Offres 2024 - 2025
Offre 1
Title: Contribution des lipases du tissu adipeux au contrôle du métabolisme et de la fonction musculaire
Contact:
Nom, Prénom : MOUISEL Etienne, etienne.mouisel@inserm.fr
MCF / Ass. Professor - Physiologie Animale
Team AdipoLive / I2MC
UMR1297 Inserm/Université Toulouse III Paul Sabatier
Laboratory:
Intitulé : Métabolisme du tissu adipeux et du foie dans les maladies métaboliques (AdipoLive)
Responsable(s) : Dominique Langin / Pierre Gourdy
Laboratoire : Institut des Maladies Métaboliques et Cardiovasculaires (I2MC) - INSERM U1297
Description of the project:
Le tissu adipeux est composé de cellules adipeuses, ou adipocytes, spécialisées dans le stockage et la restitution d’énergie sous forme d’acides gras (AG) en période de besoin comme lors du jeun ou de l’exercice physique (1, 2). La lipolyse est la voie permettant l’hydrolyse et la libération des AG stockés, via deux principales enzymes, ATGL (adipose triglyceride lipase) et HSL (hormone-sensitive lipase) (3). Nous étudions au laboratoire les conséquences systémiques d’une modulation du flux lipolytique (activation pharmacologique ou suppression génétique), autrement dit comment les adipocytes dialoguent, via les AG et d’autres produits de sécrétion, avec d’autres tissus et/ou types cellulaires clés du métabolisme. Ce projet vise à ré-évaluer la contribution de la lipolyse adipocytaire au contrôle du métabolisme musculaire. Bien que le rôle des AG sécrétés et ciblant le muscle soit connu (bénéfique/énergétique lors d’un exercice physique ou néfaste/lipotoxique en cas d’obésité), les données publiées ont été obtenues en utilisant des modèles de délétion partielle de la lipolyse, i.e. une seule enzyme (ATGL ou HSL), ou non spécifique des adipocytes.
L’objectif de ce projet de stage sera donc d’évaluer finement, via l’utilisation d’un modèle murin de double délétion adipocytaire des lipases ATGL et HSL (DaKO), les capacités fonctionnelles et métaboliques d’un muscle squelettique dépourvu d’apports d’AG.
Pour cela et selon nos données préliminaires, le/la candidat/e sera amené/e à explorer un ou plusieurs des axes expérimentaux suivants :
1- Evaluation de la capacité des souris DaKO à répondre à différents stimuli impactant le système musculaire
- i) Evaluation de la capacité à l’exercice forcé des souris DaKO
- ii) Evaluation histologique et fonctionnelle de la capacité de régénération musculaire suite à l’injection d’un agent pharmacologique dégénérant le muscle.
iii) Evaluation de la capacité musculaire des souris DaKO à faire face à un stimulus engendrant une atrophie (microgravité)
2- Evaluation de la réponse musculaire « énergétique » de souris DaKO soumises à différents stress nutritionnels
- i) Evaluation du remodelage énergétique musculaire lors du jeun. Nous soumettrons les souris DaKO à différents temps de jeun et évaluerons l’impact de ceux-ci sur la perte de masse musculaire et sur la contribution des voies de dégradation protéique du muscle.
- ii) Evaluation du remodelage énergétique musculaire suite à différents régimes gras : analyse lipidomique quantitative / qualitative des différentes espèces lipidique, coloration histologique à l’huile rouge et mesure de la quantité de glycogène. Les tissus musculaires sont d’ores et déjà disponibles au laboratoire pour cela.
L’ensemble des résultats obtenus nous permettra de :
- déterminer les substrats énergétiques et voies de signalisation privilégiés dans un muscle squelettique dont le tissu adipeux ne peut lui fournir des acides gras à jeun.
- évaluer la lipotoxicité de ces mêmes muscles lors d’un contexte de régime hypercalorique.
- révéler si la lipolyse adipocytaire est nécessaire à la performance physique, ainsi qu’au remodelage anabolique ou catabolique inhérents à une lésion aigue ou une hypoactivité chronique.
Références bibliographiques éventuelles :
- Morigny et al. Lipid and glucose metabolism in white adipocytes: pathways, dysfunction and therapeutics. Nat Rev Endocrinol, 17(5): 276-295, 2021.
- Sakers et al. Adipose-tissue plasticity in health and disease. Cell, 185(3): 419-446, 2022.
- Grabner et al. Lipolysis: cellular mechanisms for lipid mobilization from fat stores. Nat Metab, 3(11): 1445-1465, 2021.
Précisions éventuelles sur les techniques utilisées et/ou les compétences requises pour le(la) candidat(e):
Expérimentation animale
Extraction protéique et Western Blot
Extraction d’ARN er RT-qPCR
Analyses histologiques
Analyse de données
Offres 2023 - 2024
Offre 1
Title: Selective autophagy and skeletal striated muscle: identification of novel molecular mechanisms involved in myotonic dystrophy type 1 (DM1)
Contact: Dr Flavie Strappazzon & Dr Rémi Mounier
Email: flavie.strappazzon@univ-lyon1.fr et remi.mounier@univ-lyon1.fr
Phone: +33 4 26 68 82 46
Laboratoire d'accueil : Physiopathologie Génétique du Neurone et du Muscle (PGNM)
UMR5261 CNRS- U1315 INSERM
Institut NeuroMyoGène
Faculté de Médecine- 8 avenue Rockefeller
69008 LYON-FRANCE
Description of the project:
The ability of skeletal muscle to maintain its homeostasis is severely compromised in patients with neuromuscular diseases. Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease affecting skeletal striated muscle as well as cardiac muscle, resulting in myofiber atrophy and skeletal muscle weakness. DM1 is caused by a CTG expansion in the 3'UTR of the DMPK gene transcript. Toxic mRNAs, carrying CUG repeats, accumulate in the nucleus of myofibers, disrupting mRNA splicing and thus altering the expression of genes involved in muscle differentiation. In fibroblasts from patients with DM1, impaired mitochondria function results in a decrease in ATP production and in an increase of the production of reactive oxygen species. Patients with DM1 show an accumulation of mitochondria in their muscle cells. These data suggest that mitophagy, a selective form of autophagy responsible for the degradation of damaged or excessive mitochondria, is impaired in DM1 patients.
