Our project is to determine the potential contribution of diseased astrocytes in the upregulation of CRMP4 in resistant MNs in order to develop therapeutic approaches based on CRMP4 downregulation on SOD1G93A mice.

By using an astrocyte-motoneuron coculture system, we observed a significant increase in CRMP4 specifically in SOD1G93A MNs plated on SOD1G93A astrocytes or treated with culture medium conditioned by SOD1G93A astrocytes. In vivo, the silencing of mutated SOD1 expression specifically in astrocytes, abolished the increase in CRMP4 level previously observed in SOD1G93A MNs at the onset of symptoms. Using the immunodetection of Matrix Metallopeptidase 9 (MMP9) and retrograde labeling experiments, we demonsrated that the increase in CRMP4 expression occurred mainly in the RES populations of SOD1G93A MNs.

All these results demonstrate for the first time the cooperation between glial cells and motoneurons in the selective degeneration of the resistant motoneurons, and may encourage the development of new strategies based on CRMP4 reduction for ALS.

(1) Saxena S, Nat Neurosci ,2009 (2) Dirren E, Ann Clin Transl Neurol, 2015 (3) Duplan L, J. Neurosci, 2010

crmp4, astrocytes, motoneuron degeneration

P03- Animal models- N° 27 to N° 49

Animal models- #2457 P03- 27- deficient rats: A robust animal model for duchenne studies Aude LAFOUX (1), Thibaut LARCHER (2), Laurent TESSON (3), Severine REMY (3), Virginie THEPENIER (3), Caroline Le GUINER (4), Virginie FRANCOIS (4), Lydie GUIGAND (2), Gilles TOUMANIANTZ (1), Anne De Cian (5), Charlotte BOIX (5), Jean-Baptiste RENAUD (5), Yan CHEREL (2), Carine GIOVANNANGELI (5), Jean-Paul CONCORDET (5), Ignacio ANEGON (3), Corinne HUCHET (1) 1. INSERM U1087, Université Nantes, Nantes, France 2. INRA UMR 703 , ONIRIS, Nantes, France 3. INSERM U1064, Université Nantes, Nantes, France 4. Atlantic Therapies, INSERM U1089 , Université Nantes, Nantes, France 5. INSERM U1154 , Museum National Histoire Naturelle, Paris, France

Duchenne Muscular Dystrophy (DMD) is a severe muscle-wasting disorder caused by in the dystrophin gene, without curative treatment yet available. For pre-clinical evaluation of therapeutic approaches, few animal models are available. Large animal models of DMD such as dogs or pigs are expensive, difficult to handle and show important clinical heterogeneity, while mdx mice exhibit only limited chronic muscular lesions and muscle weakness. A rat model could represent a useful alternative since rats are small animals but 10 times bigger than mice and could better mimic the human .

A line of Dmd mutated-rats (Dmdmdx) was generated using TALENs. Animals showed undetectable levels of dystrophin by western-blot and less than 5 % of dystrophin positive fibers by immunohistochemistry in muscles analyzed. The results showed that Dmdmdx rats have significantly reduced body weight from 4 weeks. At 3 months, limb and diaphragm muscles displayed intense necrosis and regeneration. At 7 and 12 months, these muscles showed severe fibrosis and adipose tissue infiltration. From 6 weeks, Dmdmdx rats showed significant reduction in muscle strength associated with muscular fatigue and a decrease in spontaneous motor activity. Concerning the heart, echocardiography showed significant concentric remodeling and alteration of diastolic function at 3 months. Subsequently, the heart morphology evolved into a dilated cardiomyopathy with necrotic and fibrotic tissue. A long-term study showed that life span was reduced in Dmdmdx rats. Furthermore, cardiac insufficiency or dilated cardiomyopathy were frequently the direct cause of death of these Dmdmdx rats.

In conclusion, Dmdmdx rats represent a very promising small animal model that can be used now for pre-clinical evaluation of therapeutic approaches of DMD, in particular for testing effects on disease progression and cardiac anomalies that were difficult to assess using the current DMD animal models.

animal model, muscular dystrophy, , heart, TALENs

Animal models- #2526 P03- 28- Characterization of a DmdEGFP reporter mouse Mina V. Petkova (1), Susanne Morales-Gonzales (1), Esther Gill (1), Franziska Seifert (1), Josefine Radke (2), Werner Stenzel (2), Luis Garcia (3), Helge Amthor (3), Markus Schuelke (1) 1. NeuroCure Clinical Research Center, Charité?Universitätsmedizin, Berlin, Allemagne 2. Department of Neuropathology, Charité?Universitätsmedizin, Berlin, Allemagne 3. INSERM U1179 and LIA BAHN Centre scientifique de Monaco, Montigny-le Bretonneux, Université de Versailles St-Quentin, Saint-Quentin-en-Yvelines, France

We generated a novel DmdEGFP reporter mouse, in which an EGFP coding sequence was inserted behind exon 79 of the Dmd gene. This exon is known to be present in most dystrophin isoforms and splice variants, as well as in revertant dystrophin. To date no dystrophin reporter mice exist and imaging is only possible by indirect antibody-mediated staining ex-vivo. We characterized this mouse model and found by Western blot analysis normal dystrophin expression levels in limb muscles, diaphragm, heart, brain, and retina. We found high native EGFP fluorescence at all expected sites of dystrophin expression. Skeletal muscles showed normal as well as sarcolemmal/subsarcolemmal localization of dystrophin-EGFP and of

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 components of the dystrophin associated complex. These findings rule out a dystrophic that might have been introduced by the C-terminal EGFP-tag. Following the EGFP fluorescence under the microscope it is easy to distinguish between muscles from reporter mice and their wildtype littermates, even on the level of single isolated muscle fibers. The novel dystrophin reporter mouse provides a valuable tool for direct visualization of dystrophin expression.

