Transcriptional activator TAp63 is upregulated in muscular atrophy during ALS and induces the pro-atrophic ubiquitin ligase Trim63
- Institut national de la santé et de la recherche médicale, France
- Strasbourg University, France
- Sanford Burnham Medical Research Institute, United States
- Institut national de la santé et de la recherche médicale, Laboratoire SMN, France
Figures
Figure 1 with 2 supplements
Microarray meta-analysis highlights links between the deregulation of p53 family related genes and ALS.
(A) Representation of the number of deregulated target genes of the indicated transcription factors. Data were obtained using the indicated datasets from the Array Express database (EMBL-EBI) and …
Figure 1—figure supplement 1
Regulation of p53-family related genes in skeletal muscle of SOD1(G86R) and denervated mice.
Data were generated using a gene expression database deposited at ebi.ac.uk/arrayexpress (accession number E-TABM-195). Gastrocnemius muscle samples from male SOD1(G86R) mice with no symptoms …
Figure 1—figure supplement 2
mRNA levels from control and ALS patient deltoid muscles as by DNA microarray were correlated with the intensity of muscle injury.
Expression data were generated using a murine gene profiling database deposited at ebi.ac.uk/arrayexpress (accession number E-MEXP-3260). In the corresponding study, muscle injury was estimated …
Figure 2 with 1 supplement
p53-family target gene expression in muscles from ALS patients and in an ALS mouse model correlates with disease intensity.
(A–C) RNA from muscle biopsies of control and ALS patients (n = 8, Neuromuscular Unit [BioBank of Skeletal Muscle, Nerve Tissue, DNA and cell lines]) was extracted and analyzed by RT-qPCR. Absolute …
Figure 2—figure supplement 1
Gastrocnemius muscles from wild-type or symptomatic SOD1(G86R) (105 days) mice were dissected and weighted.
Graph represents the weight (n = 5). *p<0.01 compared to control, as calculated by a one-way ANOVA test followed by a Tukey post-test. NS: non denervated, S: denerveted as assessed by acetylcholine …
Figure 3
Expression of p53-family members in SOD1(G86R) muscles.
p53 family members, Chrna1 (Acetylcholine receptor subunit alpha) or muscle atrophy effectors Trim63 (MuRF1) and Fbxo32 (Atrogin1) mRNA levels were assayed in SOD1(G86R) mouse gastrocnemius muscle …
Figure 4 with 3 supplements
p63 protein expression in SOD1(G86R) muscle.
(A) Proteins from muscles were immuno-precipitated with a p63 antibody and then separated on a 10% SDS PAGE gel. Western blot experiment was performed using an antibody against TAp63. Each …
Figure 4—figure supplement 1
p53 and p63 protein expression in muscles of SOD1(G86R) mice.
(A) Proteins from muscles were immuno-precipitated with a p63 antibody and then separated on a 10% SDS PAGE gel. Western blot experiment was performed using an antibody against p63 total. Shows …
Figure 4—figure supplement 2
Gastrocnemius muscles from wild-type or symptomatic SOD1(G86R) (105 days) mice were cryodissected and probed for total p73 protein.
Graph represents the number of fibers per surface unit as indicated (n = 5). *p<0.01 compared to control, as calculated by a one-way ANOVA test followed by a Tukey post-test.
Figure 4—figure supplement 3
Gastrocnemius muscles from wild-type or symptomatic SOD1(G86R) (105 days) mice were cryodissected and probed for total p63 protein and nuclei (Hoechst).
https://doi.org/10.7554/eLife.10528.013
Figure 5 with 2 supplements
Expression of p63 and p53-family target genes following sciatic nerve crush, SOD1 expression of induction of stress
(A–D) WT and SOD1(G86R) mice (80 days of age) were anesthetized and the sciatic nerve crushed. Sham-operated contra limbs served as control (Ct). After 7 days, expression of TA isoforms of Trp63 (A, …
Figure 5—figure supplement 1
Regulation of p63 and Mdm2 expression by SOD1 (G86R).