Despite huge efforts, the pathophysiological mechanisms underlying DM1 still remainelusive.
AMPK kinase is a major regulator of mitophagy in muscle and AMPK signaling has been shown to be repressed in DM1 skeletal muscle. In addition, the Mounier/Courchet teams have recently observed that the absence of the kinase NUAK1 in skeletal muscle, a kinase belonging to the AMPK family, disrupts muscle fiber homeostasis. By using a combination of in vivo (mouse) and in vitro (primary culture of muscle cells) models associated with biochemical, molecular, cellular and physiological approaches mastered by the host teams, the objective of the project will be to understand what are the benefits of activation of AMPK kinase and/or NUAK1 kinase on mitochondrial dysfunction and skeletal muscle atrophy while providing insights on novel therapies for DM1.
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Offre 2
Title: Proteomics of skeletal muscle triads
Contact: Anne-Sophie NICOT
Email: nicotan@univ-grenoble-alpes.fr
Phone: +33 4 56 52 05 70
Laboratoire d'accueil :
Team: Cellular myology and pathologies (Dir. Isabelle Marty)
Grenoble Institut des Neurosciences (GIN)
Inserm U1216 , Université Grenoble-Alpes
Bât. EJ Safra, Chemin Fortuné Ferrini, 38700 La Tronche
Description of the project:
In skeletal muscle, triads are the anatomical basis for excitation-contraction coupling. A triad is composed of an invagination of the plasma membrane, the transverse tubule, flanked by two terminal cisternae of the sarcoplasmic reticulum (SR) which are calcium storage sites. Action potentials transmitted by nerves activate calcium channels at triads, leading to intracellular SR calcium release and sarcomere contraction. Calcium exchanges occurring at triads have been extensively studied but the triad structure remains mysterious in itself. Especially, the mechanisms governing the formation and the maintenance of triads remain largely unknown.
The aim of this master 2 project is to address those unresolved questions through the identification of proteins localized at triads, during their formation and, once formed, under electrical stimulation. To achieve this goal, we will use the recently developed technique of proximity-dependent biotinylation identification (BioID). BioID consists in the biotinylation of proteins surrounding an anchor protein fused to a biotin ligase. Biotinylated proteins are then enriched and identified by mass-spectrometry. The master student will learn the technics and test proteins specifically localized at triads as anchors for BioID. The optimal fusion protein will be expressed in cultured muscle cells (1) at different time points of differentiation, (2) in differentiated muscle cells stimulated or not to mimic nerve stimulation. Biotinylated proteins will then be identified by mass spectrometry.
The data obtained during the master internship will allow the identification of pathways potentially involved in the formation and maintenance of triads. This will bring new insight in the understanding of the triads, crucial structures for the function of skeletal muscles.
Methods: molecular biology, cell culture and microscopy in muscle cells, biochemistry
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Offre 3
Title: Complications following rheumatoid arthritis: elucidating the distorted glucose homeostasis in skeletal muscle
Contact: Dr. Baptiste Jude and Dr. Johanna T Lanner
Laboratory: Karolinska Institutet, Molecular Muscle Physiology & Pathophysiology Group, Dept of Physiology & Pharmacology, Biomedicum QC5, Tomtebodav 16, 17177 Stockholm, Sweden
Description of the project:
Rheumatoid arthritis (RA) is an autoimmune chronic inflammatory disease that predominantly affects women with a prevalence of 1%. In addition to primarily affecting the joints and causing swelling and pain, RA is linked to skeletal muscle dysfunction. Skeletal muscle is essential for our ability to move and is the body’s primary organ for insulin-mediated glucose disposal. Intriguingly, patients with RA have twice as high a risk to develop type 2 diabetes (T2D) as compared with the healthy population. However, the mechanisms responsible for the metabolic alteration in RA remain unknown.
Using a mice model of RA (Complete Freund’s adjuvant-induced arthritis), we observed that skeletal muscle expression of the glucose transporter 4 (GLUT4), responsible for the muscle glucose uptake following insulin stimulation and exercise, was dramatically increased by ~200%. However, when we measured ex vivo the specific glucose uptake, no increase in glucose was observed. Further, in vivo glucose uptake into skeletal was drastically reduced in these animals, as compared with healthy controls. Thus, there is a mismatch between the amount of GLUT4 available and their capacity to facilitate glucose uptake which implies and impaired GLUT4 trafficking in skeletal muscle afflicted by RA.
Here we aim to elucidate how rheumatoid arthritis alters skeletal muscle glucose disposal and metabolism.
Our preclinical model of RA in combination with transgenic and knock-out mice models targeting genes of interest will be used to analyze glucose disposal and tolerance tests in vivo and ex vivo contraction-mediated glucose uptake. Moreover, we will dissect the pathways responsible for GLUT4 trafficking, glucose uptake, and glycolysis. By using molecular and biochemical technics we will investigate the gene expression, the protein expression and regulation (i.e. phosphorylation), and also the GLUT4 localization by protein fractionation, to understand the function and the regulation of this abnormal GLUT4 expression in skeletal muscle.