Animal models- #2530 P03- 29- Importance of alum particle size for delayed neurotoxic effects of intramuscular injections in CD1 mice. Guillemette Crepeaux (1), Housam Eidi (2), Eleni Tzavara (3), Jessie Aouizerate (1), Marie-Odile David (4), Bruno Giros (3), Christopher Exley (5), Christopher Shaw (6), François-Jérome Authier (1), Josette Cadusseau (1), Romain Gherardi (1) 1. BNMS, IMRB, creteil, France 2. BNMS, IMRB, creteil, 3. UPMC UM CR18, UPMC, Paris, France 4. Inserm U1204, University of Evry, Evry, France 5. Keele University, Keele University, Staffordshire, Royaume Uni 6. University of British Columbia, University of British Columbia, Vancouver, Canada

Vaccines have allowed the eradication or containment of several severe infectious . Adjuvants such as aluminium hydroxide particles (alum) have long been added to vaccines to initiate and enhance the immune response. Concerns linked to the use of alum particles emerged following recognition of their unexpectedly long-lasting biopersistence within immune cells of patients with chronic fatigue, cognitive dysfunction, myalgias and autoimmune/inflammatory features. Previous experimental reports have documented slow translocation of alum particles from the injected muscle to brain. The present study aimed at evaluating mouse behaviour and Al cerebral concentrations long after intramuscular injections of various doses of alum.

Alum adjuvant particles (Alhydrogel®) were injected in the tibialis anterior muscle in adult female CD1 mice at different doses ranging from 133 to 800 µg Al/kg of body weight. Eight validated tests were performed to evaluate behaviour and motor performances 180 days after injection. Brains removed after test session allowed determination of Al levels by atomic furnace absorption spectrometry.

A very unusual neuro-toxicological pattern limited to lower doses of alum was observed. Neurobehavioural changes, including decreased activity levels and depressed adaptative anxiety-like behaviour, were documented in animals exposed to the two lowest doses (133 and 200 µg Al/kg) but not at the highest dose (800 µg Al/kg), compared to controls. Consistently, cerebral Al levels were increased in animals exposed to the lowest doses.

This study shows that, depending on the experimental conditions, alum particles injected in muscle may induce neurotoxic effects and Al cerebral accumulation several months after injection. Above all, it appears that particle properties, such as agglomeration and particle size, are critical parameters influencing their toxicity. Indeed in vivo neurotoxic effects are presently restricted to smaller particles and lower doses, showing non-monotonic ?U? dose-response curves. In contrast to common belief that increasing the dose makes the poison, futures studies on potential toxic effects of aluminum-based adjuvants should specifically focus on small-size or low-dose conditions.

Al hydroxyde, vaccine, MMF, neurotoxicity, behaviour, mice, non-monotonic dose response

Animal models- #2578 P03- 30- Inflammation, muscular loss and ubiquitin ligases up-regulation: a new murine model of septic cachexia Damien Restagno (1), Ludovic Freyburger (1), Christian Paquet (1), Jeanne-Marie Bonnet (1), Vanessa Louzier (1) 1. APCSE Agressions Pulmonaires et Circulatoires dans le Sepsis, VetAgro Sup, Marcy l'Etoile, France

Background Severe injury and sepsis are associated with metabolic changes as catabolic response in skeletal muscle and result in muscle atrophy. Massive inflammation as occurring during sepsis contributes to weight and muscular loss. Indeed, expression of pro- inflammatory cytokines such as TNF? and IL-6 in the early phase of sepsis is known to trigger cachexia. Pro-inflammatory cytokines are responsible for the induction of several atrogenes among which MuRF1 and MAFbx, two ubiquitin ligases, are the most studied. Failure in skeletal muscle induces weakness and fatigue, prevents ambulation and delays recovery in septic patients. There is to date no animal model of long term muscle wasting due to sepsis. The aim of this study was to create and characterize a murine model of septic cachexia.

Material and methods Sepsis was induced by a mild CLP (survival ? 80%). C57BL/6 mice (male, 20-25g, 6-8 weeks) were anesthetized, caecum was isolated, ligated (1/3), punctured twice (21G needle). For Sham animals, a laparotomy was performed without ligation nor puncture. Mice weight was followed every day, blood collections and hindlimb muscles dissection were realized 2h, 6h, 1, 2, 3, 5 and 13 days after CLp. Plasmatic cytokines (TNF?, IL-6) were evaluated via Luminex technique and muscles were weighted. Muscles from the other hindlimb were used to assess ubiquitin ligases.

Results After surgery, weight loss in CLP mice increased from D1 to D11 with a peak at D3 (20% versus 10% for the Sham, p>0.05). On D12, septic mice reached their original weight which became identical to the Sham on D13. Despite a similar weight, CLP mice muscles (tibialis anterior, extensor digitorum longus and gastrocnemius) were lighter on the thirteenth day (p>0.05). Circulating TNF? and IL-6 levels increased 2h after CLP and remained higher than for the Sham operated mice (for 24h and 13 days respectively, p>0.05). On the second day post-CLP, in the tibialis anterior, a dozen of ubiquitin ligases were significantly up- regulated among which MuRF1 and MAFbx (29 and 18 fold up regulation, respectively).