(A) Protein were extracted from C2C12 myoblasts expressing WT or SOD1(G86R) after 5-days puromycin selection. Western blot analysis revealed TAp63, Bax or SOD1 expression. Actin was used as loading …
Figure 5—figure supplement 2
Functional interaction between members of the p53 family and ER or mitochondrial stress.
(A) Quantification: Proteins were extracted from C2C12 myoblasts treated with compounds: FCCP, Tunicamycin (Tun), Etoposide (Eto), menadione (Men). Western blot analysis revealed TAp63 expression. …
Figure 6
Effects of p53-family expression on Trim63 and p53-family target genes.
(A) Schematic representation of the Trim63 promoter indicating the location of putative p53/p63 binding sites. (B, C) C2C12 myoblasts were transfected (inserted panel: western blot) with various …
Figure 7 with 2 supplements
Regulation of Trim63 promoter by p63.
(A, B) Trim63 promoter reporter constructs were co-transfected with pCDNA3 (Ct) or TAp63 into C2C12 cells and luciferase activity was assessed 16 hr later. pGL3 was used as a negative control. Bars …
Figure 7—figure supplement 1
Regulation of Trim63 by p53 and p73 proteins.
(A) mRNA levels of Trim63 in C2C12 cells following transfection with siRNA control and siRNA directed against p73, p53 and a mix of siRNA against P53, and the TA isoforms of Trp63 and P73 (siMIX). Ba…
Figure 7—figure supplement 2
Impact of p63 on C2C12 cell survival.
(A, B) C2C12 were transfected with a GFP expression vector and either TAp63γ or ΔNp63γ expression vectors. After 24 hr, cells were left untreated (Ct) or treated with FCCP (1 µM) or menadione (1 µM) …
Tables
Table 1
Fold induction of p53-related genes in the ALS model SOD1 (G86R).
| Gene name | Function | 90 d. | 105 d. |
|---|---|---|---|
| p53-family target genes | |||
| Cdkn1a (p21) | Cell cycle arrest | 4 | 13 |
| Gadd45a | Cell cycle arrest | 5,6 | 21 |
| Peg3 | Apoptosis inducing | 3 | 7 |
| Perp | Cell cycle arrest | 4 | 12 |
| Pmaip1 | Apoptosis effector | 5 | 12 |
| Bax | Apoptosis effector | 3 | 8 |
| Siva | Apoptosis inducing | 3 | 5 |
| Zmat3 | Growth regulation | 1,6 | 1,1 |
| Eda2R | NF.Kb/JNK pathway | 3,4 | 9,4 |
| Tigar | Glucose metabolism | 0,75 | 0,2 |
| Sens1 | ROS homeostasis | - | 16,3 |
| Sens2 | ROS homeostasis | 1,27 | 1,46 |
| Sco2 | Glucose metabolism | 1,18 | 0,91 |
| Ddit3 (Chop) | ER stress | 1,14 | 0,35 |
| Bip (Grp78) | ER stress | 1,25 | 1,08 |
| Xbp1 | ER stress | 2 | 2,51 |
| p53-family regulators | |||
| Mlf1 | Cell cycle arrest/differentiation | 0,9 | 0,2 |
| Myf6 | Differentiation | 4 | 7 |
| Mdm2 | p53 degradation | 4 | 6 |
| Txn1 | Oxidative stress response | 4 | 6 |
| Id2 | Inhibition of differentiation | 2 | 3,1 |
| p53-family members | |||
| P53 | 4 | 3 | |
| TAp63 | 4 | 12 | |
| ∆Np63 | 0,5 | 0,3 | |
| TAp73 | 2 | 3 | |
| ∆Np73 | 0,9 | 0,8 | |
| Denervation/atrophy markers | |||
| Chrna1 (ACh Receptor alpha) | Neuromuscular junction | 4,2 | 12,4 |
Additional files
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Supplementary file 1
- https://doi.org/10.7554/eLife.10528.021
-
Supplementary file 2
- https://doi.org/10.7554/eLife.10528.022