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Offre 4
Title: Single-cell functional analysis of skeletal muscle regeneration
Contact: Fabien Legrand (0667537091)
Laboratory: Equipe "Signaling Pathways and Skeletal Muscles"
Institut NeuroMyoGène – Pathophysiology and Genetics of Neuron and Muscle (INMG-PGNM)
UCBL - CNRS UMR5261 - INSERM U1315
Faculté de Médecine et de Pharmacie, 8 Avenue Rockefeller, 69008 LYON
Description of the project:
Adult skeletal muscle is a complex structure endowed with remarkable regenerative potential. This ability relies on the orchestrated interplay between muscle stem cells, called satellite cells (MuSCs), and the multiple muscle resident populations. Conventional techniques have been widely used to study skeletal muscle regeneration. Unfortunately, limitations on the isolation strategies and the use of bulk-scale methods greatly restrained our comprehension of this process. We recently used a combination of single-cell RNA-sequencing (scRNA-seq) and mass cytometry (CyTOF) to define the blueprint of muscle tissue organization at the single-cell resolution (PMID: 30922843). We now intent to elucidate the framework of the cellular events underlying the different steps of muscle tissue repair at the single-cell level.
To this aim the master student will first learn how to perform scRNA-seq assay on adult skeletal muscle tissue. Next, high-resolution phenotypic and functional profiles of single cells will be generated. Then, using computer-generated topological maps the applicant will delineate the different cell populations that pre-exist and arise during muscle tissue repair and identify the novel cellular subsets involved in this process. Validation of newly classified cell populations will then be achieved by using classical techniques mastered in the lab such. Depending on the results, bulk RNA-sequencing of FACS-isolated cells may also be conducted.
The data obtained during the master internship will bring insight in MuSC hierarchy and help identify cellular sub-populations responsible for extra-cellular matrix remodeling during tissue repair. The long-term goal of this research project, extending beyond the master internship, is to identify disease-specific cell subsets in animal models of myopathy.
Technologies utilisées :Single cell RNA-seq, bulk RNA-seq, immunolocalization on tissue sections, culture of FACS-sorted cells, Western-Blot/qRT-PCR,
Mots clés : Muscle squelettique, Régénération, Voies de signalisation ; communication cellule-cellule
Publications d’intérêt :
1: Rudolf A, Schirwis E, Giordani L, Parisi A, Lepper C, Taketo MM, Le Grand F. β-Catenin Activation in Muscle Progenitor Cells Regulates Tissue Repair. Cell Reports. 2016 May 10;15(6):1277-90. doi: 10.1016/j.celrep.2016.04.022. PMID: 27134174
2: Giordani L, He GJ, Negroni E, Sakai H, Law JYC, Siu MM, Wan R, Corneau A, Tajbakhsh S, Cheung TH, Le Grand F. High-Dimensional Single-Cell Cartography Reveals Novel Skeletal Muscle-Resident Cell Populations. Molecular Cell. 2019 May 2;74(3):609-621.e6. doi: 10.1016/j.molcel.2019.02.026. PMID: 30922843
3: Girardi F, Taleb A, Ebrahimi M, Datye A, Gamage DG, Peccate C, Giordani L, Millay DP, Gilbert PM, Cadot B, Le Grand F. TGFβ signaling curbs cell fusion and muscle regeneration. Nature Communications. 2021 Feb 2;12(1):750. doi: 10.1038/s41467-020-20289-8. PMID: 33531466
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Offre 5
Title: Determinants of muscle cell fusion and syncytium formation
Contact: Fabien Legrand (0667537091)
Laboratory:Equipe "Signaling Pathways and Skeletal Muscles"
Institut NeuroMyoGène – Pathophysiology and Genetics of Neuron and Muscle (INMG-PGNM)
UCBL - CNRS UMR5261 - INSERM U1315
Faculté de Médecine et de Pharmacie, 8 Avenue Rockefeller, 69008 LYON
Description of the project:
Skeletal muscles are composed of multinucleated myofibers formed through the fusion of muscle progenitor cells during development and postnatal growth. In the adult, cell fusion is crucial for muscle regeneration after injury, and it relies on the action of satellite cells, the adult muscle stem cells (MuSCs) that form myoblasts and differentiating myocytes.
We recently demonstrated that TGFβ (SMAD2/3-dependent) signaling acts as a molecular brake on muscle fusion during mouse muscle regeneration (PMID: 33531466). The goal of the M2 internship is to identify transcriptional effectors mediating the inhibitory effect of TGFβ signaling on myoblast fusion.
To this aim, the applicant will undertake a multi-level, genome-wide approach on primary myocytes derived from FACS-sorted MuSCs. He/she will perform RNA-sequencing of myocytes with gain-of-function and loss-of-function for TGFβ signaling. Then, he/she will use Principal component analysis (PCA) to make predictive models; GO and KEGG gene annotation schemes to identify significant co-clustering of genes with similar functional properties. Finally pathway enrichment analysis and visualization will be performed using g:Profiler, GSEA, Cytoscape and EnrichmentMap. The result of these approaches will provide a gene signature of muscle cells exposed to TGFβ signaling. In addition, direct targets of TGFb signaling will be identified by CUT&TAG to analyze the binding of SMAD2/3 to DNA throughout the genome. Candidate genes that display expression patterns compatible with a role in myoblast fusion will then be functionally tested.
In summary, this project will characterize novel aspects of the machinery regulating MuSC fusion through the identification of novel molecules and the characterization of their function during this process.