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 Conclusion We finalized a model of septic cachexia. Indeed we evidenced in our CLP mice model: wasting syndrome, inflammation and ubiquitin ligases up regulation (which accounts for an activation of the proteasomal degradation pathway) which are the main conditions for cachexia.

Sepsis, cachexia, inflammation, ubiquitin ligases, atrophy

Animal models- #2601 P03- 31- Pathological mechanisms of autosomal recessive centronuclear Ivana Prokic (1), Belinda S. Cowling (1), Catherine Koch (1), Christine Kretz (1), Christos Gavriilidis (1), Vincent Gache (2), Arnaud Ferry (3), Jeanne Laine (4), Jocelyn Laporte (1) 1. Dpt Translational Medicine and Neurogenetics, IGBMC, Illkirch, France, Illkirch, France 2. INSERM U1217, UCBL-1/ Institut NeuroMyoGene (INMG)/ Muscle Nuclear & Architecture Team (MNCA), ENS Lyon, 46 Allée d'Italie, 69364 LYON cedex 07, France, Lyon, France 3. Université Paris 6 UR76, Inserm UMR 974, CNRS UMR 7215, Institut de Myologie, Unité de Morphologie Neuromusculaire, Paris, France 4. Sorbonne Universités, UPMC Univ Paris 06, INSERM UMRS974, CNRS FRE3617, Center for Research in Myology, F- 75013, Paris, France, Paris, France

Centronuclear (CNM) are a group of congenital disorders characterized by and typical skeletal muscle show small rounded fibres with central nuclei. Autosomal recessive CNM (ARCNM) is due to loss-of-function mutations in amphiphysin 2 (BIN1), including mutations inducing misplicing of the in-frame muscle specific exon 11 or causing truncation of the C-terminal SH3 domain. Recently BIN1 gain-of-function mutations have been found as well, causing autosomal dominant CNM (ADCNM).

The aim of this research is to better understand the role of amphyphysin2 / BIN1 in healthy muscle and in the of CNM. Therefore we characterized constitutive (CMV) knockout mice lines generated by targeted of exon 11 (muscle specific exon) and constitutive (CMV) and muscle-specific (HSA) knockout mice lines generated by targeted deletion of exon 20 coding for part of the SH3 domain. The SH3 domain is involved in interactions with different , among which 2 (DNM2; also mutated in the ADCNM).

Our results showed that both KO of exon 20 in all tissues and muscle-specific KO of exon 20 lead to perinatal lethality, emphasizing the importance of BIN1 in skeletal muscle maturation. Most probably due to feeding inability, mice are hypoglycemic and die within 12 hours from birth. This phenotype parallels a major disruption in T-tubule and organization and positioning. Our work suggests that BIN1 and probably its SH3 domain are indispensable for correct skeletal muscle formation.

Surprisingly our results demonstrate that the muscle specific isoform (containing exon 11) is not necessary for skeletal muscle formation or maintenance. However, its absence causes defects during muscle regeneration. Therefore for the first time we identify that the muscle specific isoform of BIN1 plays an important function in skeletal muscle regeneration.

This work provides a better comprehension of the normal role of BIN1 in skeletal muscle, and the pathological mechanisms of autosomal centronuclear myopathies.

CNM, BIN1,amphiphysin 2, centronuclear myopathies, mouse model, ARCNM, knockout, regeneration, development

Animal models- #2605 P03- 32- Automated system for hanging wire test enables researchers to use more accurate scores for estimation of muscle activity Ekaterina Shestimerova (1), Dmitry Vlodavets (2), Denis Reshetov (1), Anna Zybina (1) 1. Marlin Biotech LLC, Moscow, Russie 2. Pirogov Russian National Research Medical University, Moscow, Russian Federation, Moscow, Russie

We have created and validated an automated system for using hanging wire test as a tool in myology research practice for estimation of muscle activity in mice. The test was adopted from classic experiment ?hanging wire test?. Our system consists of software, computer, camera, box and a platform on the top 35 sm height. The 55 long and 2 mm in diameter wire is stretched in the middle of the top platform.

A researcher should pick up a mice over fore paws and put it onto the wire's center. There is the soft surface at the bottom of the box for mouse's safe fall. Mouse should hang on the wire by itself. Mouse can freely move on the wire within 5 minutes. In the classic experiment a researcher counts the quantity of failings (falls score) and successful reaching the platform in the end of the wire (reaches score). Our system calculates this scores automatically.

?Falls score? and ?reaches score? can't fully describe mouse's activity. For example, the mouse constantly moves along the wire without reaching the ends by changing its path. In such cases the ?total motor movement? of a mouse or ?work? performed by mouse are a good indicators of its activity.

We have applied this automated system for monitoring mdx mice activity during 5 days and found that ?total motor movement? of a mouse and ?work? performed by mouse more thoroughly describe mouse muscle activity. The automated system is available as a commercial product through Marlin Biotech LLC.

