_013642.3 (Dusp1), NM_010090.2 (Dusp2), NM_176933.4 (Dusp4), NM_001085390.1 (Dusp5), NM_026268.3 (Dusp6), NM_153459.4 (Dusp7), NM_008748.3 (Dusp8), NM_022019.6 (Dusp10) and NM_130447.3 (Dusp16). Base numbers indicate the location of the primer sequences in the corresponding mRNA; primers for Dusp1 were designed in the region of exon 2.
- https://doi.org/10.7554/eLife.39159.018
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Transparent reporting form
- https://doi.org/10.7554/eLife.39159.019