Technologies utilisées : bulk RNA-seq, immunolocalization, culture of FACS-sorted cells, Western-Blot/qRT-PCR, Cut&Tag
Mots clés : Muscle squelettique, Régénération, Voies de signalisation; Fusion cellule-cellule
Publications d’intérêt :
1: Rudolf A, Schirwis E, Giordani L, Parisi A, Lepper C, Taketo MM, Le Grand F. β-Catenin Activation in Muscle Progenitor Cells Regulates Tissue Repair. Cell Reports. 2016 May 10;15(6):1277-90. doi: 10.1016/j.celrep.2016.04.022. PMID: 27134174
2: Giordani L, He GJ, Negroni E, Sakai H, Law JYC, Siu MM, Wan R, Corneau A, Tajbakhsh S, Cheung TH, Le Grand F. High-Dimensional Single-Cell Cartography Reveals Novel Skeletal Muscle-Resident Cell Populations. Molecular Cell. 2019 May 2;74(3):609-621.e6. doi: 10.1016/j.molcel.2019.02.026. PMID: 30922843
3: Girardi F, Taleb A, Ebrahimi M, Datye A, Gamage DG, Peccate C, Giordani L, Millay DP, Gilbert PM, Cadot B, Le Grand F. TGFβ signaling curbs cell fusion and muscle regeneration. Nature Communications. 2021 Feb 2;12(1):750. doi: 10.1038/s41467-020-20289-8. PMID: 33531466
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Offres 2022-2023
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Offre 1
Title: Therapeutic potential of GDF5-based treatments during immobilization and recovery in mice
Contact:
/ france.pietri-rouxel@upmc.fr
Phone: +33 +33 (1) 40 77 96 35
Laboratoire d'accueil:
Team: Gene therapy for DMD and pathophysiology of skeletal muscle
Center of Research in Myology
Sorbonne Université-UMRS974-Inserm-Institut de Myologie
Faculté de Médecine de la Pitié Salpêtrière
105 boulevard de l’Hôpital
75013 Paris France
https://recherche-myologie.en
Description of the project:
Muscle atrophy results from a significant degradation of muscle proteins which leads to a loss of muscle mass and function. However, skeletal muscle is able to induce a molecular compensatory response aimed to limit mass loss. One of the components of this compensatory response, identified in mice, is GDF5 (Growth and Differentiation Factor 5), a member of the BMPs (Bone Morphogenic Proteins) family which plays a key role in muscle maintenance following denervation (1). GDF5 induction after reversible nerve damage has been shown as fundamental to ensure the correct re-innervation of muscle fibers (2). Furthermore, our recent work demonstrated that GDF5 overexpression in aging muscle is able to prevent age-related muscle wasting, in mice (3).
One of the major problems in considering a long space expedition in the International Space Station is the loss of muscle mass induced by microgravity. This muscle atrophy occurs rapidly, from the onset of microgravity exposure, and then worsens over time. The loss of muscle mass and quality is associated with a decrease in strength which can be a problem when returning to gravity. Thus, deciphering the role of these compensatory mechanisms in atrophying conditions such as muscle loss linked to microgravity, is a promising avenue to identify new therapeutic strategies needed for the development of projects for the future stay in space.
Objective: Our project proposes to evaluate the therapeutic potential of the use of the rGDF5 protein during immobilization protocols mimicking the atrophic effect of microgravity and on recovery in mice.
Expected results. We hypothesize that the precocity and severity of muscle mass loss is related to the extent of activation of molecular mechanisms of the compensatory response to muscle atrophy in humans. Thus, by implementation with rGDF5 muscle atrophy would be reduced and functional recovery promoted. These results will have an impact on the understanding of the compensatory responses to muscle mass loss, in particular in atrophy linked to microgravity condition but also for many clinical situations as ageing.
Related Publications
- R. Sartori, E. Schirwis, B. Blaauw, S. Bortolanza, J. Zhao, E. Enzo, A. Stantzou, E. Mouisel, L. Toniolo, A. Ferry, S. Stricker, A. L. Goldberg, S. Dupont, S. Piccolo, H. Amthor, M. Sandri, BMP signaling controls muscle mass. Nature Genetics. 45, 1309–1318 (2013).
- P. C. D. Macpherson, P. Farshi, D. Goldman, pahim. Development. 142, 4038–4048 (2015).
- M. Traoré, C. Gentil, C. Benedetto, J.-Y. Hogrel, P. D. la Grange, B. Cadot, S. Benkhelifa-Ziyyat, L. Julien, M. Lemaitre, A. Ferry, F. Piétri-Rouxel, S. Falcone, An embryonic CaVβ1 isoform promotes muscle mass maintenance via GDF5 signaling in adult mouse. Science Translational Medicine. 11 (2019), doi:10.1126/scitranslmed.aaw1131.
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Offre 2
Title: Single-cell functional analysis of skeletal muscle regeneration
Contact:
Fabien Le Grand
Institut NeuroMyoGène
https://www.inmg.fr/le-grand/
fabien.le-grand@inserm.fr
Laboratoire d'accueil:
Team: Signaling pathways and striated muscles
Institut NeuroMyoGène, Faculté de Médecine Rockefeller, 8 avenue Rockefeller, 69008 Lyon
Description of the project:
Adult skeletal muscle is a complex structure endowed with remarkable regenerative potential. This ability relies on the orchestrated interplay between muscle stem cells, called satellite cells (MuSCs), and the multiple muscle resident populations. Conventional techniques have been widely used to study skeletal muscle regeneration. Unfortunately, limitations on the isolation strategies and the use of bulk-scale methods greatly restrained our comprehension of this process. We recently used a combination of single-cell RNA-sequencing (scRNA-seq) and mass cytometry (CyTOF) to define the blueprint of muscle tissue organization at the single-cell resolution (PMID: 30922843). We now intent to elucidate the framework of the cellular events underlying the different steps of muscle tissue repair at the single-cell level.