DMD, mdx, hanging wire test

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Animal models- #2714 P03- 33- Gait defects in model mice of dysferlinopathies Marie Chapoton (1), Christophe Scajola (1), Marc Bartoli (1) 1. Myologie Translationnelle, Aix Marseille University, Marseille, France

Dysferlinopathies are autosomal recessive muscular dystrophies caused by DYSF mutations, which lead to a reduced amount or a complete lack of . Dysferlin is a multimeric transmembrane protein involved in muscle membrane homeostasis, in particular in the repair process. Several models animals have been created and analyzed. In this study we aim at describing gait changes in model mice of dysferlinopathies. We used two different strain of dysferlin deficient mice. A mice we created with a nonsense in exon 32. The mutation introduced is the c.3477C>A (p.Tyr1159X) variation identified in a patient affected with dysferlinopathy. We also used the well characterized Bla/J mice created by the team of I. Richard. Gait analysis in mice was performed with an automated computer assisted method (GaitLab, ViewPoint) according to manufactures instructions. The equipment consists of an elevated glass plate illuminated from the side that can detect paw contact due to changes in refractive index. A high speed camera underneath the plate captures images which are first to extract new components of mouse locomotor/gait behavior when running on the glass plate. Cumulative running activity (6-10 times) for each single mouse was considered for analysis. Parameters included in the PCA were: Stride Frequency, Stride Length, Swing Time, Pair Lag, Fore Gap, Hind Gap, Width Length, Base Of Support? Thus, a total number of twenty-three variables were included in the PCA. A total of 65 mice, covering all controls and mutants tested, were included in the analysis (5-6 mice of each genotype at 3, 6, 9 and 12 months). PCA gave rise to a two-component solution accounting for ~40% of total variance. Dimension 1 has been interpreted as ?general stride frequency'. Dimension 2 accounted for ~30% of total variance and it was interpreted as ?the flattening of mice'. As presented in the figure, statistical analysis demonstrated a significant effect size when comparing all groups of mice (wt and KI in the figure). In conclusion, gait analysis is a promising tool to assess dysferlin deficiency in mice and could be used to monitor therapeutic strategies at a global level.

Gait, dysferlin

Animal models- #2778 P03- 34- coordinated development of tendons and muscles in drosophila leg lilia laddada (1), cedric soler (1), krzysztof jagla (2) 1. Laboratoire GRED, université d'Auvergne, clermont ferrand, France 2. Laboratoire GRED, INSERM, clermont ferrand, France

Muscle and tendon development in Drosophila provides an attractive model system for studying complex tissue formation and organization. In Drosophila leg, muscles derive from myoblasts associated with the leg imaginal disc. The leg disc is a flat epithelial sheet of cells which during metamorphosis evaginates in a telescopic way, to give rise to the different segments of the leg. Leg muscles are composed of several muscle fibres. They are associated with each other by the attachment to long internal tendons connected to the exoskeleton allowing precise movement of the articulated appendage. Drosophila tendon originate from ectodermic cells characterized by the expression of the Stripe (Sr) an Egfr-like transcription factor which is necessary and sufficient for their specification and differentiation.

During Drosophila leg development, a specialized subpopulation of Adult Muscle Precursors (AMPs) called founder cells (FCs) segregates close to tendon precursors, and then keep contact with the invaginating internal tendons to reach the position at which the corresponding muscle fibres will develop.

Gaining insights into the genetic control of leg muscle patterning in Drosophila could improve our understanding of how appendicular is regulated in general. Our aim is to characterize potential early interactions between muscle and tendon precursors and to identify the actors involved in the establishment of their final organization. A transcriptomic study will be performed using cell-specific approaches in order to identify allowing the acquisition of the identity of the different muscles and tendons.

drosophila, leg muscle development, tendon, tissue interaction

Animal models- #2867 P03- 35- Drosophila Nesprin1 is involved in organization Véronique Morel (1), Simon Lepicard (2), Alexandre Rey (1), Marie-Laure Parmentier (2), Laurent Schaeffer (3) 1. CNRS, UMR5239, ENS-Lyon, Lyon, France 2. CNRS, UMR 5203, IGF, Montpellier, France 3. Université Lyon 1, ENS-Lyon, Lyon, France

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 Nesprin1 is a large protein expressed in muscles and characterized by a nucleus anchoring domain in Cter, the KASH, and two association domains in Nter. KASH domain deletion has been associated to several diseases, including Emery Dreifuss Muscular Dystrophy, and was shown to induce myonuclei disorganization and muscle impairment.

We show here that disconnecting Drosophila Nesprin-1, Msp300, from nuclei by its KASH domain deletion (Msp300DKASH) leads to locomotion defects in larvae. Electrophysiological analysis of Msp300DKASH larvae reveals a synaptic malfunction, which we prove to be mainly post-synaptic. Two moieties of glutamate receptors, containing either GluRIIA or GluRIIB sub-units, are present at the neuromuscular junction. Synaptic organization investigation reveals that although Msp300DKASH larvae present the same glutamate receptor density as wild-type larvae, they have a lower GluRIIA synaptic density, explaining the locomotor observed.

We are currently investigating the mechanisms of synaptic glutamate receptor composition control by Msp300.

Our work provides the first molecular evidence for the locomotion phenotype associated with Msp-300 disconnection from nuclei, thus opening a new experimental field to explain how Nesprin-1 KASH domain deletion leads to severe muscle impairment in Emery Dreifuss patients.

neuromuscular junction, Nesprin1, drosophila, glutamate receptors

Animal models- #2914 P03- 36- Identification of a transcription factor as determinant of muscle aging in Caenorahbditis elegans Adeline Mergoud dit Lamarche (1), Laurent Molin (1), Kathrin Gieseler (1), jean-louis Bessereau (1), Florence Solari (1) 1. Equipe Bessereau, CGphiMC / UMR5534, Lyon, France

Aging is accompanied by a progressive loss of muscle mass and function termed sarcopenia. In human, sarcopenia is responsible for a decrease in mobility, leading to a reduction in the quality of life. Epidemiological studies further suggest that skeletal muscle aging is also a risk factor for the development of several age-related diseases such as diabetes, cancer, Alzheimer's disease, and Parkinson's disease.