To this aim the master student will first learn how to perform scRNA-seq assay on adult skeletal muscle tissue. Next, high-resolution phenotypic and functional profiles of single cells will be generated. Then, using computer-generated topological maps the applicant will delineate the different cell populations that pre-exist and arise during muscle tissue repair and identify the novel cellular subsets involved in this process.
Validation of newly classified cell populations will then be achieved by using classical techniques mastered in the lab such as immunolocalization on tissue sections, culture of FACS-sorted cells, and Western-Blot/qRT-PCR. Depending on the results, bulk RNA-sequencing of FACS-isolated cells may also be conducted.
The data obtained during the master internship will bring insight in MuSC hierarchy and help identify cellular sub-populations responsible for extra-cellular matrix remodeling during tissue repair. The long-term goal of this research project, extending beyond the master internship, is to identify disease-specific cell subsets in animal models of myopathy.
_____________________________________________________________________
Offre 3
Title: Determinants of muscle cell fusion
Contact:
Fabien Le Grand
Institut NeuroMyoGène
https://www.inmg.fr/le-grand/
fabien.le-grand@inserm.fr
Laboratoire d'accueil:
Team: Signaling pathways and striated muscles
Institut NeuroMyoGène, Faculté de Médecine Rockefeller, 8 avenue Rockefeller, 69008 Lyon
Description of the project:
Skeletal muscles are composed of multinucleated myofibers formed through the fusion of muscle progenitor cells during development and postnatal growth. In the adult, cell fusion is crucial for muscle regeneration after injury, and it relies on the action of satellite cells, the adult muscle stem cells (MuSCs) that form myoblasts and differentiating myocytes.
We recently demonstrated that TGFβ (SMAD2/3-dependent) signaling acts as a molecular brake on muscle fusion during mouse muscle regeneration (PMID: 33531466). The goal of the M2 internship is to identify transcriptional effectors mediating the inhibitory effect of TGFβ signaling on myoblast fusion.
To this aim, the applicant will undertake a multi-level, genome-wide approach on primary myocytes derived from FACS-sorted MuSCs. He/she will perform RNA-sequencing of myocytes with gain-of-function and loss-of-function for TGFβ signaling. Then, he/she will use Principal component analysis (PCA) to make predictive models; GO and KEGG gene annotation schemes to identify significant co-clustering of genes with similar functional properties. Finally pathway enrichment analysis and visualization will be performed using g:Profiler, GSEA, Cytoscape and EnrichmentMap. The result of these approaches will provide a gene signature of muscle cells exposed to TGFβ signaling. In addition, direct targets of TGFb signaling will be identified by CUT&TAG to analyze the binding of SMAD2/3 to DNA throughout the genome. Candidate genes that display expression patterns compatible with a role in myoblast fusion will then be functionally tested.
In summary, this project will characterize novel aspects of the machinery regulating MuSC fusion through the identification of novel molecules and the characterization of their function during this process.
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Offre 4
Title: Organisation des noyaux dans les fibres musculaires
Contact:
Vincent Gache
Tel. : 04 78 77 75 51
e-mail : vincent.gache@univ-lyon1.fr
Laboratoire d'accueil:
INMG CNRS UMR 5310 - INSERM U1217 - UCBL1-Université de Lyon
8 avenue Rockefeller
69008 Lyon
Equipe : Architecture nucléaire et du cytosquelette
Description du projet :
Au cours du développement musculaire, les noyaux des fibres musculaires se positionnent activement et précisément. In fine, cette organisation nucléaire permet l’établissement de domaines nucléaires où chaque noyau garantit l’intégrité fonctionnel de la fibre musculaire. Les mécanismes moléculaires impliqués dans le positionnement et l’activité transcriptionnelle des noyaux au cours de la myogenèse restent encore peu connus.
Notre équipe a identifié 1 nouvelle protéine, lié au réseau microtubulaire qui impacte fortement l’établissement de ces domaines nucléaires. Notre équipe développe un modèle Murin, invalidé pour le gène codant pour cette protéine spécifiquement dans les muscles squelettiques.
Objectif 1: Analyser la contribution in vivo de la perte de cette protéine sur les étapes de formation/maturation du muscle (approche immuno-histochimique sur coupe de muscle, extraction de fibres musculaires, microscopie (confocales & STED).
Objectif 2: Compréhension des modifications liées à la perte cette protéine dans les fibres musculaires en terme de dynamique des cytosquelettes (Actine et microtubules) avec des marqueurs spécifiques de la dynamique des microtubules (EB-1(-3)-GFP, CLIP170-mCherry et SiR-tubulin®) et du réseau d’actine (actin-Chromobody®, actin-RFP, SiR-Actin®). Ces marqueurs, couplées à de la vidéo-microscopie, permettront de quantifier différents paramètres inhérents à la dynamique de ces réseaux de cytosquelettes.
Publications d’intérêt :
- SH3KBP1 scaffolds endoplasmic reticulum and controls skeletal myofibers architecture and integrity. Guiraud A, Christin E, Couturier N, Kretz-Remy C, Janin A, Ghasemizadeh A, Durieux AC, Arnould D, Romero NB, Bui MT, Buchman VL, Julien L, Bitoun M, Vincent Gache. (2020) BioXriv. Article-1
- Skeletal muscle MACF1 maintains myonuclei and mitochondria localization through microtubules to control muscle functionalities. Alireza Ghasemizadeh, Emilie Christin, Alexandre Guiraud, Nathalie Couturier, Valérie Risson, Emmanuelle Girard, Christophe Jagla, Cedric Soler, Lilia Laddada, Colline Sanchez, Francisco Jaque, Audrey Garcia, Marine Lanfranchi, Vincent Jacquemond, Julien Gondin, Julien Courchet, Laurent Schaeffer and Vincent Gache. bioRxiv 636464; Doi: Article-2.