Several cell autonomous mechanisms have been proposed to be involved in muscle aging including mitochondria default [1], apoptosis [2] and alteration of muscle protein turnover [3]. However, those data have been essentially obtained in models of experimentally induced muscle atrophy. Thus the question of their importance and kinetic in the context of physiological aging remains unanswered.We are using C. elegans to identify genetic pathways involved in muscle aging. Previous studies have shown that worms exhibit loss of mobility [4], filament disorganization and change in muscle nuclei shape with age [5]. More recently, the Xu laboratory reported a functional decline of motor neurons that precedes alteration of [6]. However those studies did not allowed to identify genuine muscle biomarker of sarcopenia that could be used to screen for genetic modifiers of sarcopenia. For this purpose, we first aimed to characterize the time course of cellular and molecular changes that take place during muscle aging.

We have shown that muscle aging is firstly characterized by a decrease in the expression of some but not all muscle genes, followed by a change in mitochondria morphology and an impairment of muscular proteostasis. As the decrease of muscle genes expression is the earliest event, we hypothesized that it may play a causal role in sarcopenia. Further genetic, cellular and molecular investigations allowed us to identify a transcription factor, which modulates different biomarkers of muscle aging and which function may be conserved in mammals.

1. Romanello V et al. EMBO J. 2010;29: 1774-1785. doi:10.1038/emboj.2010.60 2. Marzetti E et al. Gerontology. 2012;58: 99-106. doi:10.1159/000330064 3. Masiero E et al. Autophagy. 2010;6: 307-309. 4. Huang C et al. Proc Natl Acad Sci U S A. 2004;101: 8084-8089. doi:10.1073/pnas.0400848101 5. Herndon LA et al. Nature. 2002;419: 808-814. doi:10.1038/nature01135 6. Liu J et al. Cell Metab. 2013;18. doi:10.1016/j.cmet.2013.08.007

aging, C.elegans,

Animal models- #2917 P03- 37- Towards generating new dystrophic mouse mutants by homologous recombination using dimeric RNA-guided FokI-dCas9 nucleases. Laurence A. Neff (1), Leonardo Scapozza (1), Olivier M. Dorchies (1) 1. Pharmaceutical biochemistry, School of Pharmaceutical Sciences, University of Geneva, Geneva, Suisse

Duchenne muscular dystrophy (DMD) is due to the absence of dystrophin. It is characterized by progressive muscle wasting and premature death. The evaluation of therapeutic options relies mostly on dystrophic mice such as the mdx mouse and allelic variants, of which the mdx5Cv that we use in our laboratory. However, these mice develop a mild disease compared to DMD patients. DKO mice are mdx mice that constitutively lack utrophin, a dystrophin homologue. DKO mice manifest a very severe phenotype that complicates breeding schemes and is obscured by the absence of utrophin from non-muscle tissues.

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 Our lab showed that tamoxifen, a selective estrogen receptor modulator, greatly improves muscle function and structure in mdx5Cv mice. Constitutive lack of estrogen receptors ER? and ER? in mice causes sterility, which complicates investigating their roles in dystrophic disease and in tamoxifen-mediated protection, not mentioning the confounding effects of their absence from non-muscle tissues. ER? exists as 2 isoforms, ER?1 and ER?2, whose physiological roles depend on the in-frame exon 7.

We reasoned that dystrophin mice carrying floxed utrophin or floxed ER? (exon 7) alleles would allow aggravating dystrophic phenotype or preventing expression of ER?2, respectively, without the limitations of constitutive mutants. Crossing these mice with doxycycline-dependent muscle-specific Cre recombinase mice (which we have in our laboratory) would allow restricting the genetic ablation to skeletal muscles only and in a time-controlled manner.

To develop these new transgenic mice, we took advantage of the rapidly growing genome editing tool box. Because the regular Cas9-driven genome editing is associated with a high risk of off-target cleavage, we opted for dimeric gRNA-guided FokI-dCas9 nucleases. This system uses a pair of Cas9 variant, dCas9, that is unable to cleave DNA and is used to target 2 FokI subunits to a specific DNA location thanks to a pair of gRNA oligos. In order to increase the specificity even more, we mutated FokI to restrict nuclease activity to FokI heterodimers only.

The nuclease activity has been verified in HEK cells harbouring out-of-frame EGFP mutants that turn green when DNA is cleaved. Co-injection of the nucleases and targeting vectors are being performed to generate C2C12 myoblast lines that carry floxed utrophin or floxed ER? alleles. If successful, this approach will be applied to fertilized mouse oocytes for generating the new transgenic mice.

Duchenne Muscular Dystrophy, genome editing, transgenic mice, utrophin, estrogen receptor

Animal models- #2922 P03- 38- Lack of estrogens aggravates the pathology in dystrophic mice. Elinam Gayi (1), Hesham M Ismail (1), Laurence A. Neff (1), Ophélie Patthey-Vuadens (1), Katsumi Toda (2), Toshiji Saibara (2), Urs T. Ruegg (1), Leonardo Scapozza (1), Olivier M. Dorchies (1) 1. School of Pharmaceutical Sciences, University of Geneva, Geneva, Suisse 2. Department of Biochemistry, School of Medicine, Kochi University, Nankoku, Japon

Duchenne muscular dystrophy (DMD) is a severe disorder that results in progressive muscle wasting and causes death in the early adulthood. As part of our search for compounds to treat DMD, we have found that the inexpensive and safe anticancer drug tamoxifen (TAM) efficaciously counteracted dystrophic symptoms in the mdx5Cv mouse, a model of DMD. These effects were mimicked by several selective estrogen receptor modulators (SERMs) structurally related to TAM. We also found that TAM protection was abolished by fulvestrant, a pure anti-estrogenic compound. Moreover, female dystrophic mice were more resistant to muscle fatigue than males. Collectively, our findings point to a protective role of estrogen agonists in dystrophic muscle and suggest that enhancement of estrogenic signalling in muscle by SERMs might be a therapeutic approach to treat DMD.