- Microtubule motors involved in nuclear movement during skeletal muscle formation. Gache V, Gomes ER, Cadot. MBoC. 2017.Apr 1;28(7):865-874.
- MAP and kinesin-dependent nuclear positioning is required for skeletal muscle function. Gache V*, Metzger T*, Xu M, Cadot B, Folker ES, Richardson BE, Gomes ER, Baylies MK. Nature. 2012 Mar 18;484(7392):120-4.
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Offre 5
Title: The role of metabolic signalling in the regulation of postnatal myonuclear accretion
Contact:
Rémi Mounier
Anita Kneppers
Laboratoire d’accueil:
Team: Muscle stem cell environment and striate skeletal muscle homeostasis
Institut NeuroMyoGène, Faculté de Médecine Rockefeller, 8 avenue Rockefeller, 69008 Lyon
Description of the project:
Maintenance of skeletal muscle quantity and quality is crucial for healthy aging, and is facilitated by a remarkable tissue plasticity. Muscle-resident stem cells (MuSC) provide an important contribution to this plasticity by differentiation and subsequent fusion with the myofiber – a process called myonuclear accretion. The progression of this process is characterised by distinct MuSC metabolic requirements, and seems to depend on the myofiber metabolic state. We therefore anticipate a role of metabolism – and specifically, the metabolic regulator AMPKalpha2 – in myofiber to the MuSC signalling, directing MuSC fate towards myonuclear accretion. In this project, we will explore the role of AMPKa2 in the metabolic signalling from the myofiber to the MuSC, directing MuSC fate towards myonuclear accretion (i.e. differentiation and fusion with a pre-existing myofiber).
Objective: To test the causal role of AMPKa2 in the myofiber versus MuSC in the regulation of myonuclear accretion.
To achieve this objective, we will perform in vitro and in vivo experiments (neuromuscular electrical stimulation, primary MuSC culture, extraction of muscle fibers, immunohistochemistry, (live) microscopy) to trace the MuSC fate upon myofiber metabolic activation.
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Offre 6
Title : Therapeutic development for RyR1-related myopathies using a mouse model
Contact: PETIOT Anne
Email: anne.petiot@univ-grenoble-alpes.fr
Phone : 33 4 56 52 05 71
Laboratoire d'accueil : Grenoble Institut des Neurosciences (GIN)
Inserm U1216 – Eq. 4 « Myologie cellulaire et pathologies"
Bat. EJ Safra Chemin Fortuné Ferrini 38700 La Tronche
Description of the project:
Congenital myopathies are characterized by impairment of skeletal muscles calcium release leading to muscle weakness. An inducible and muscle-specific mouse model of congenital myopathy has been established in our laboratory to better understand the physio-pathology and especially the importance of the Ryanodine receptor-1 (RyR1) in these myopathies. RyR1 is the calcium channel responsible for the release of calcium from the sarcoplasmic reticulum to the cytosol, and so the muscle contraction. Our previous results show that our mouse model presents close similarities with human disease, characterized by loss of muscle strength and weight, and constitutes so a good model to test therapeutic approaches. The purpose of this work is to study ”in vivo” as well as “in vitro” the effect of different therapeutic chemicals and more specifically the antioxidant N Acetyl Cysteine, in order to offer therapeutic perspectives for congenital myopathies.
Methods: In vivo animal experimentation, primary muscle cell culture, immuno-fluorescence, western blot, immunoprecipitation , biochemical assays, functional assay (calcium imaging).
Références bibliographiques :
In vivo RyR1 reduction in muscle triggers a core-like myopathy. Pelletier L, Petiot A, Brocard J, Giannesini B, Giovannini D, Sanchez C, Travard L, Chivet M, Beaufils M, Kutchukian C, Bendahan D, Metzger D, Franzini Armstrong C, Romero NB, Rendu J, Jacquemond V, Fauré J, Marty I. Acta Neuropathol Commun. 2020 Nov 11;8(1):192.
Dynamics of triadin, a muscle-specific triad protein, within sarcoplasmic reticulum subdomains. Muriel Sébastien, Perrine Aubin, Jacques Brocard, Julie Brocard, Isabelle Marty, Julien Fauré. Mol Biol Cell. 2020 Feb 15; 31(4): 261–272. doi: 10.1091/mbc.E19-07-0399.
Dusty core disease (DuCD): expending morphological spectrum of RyR1 recessive myopathies. Matteo Garibaldi, John Rendu, Julie Brocard, Emmanuelle Lacene, Julien Fauré, Guy Brochier, Maud Beuvin, Clemence Labasse, Angeline Madelaine, Edoardo Malfatti, Jorge Alfredo Bevilacqua, Fabiana Lubieniecki, Soledad Monges, Ana Lia Taratuto, Jocelyn Laporte, Isabelle Marty, Giovanni Antonini, Norma Beatriz Romero. Acta Neuropathol Commun. 2019; 7: 3. Published online 2019 Jan 5.
Functional Characterization of a Central Core Disease RyR1 Mutation (p.Y4864H) Associated with Quantitative Defect in RyR1 Protein. Cacheux M, Blum A, Sébastien M, Wozny AS, Brocard J, Mamchaoui K, Mouly V, Roux-Buisson N, Rendu J, Monnier N, Krivosic R, Allen P, Lacour A, Lunardi J, Fauré J, Marty I. J Neuromuscul Dis. 2015 Nov 20;2(4):421-432.