In order to investigate the roles of estrogen deficiency in dystrophic muscle, we have generated dystrophic (mdx5Cv) mice lacking aromatase, the rate-limiting in the biosynthesis of estrogens from androgens. Longitudinal analysis of mice from 2 months to 20 months of age, revealed that lack of estrogens due to aromatase deficiency greatly decreased locomotor activity in both male and female dystrophic mice. In young mice, aromatase deficiency caused a significant increase in plasma creatine kinase. At 2 years of age, aromatase deficiency decreased force output and aggravated muscle fatigue in dystrophic males, and caused prominent fibrosis in the diaphragm and soleus muscle of dystrophic females. Other motor and structural features of aromatase deficient dystrophic mice are being analyzed.

These findings highlight the important contribution of estrogens, not only in female but also in male dystrophic mice and warrant further investigation of SERMs as adjuvant therapy for DMD.

dystrophic mouse, estrogen, aromatase, force, fibrosis

Animal models- #2925 P03- 39- The mdx5Cv dystrophic mouse: first in depth longitudinal phenotyping. Olivier M. Dorchies (1), Elinam Gayi (1), Hesham M Ismail (1), Laurence A. Neff (1), Ophélie Patthey-Vuadens (1), Urs T. Ruegg (1), Leonardo Scapozza (1) 1. School of Pharmaceutical Sciences, University of Geneva, Geneva, Suisse

The mdx mouse is the most commonly used animal model for Duchenne muscular dystrophy. The mdx5Cv mouse, an allelic variant of mdx, is in the C57BL/6J background like most transgenic mice, exhibits a more severe phenotype than the mdx mouse, and does not display revertant fibres, a valuable feature for testing dystrophin restoration therapies. In spite of these advantages, the mdx5Cv phenotype has never been described in detail.

We have started the systematic characterization of male and female mdx5Cv mice, and their wild type counterparts, at the age of 2, 3, 4, 5, 8 weeks, and 8, 16, and 24 months. These time points encompass all the critical stages of the murine disease: the asymptomatic stage, necrosis and regeneration of young individuals, low intensity chronic disease of adults, and late muscle defects of elderly, including prominent fibrosis and cardiac dysfunction.

To date, around 350 mice have been investigated with respect to non-invasive locomotor activity, skeleton structure and quality (using X-ray tomography), isometric contraction of triceps surae muscle in situ, blood biomarker levels, muscle weights and histological characteristics. The following parameters were assessed: Locomotor activity score, body weight and length, ano- genital distance, kyphosis index, ectopic calcifications of the Achilles tendon, density, weight of diaphragm, triceps, and

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 heart muscles, phasic and tetanic forces, kinetics of contraction and relaxation, force-frequency relationship, muscle resistance to repeated tetanization, plasma CK and other biomarkers, amount of necrosis, centronucleated fibres, fibrosis, etc. Our data should allow setting reference values and help designing optimal procedures for testing therapeutic interventions in mdx5Cv mice. Our large collection of samples shall be available for analysis by other scientists through collaborations.

Dystrophic mouse, Duchenne muscular dystrophy, phenotyping, force, biomarker

Animal models- #2927 P03- 40- PCR-restriction-based strategies allow genotyping the mdx mutation and several allelic variants that carry point mutations without sequencing. Laurence A. Neff (1), Elinam Gayi (1), Leonardo Scapozza (1), Olivier M. Dorchies (1) 1. Pharmaceutical biochemistry, School of Pharmaceutical Sciences, University of Geneva, Geneva, Suisse

Duchenne muscular dystrophy (DMD) is due to the absence of dystrophin. It is characterized by progressive muscle wasting and premature death. The exploration of the pathogenic mechanisms and the evaluation of therapeutic options rely mostly on dystrophic mice.

The mdx mouse originates from a spontaneous mutation that arose in a colony of C57BL/10 mice in the 1980's. Since then, it has become the most commonly used animal model of DMD.

In the 1990's, several allelic variants of the mdx mouse, namely the mdx2Cv, mdx3Cv, mdx4Cv, and mdx5Cv, have been recovered from chemical mutagenesis in the C57BL/6J background. In some circumstances, these dystrophic mice have advantages over the original mdx. First, the C57BL/6J genetic background is commonly used for generating transgenic mice, which facilitates the construction of multiple mutants. Second, the mutations are located on different exons. These features allow differential expression of naturally occurring shorter isoforms (C-terminus truncated) of dystrophin in a tissue-dependent manner: The mdx2Cv, mdx3Cv and mdx4Cvstrains are useful strains for studying the roles of dystrophin and dystrophin- associated glycoprotein complex in the retina, kidney, and brain. Third, these strains are associated with different levels of ?revertant fibres?, in which dystrophin is expressed secondary to spontaneous skipping of the mutated exons. Interestingly, the mutation carried by the mdx5Cv mouse is such that revertant fibres are virtually absent, an advantage for evaluating gene-, virus, or cell-mediated dystrophin restoration.

The mdx, mdx2Cv, mdx3Cv, mdx4Cv, and mdx5Cv strains all carry point mutations. Conventional genotyping involves PCR amplification, leading to amplicons of equal sizes, followed by allele identification by sequencing. Sequence analysis revealed that the mdx3Cv and mdx5Cv alleles bear restriction sites for AluI, and HphI, respectively, which are absent from the wild type allele. The mdx4Cv mutation disrupts a restriction site for BsmAI. Based on these findings, we designed new strategies for genotyping these dystrophic strains using PCR amplification followed by enzymatic restriction of the amplicon. Compared to strategies proposed by others, our procedure is time and cost effective: It is achieved within a day using standard molecular biology techniques, does not make use of extended primers requiring complicated PCR protocols, and eliminates extra cost for sequencing.

Dystrophic mouse, Duchenne muscular dystrophy, genotyping, PCR, method

Animal models- #2975 P03- 41- A mutant form of Mitofusin commonly associated with CMT2A neuropathy triggers neuronal alterations by enhancing mitochondrial fusion NAJLA EL FISSI (1), AICHA AOUANE (1), MANUEL ROJO (2), JULIEN ROYET (1), THOMAS RIVAL (1) 1. IBDM, Marseille, France 2. IBGC, Bordeaux, France

Charcot-Marie-Tooth type 2A disease (CMT2A) is a dominant hereditary neuropathy triggers by mutations in the mitofusin MFN2. Several pathogenic mutations, targeting in particular the GTPase domain, have lost their mitochondrial fusion activity, and were defined as dominant negative forms. However, some CMT2A alleles, affecting different domains, mysteriously encodes functional proteins. Indeed, we showed that MFN2 carrying the highly prevalent R364W substitution restores normal mitochondrial morphology in MFN1/MFN2 double knock out cells. To further characterize the pathogenicity of the R364W allele in vivo, we introduce the corresponding substitution within the drosophila mitofusin Marf, and expressed this construct in fly neurons. Consequently, flies developed locomotor defects associated with mitochondria depletion at neuromuscular junctions (NMJ). Very intriguingly, by analysing mitochondrial morphology in motor neuron, we reveal the presence of giant mitochondria in their soma. This contrasts with the aggregates of mitochondria found in fly neurons expressing CMT2A-like alleles previously characterised as dominant negative (R94Q, T105M). To test whether the giant mitochondria phenotype was a consequence of enhanced mitochondrial fusion, we overexpressed the fission effector DRP1 in R364W-mimetic flies. This rescues mitochondrial morphology, mitochondrial transport at NMJ, and locomotion demonstrating that excessive mitochondrial fusion was responsible of these phenotypes. Finally, we showed that our CMT2A flies accumulate more mtDNA mutations. These defects were rescued by overexpressing DRP1 or PARKIN showing that hyperfusion might impair the removal of defective mitochondria. In conclusion, we showed for the first time that enhanced mitochondrial fusion activity can drive CMT2A neupathy providing new tracks for future therapies.

Animal models- #2977 P03- 42- Assessment of cardiac defects in Drosophila models of Steinert disease and identification of associated candidate genes by a cell-specific approach: TU-tagging Emilie Plantié (1), Lucie Picchio (1), Yoan Renaud (1), Krzysztof Jagla (1) 1. Laboratoire GReD, Université d'Auvergne, Clermont-Ferrand, France

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The most common muscular dystrophy in adults, Steinert disease or Type 1 (DM1) is caused by an unstable CTG repeat expansion in the 3' untranslated region of the Dystrophia Myotonica Protein Kinase (DMPK) gene. This multisystemic disease, affecting particularly skeletal muscles and the heart is called a spliceopathy because it involves the sequestration of the MBNL1 splicing factor by the abnormal CUG repeats of mutated dmpk transcripts and the stabilization of the CELF1 splicing factor. The deregulation of the balance between these two factors is responsible for disease symptoms.

Cardiac symptoms occur in 80% of DM1 patients, ranging from conduction defects to arrhythmia and are the second cause of death, mainly due to heart block. Moreover, a positive correlation between CTG repeats number and cardiac involvement has been reported [1]. To better understand the causes of cardiac symptoms, we took advantage of our DM1 Drosophila models [2], performing phenotypic analyses on adult heart of DM1 flies which express specifically in the heart: i) 960 interrupted CTG, ii) a RNAi construct for Mbl (MBNL1 orthologue), iii) a gain-of-function for bru3 (CELF1 orthologue). These heart analyses showed similar symptoms, as observed in patients, such as dilated cardiomyopathy, fibrillation or conduction defects. To identify new molecular actors responsible for the DM1 associated heart defects, we performed cardiac cell-specific transcriptional analyses by RNA-sequencing, using TU-TAGGING technique [3]. We are currently selecting deregulated candidate genes, differently spliced transcripts but also modified non-coding RNAs that could be linked to the particular observed phenotypes. The identified candidates, ranked depending on their conservation and deregulation level will be validated in a near future.

Then, we would like to test whether identified candidates are deregulated as well in DM1 patients displaying cardiac abnormalities.