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Offre 7
Title: Cellular interactions involved in physiological muscle repair
Contact:
Julien GONDIN, julien.gondin@univ-lyon1.fr (CR CNRS, HDR)
Laboratoire d’accueil:
Team: Muscle stem cell environment and striate skeletal muscle homeostasis (B. Chazaud; http://musclestem.com/)
Institut NeuroMyoGène, Faculté de Médecine Rockefeller, 8 avenue Rockefeller, 69008 Lyon
Description of the project:
Skeletal muscle is a highly dynamic tissue that regenerates ad integrum after an injury. We previously demonstrated that the different stages of skeletal muscle repair greatly rely on the dynamic interplay between muscle stem cells (MuSCs) and their environment including immune cells (Arnold et al., 2007) (i.e., macrophages), fibro-adipogenic progenitor (FAPs)/fibroblasts (Mackey et al., 2017) and endothelial cells (Latroche et al., 2017). However, these mechanisms have been essentially described using non-physiological models of injury (e.g., injection of toxin) that damage all the myofibers but remain totally unknown when the muscle is “mechanically” damaged by different strenuous exercises (i.e., from a mild to a severe injury as this occurs in Human (Fouré and Gondin, 2021)).
This project will investigate how inflammatory, vascular and connective cells control skeletal muscle repair in a physiological model of exercise-induced muscle injury.
The cellular interactions involved in muscle repair will be investigated using 3D histology, flow cytometry and in vivo live imaging (Lau et al., 2016) with mouse strains allowing MuSCs and macrophage tracking. We will focus on tissue resident macrophages versus monocyte-derived macrophages to define their roles according to the magnitude of muscle injury. We will also evaluate the effects of non-pharmacological approaches on the recovery of muscle homeostasis.
This project will provide the first cellular (and molecular) analysis of muscle regeneration after a physiological injury and illustrate the potential benefits of therapeutic interventions.
Techniques: Histology, immunostaining, cell biology, flow cytometry, in vivo live imaging (biphoton microscopy).
References
Arnold, L., Henry, A., Poron, F., Baba-Amer, Y., van Rooijen, N., Plonquet, A., et al. (2007). Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis. J. Exp. Med. 204, 1057–1069. doi:10.1084/jem.20070075.
Fouré, A., and Gondin, J. (2021). Skeletal Muscle Damage Produced by Electrically Evoked Muscle Contractions. Exerc Sport Sci Rev 49, 59–65. doi:10.1249/JES.0000000000000239.
Latroche, C., Weiss-Gayet, M., Muller, L., Gitiaux, C., Leblanc, P., Liot, S., et al. (2017). Coupling between Myogenesis and Angiogenesis during Skeletal Muscle Regeneration Is Stimulated by Restorative Macrophages. Stem Cell Reports 9, 2018–2033. doi:10.1016/j.stemcr.2017.10.027.
Lau, J., Goh, C. C., Devi, S., Keeble, J., See, P., Ginhoux, F., et al. (2016). Intravital multiphoton imaging of mouse tibialis anterior muscle. Intravital 5, e1156272. doi:10.1080/21659087.2016.1156272.
Mackey, A. L., Magnan, M., Chazaud, B., and Kjaer, M. (2017). Human skeletal muscle fibroblasts stimulate in vitro myogenesis and in vivo muscle regeneration. J. Physiol. (Lond.) 595, 5115–5127. doi:10.1113/JP273997.
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Offre 8
Title : Proteomics of skeletal muscle triads
Contact: Anne-Sophie NICOT
Email: nicotan@univ-grenoble-alpes.fr
Phone : +33 4 56 52 05 70
Laboratory:
Team : Cellular myology and pathologies (Dir. Isabelle Marty)
Grenoble Institut des Neurosciences (GIN)
Inserm U1216 , Université Grenoble-Alpes
Bât. EJ Safra, Chemin Fortuné Ferrini, 38700 La Tronche
Description of the project:
In skeletal muscle, triads are the anatomical basis for excitation-contraction coupling. A triad is composed of an invagination of the plasma membrane, the transverse tubule, flanked by two terminal cisternae of the sarcoplasmic reticulum (SR) which are calcium storage sites. Action potentials transmitted by nerves activate calcium channels at triads, leading to intracellular SR calcium release and sarcomere contraction. Calcium exchanges occurring at triads have been extensively studied but the triad structure remains mysterious in itself. Especially, the mechanisms governing the formation and the maintenance of triads remain largely unknown.
The aim of this master 2 project is to address those unresolved questions through the identification of proteins localized at triads, during their formation and, once formed, under electrical stimulation. To achieve this goal, we will use the recently developed technique of Turbo proximity-dependent biotinylation identification (TurboID). TurboID consists in the biotinylation of proteins surrounding an anchor protein fused to a biotin ligase. Biotinylated proteins are then enriched and identified by mass-spectrometry. The master student will learn the technics and test proteins specifically localized at triads as anchors for TurboID. The optimal fusion protein will be expressed in cultured muscle cells (1) at different time points of differentiation, (2) in differentiated muscle cells stimulated or not to mimic nerve stimulation. Biotinylated proteins will then be identified by mass spectrometry.
The data obtained during the master internship will allow the identification of pathways potentially involved in the formation and maintenance of triads. This will bring new insight in the understanding of the triads, crucial structures for the function of skeletal muscles.
Methods: molecular biology, cell culture and microscopy in muscle cells, biochemistry
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Offre 9
Title: Therapy by allele-specific silencing of Dynamin 2
Contact: Delphine Trochet
Email: d.trochet@institut-myologie.org
Phone: +33 1 42 16 57 08
Laboratory:
Centre de recherche en Myologie
Sorbonne Université - UMRS974 - Inserm - Institut de Myologie
105 boulevard de l’Hôpital
75013 Paris, France.
https://recherche-myologie.fr/
Description of the project:
Autosomal dominant centronuclear myopathy (AD-CNM, MIM #160150) is a rare congenital myopathy characterized by progressive diffuse muscle weakness with variable severity ranging from severe-neonatal to mild-adult forms. This condition results from heterozygous mutations in the DNM2 gene which encodes dynamin 2 (DNM2), a GTPase crucial for membrane vesicle formation that also plays an important role in actin and microtubules cytoskeleton regulations. Mutations or overexpression of the DNM2 gene are also involved in others diseases such as Charcot-Marie-Tooth, hereditary spastic paraplegia, others centronuclear myopathies and cancers.
One objective of our team is to study the pathomechanisms and develop therapies for AD-CNM. We have previously achieved the proof-of-concept of mutant-specific RNA silencing as efficient therapy for AD-CNM by targeting the most frequent missense mutation. We have demonstrated functional rescue in patient-derived cells using allele-specific siRNA. In a AD-CNM mouse model the expression in the muscle of allele-specific sh-RNA using adeno-Associated virus (AAV) rescued the muscle phenotype in young mice. However, in older mice the muscle phenotype is only partially improved and we have linked this partial effect to a viral transduction defect. Our objectives are now to pursue the preclinical development in the mouse model by studying the benefit of a systemic delivery in young mice, improve the late treatment administration and extend the therapy to others DNM2 mutations or overexpression.
In this context, the project will focus on one or several of the followings items i) assess the effect on muscle and others organs of systemic delivery of the therapeutic sh-RNA, ii) evaluate different carriers (AAV or nanoparticles) for their efficacy to bring the therapeutic allele-specific siRNA in vitro and in vivo, iii) test versatile DNM2 allele-specific siRNAs developed by the team in different DNM2-related diseases context.
To achieve these goals the methods used include molecular (acid nucleic extraction, RT-qPCR) and cellular (cell culture, transfection, immunostaining) biology, western Blot, histology.
References
Bitoun, M. et al. Mutations in dynamin 2 cause dominant Centronuclear Myopathy. Nature Genet 37, 1207-1209 (2005).
Zuchner, S. et al. Mutations in the pleckstrin homology domain of dynamin 2 cause dominant intermediate Charcot-Marie-Tooth disease. Nat Genet 37, 289-294 (2005).
Durieux, A., Prudhon, B., Guicheney, P. & Bitoun, M. Dynamin 2 and Human diseases. J Mol Med 88, 339-350 (2010).
Sambuughin, N. et al. Adult-onset autosomal dominant spastic paraplegia linked to a GTPase-effector domain mutation of dynamin 2. BMC Neurol 15, 223 (2015).
Zhao, M. et al. Dynamin 2 (DNM2) as Cause of, and Modifier for, Human Neuromuscular Disease. Neurotherapeutics. 2018 Oct;15(4):966-975.
Trochet, D. et al. Allele-specific silencing therapy for Dynamin 2-related dominant centronuclear myopathy. EMBO Mol Med 10, 239-253, doi:10.15252/emmm.201707988 (2018).
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Offre 10
Titre : Identification and validation of novel genes for myopathies
Contact: Jocelyn Laporte
Email: jocelyn@igbmc.fr
Phone: 0388653412
Laboratory:
Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) - CNRS UMR 7104 - Inserm U 1258
Equipe Physiopathologie des maladies Neuromusculaires
1 rue Laurent Fries / BP 10142 / 67404 Illkirch
Site web: www.igbmc.fr\Laporte
Description of the project:
Congenital myopathies are severe genetic diseases that are characterized by muscle weakness at birth. Main bottlenecks are that half of patients lack a genetic diagnosis, the pathological mechanisms are not understood, and there is no specific cure for most of them. Thus we aim to identify novel myopathy genes that will represent novel therapeutic targets. We will use exome and genome sequencing of patients DNA to identify novel implicated genes. Comparison of variants with patient phenotypes will highlight the potential mutations. The impact of mutations in candidate genes will be confirmed by in vitro assays with recombinant proteins and transfected and differentiated muscle cells followed by confocal and timelapse microscopies. The importance of the novel genes for muscle will be further assessed in mice upon gene modulation with RNA interference or CRISPR/Cas9, with help of adeno-associated virus. Overall, the success of the project will lead to a better diagnosis and patient care, and the identified genes will increase our understanding of the pathomechanisms and represent novel therapeutic targets.
References :
Lornage X, Romero NB, Grosgogeat CA, Malfatti E, Donkervoort S, Marchetti MM, Neuhaus SB, Foley AR, Labasse C, Schneider R, Carlier RY, Chao KR, Medne L, Deleuze JF, Orlikowski D, Bönnemann CG, Gupta VA, Fardeau M, Böhm J, Laporte J. ACTN2 mutations cause "Multiple structured Core Disease" (MsCD). Acta Neuropathol. 2019 Mar;137(3):501-519.
Lionello VM, Nicot AS, Sartori M, Kretz C, Kessler P, Buono S, Djerroud S, Messaddeq N, Koebel P, Prokic I, Herault Y, Romero NB, Laporte J, Cowling BS. Amphiphysin 2 (BIN1) modulation rescues MTM1 centronuclear myopathy and prevents focal adhesion defects. Sci Transl Med. 2019 Mar 20;11(484).