[1] Petri H, Vissing J, Witting N, Bundgaard H, Køber L. Cardiac manifestations of myotonic dystrophy type 1. Int J Cardiol. 2012 [2] Picchio, L., Plantié, E., Renaud,Y., Poovthumkadavil, p. and Jagla, K. Novel Drosophila model of myotonic dystrophy type 1: phenotypic characterization and genome-wide view of altered . Hum.Mol.Genet. 2013 [3] Miller MR, Robinson KJ, Cleary MD, Doe CQ. Tu-tagging: Cell type-specific RNA isolation from intact complex tissues. Nat Meth. 2009

Steinert, DM1, heart, Drosophila, animal model, fibrillation, dilated cardiomyopathy, conduction, TU-tagging, RNA-sequencing

Animal models- #3013 P03- 43- Lack of TRPC1 cation channels enhances muscle fatigue in mdx5Cv dystrophic mice Hesham Ismail (1), Nadège Zanou (2), George Shapovalov (3), Elinam Gayi (1), Miriam Kovacs-Strosova (3), Julie Reutenauer-Patte (3), Emmanuelle Roulet (3), Alexander Dietrich (4), Lutz Birnbaumer (5), Leonardo Scapozza (1), Philippe Gailly (2), Urs Ruegg (6), Olivier Dorchies (1) 1. School of Pharmaceutical Sciences, Pharmaceutical Biochemistry, University of Geneva, Geneva, Suisse 2. Institute of Neuroscience, Université Catholique de Louvain, louvain, Belgique 3. School of Pharmaceutical Sciences, University of Geneva, Geneva, Suisse 4. Walther-Straub-Institute for Pharmacology and Toxicology, University of Munich, Munich, Allemagne 5. Laboratory of Signal Transduction, NIH, Research Triangle Park, North Carolina, Etats-Unis 6. School of Pharmaceutical Sciences, Pharmacology, University of Geneva, Geneva, Suisse

Calcium dysregulation has been shown as one of the triggering events causing muscular degeneration in Duchenne muscular dystrophy (DMD). The increased calcium influx could result from transient membrane lesions or from influx through overactive cationic channels such as store operated channels or stretch activated channels. The transient receptor potential, canonical type 1 (TRPC1) cationic channel has been reported to contribute to such influxes and to be involved in skeletal muscle function. After crossing mdx5Cv dystrophic mice (a model of DMD) with TRPC1 knock-out mice, the progeny was further crossed to generate wild-type mice (WT), dystrophic mice (Dys), TRPC1KO mice (C1), and double mutant mice (DysC1).

The lack of TRPC1 on calcium regulation and muscle function was examined in mice with normal and dystrophic muscle.

The phenotype of C1 and DysC1 mice was compared to that of WT and Dys mice using a combination of functional, biochemical, and molecular approaches in vivo, ex vivo, and in vitro. Muscle function was measured using a locomotor activity assay, grid hanging tests, and isometric and eccentric recordings of muscle mechanical properties. Alteration of calcium handling was assessed using electrophysiology recordings and fluorescent measurement of calcium fluxes in isolated muscle fibres, calcium uptake in whole muscles, and SERCA activity on muscle homogenates. The absence of TRPC1 modified neither the generation of phasic and tetanic force measured isometrically, nor the loss of force mediated by eccentric contractions. However, lack of TRPC1 slightly altered muscle structure, increased plasma CK, decreased score in a grid hanging test, diminished wheel running capacity, and enhanced muscle fatigue induced by repeated tetanic contractions. Overall, these effects were more intense in dystrophic than in wild type mice. Molecular assays revealed that the lack of TRPC1 correlated with over-expression of other cationic channels, of which TRPV2.

Altogether, our results show that the lack of TRPC1 from WT and Dys muscles spares force generation but aggravates muscle fatigue, probably secondary to an altered capacity of the muscles to reconstitute intracellular calcium stores. Although TRPC1 seems to play a role in the pathogenesis of dystrophic mice, understanding its roles is complicated by concomitant up-regulation of other cationic channels.

Duchenne, calcium channels, TRPC, fatigue

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 Animal models- #3045 P03- 44- Quantitative muscle ultrasound as a follow-up tool in the GRMD dog model of DMD Inès Barthélémy (1), Olivier Piperno (1), Xavier Cauchois (1), Isabel Punzon (1), Stéphane Blot (1) 1. UMR U955 Equipe 10 BNMS Neurobiologie, Université Paris Est, Ecole nationale vétérinaire d'Alfort, Maisons-Alfort, France

The dystrophin-deficient dog is a clinically and histopathologically relevant model of Duchenne muscular dystrophy (DMD) to assess a therapeutic effect, because it shares numerous similarities with patients in its disease course. In DMD patients quantitative muscle ultrasound has proven to be a promising tool to follow-up the dystrophic process, an increased echointensity being correlated with disease progression. This study aimed to assess the potential interest of this tool in the GRMD (Golden retriever muscular dystrophy) dog model of DMD.

Ultrasound assessments of the cranial sartorius and biceps femoris muscles were performed iteratively in ten GRMD and five heathy littermates followed-up between 2 and 12 months of age. Longitudinal images of the muscles were acquired using a 12.5-5 MHz linear transducer under general anaesthesia. The echointensity of the muscles was measured using a gray scale analysis performed in the Image J software, and the muscle section was measured on the ultrasound scanner (Philips HD7). Both muscles showed increased echointensity progressing with age, and this was especially the case of the sartorius muscle. The section of this latter muscle was markedly increased confirming the already described hypertrophy of this muscle. Interestingly the progression of this hypertrophy was correlated to the disease severity, corroborating the hypothesis following which hypertrophy pathways may be implicated in the disease modulation in dystrophin-deficiency.

Complementary studies are ongoing to study inter- and intra-operator variability in the image acquisition, and the effects of sedation on this variability. Despite the probable lack of specificity of the proposed indices to the DMD pathology, this study reveals that quantitative muscle ultrasound may represent a possible new outcome measure to add to the evaluation tool panel developed for GRMD dogs, as a non-invasive, simple to perform, and translatable test to iteratively follow-up the dystrophic process in this preclinical model.

DMD, GRMD dog, muscle ultrasound

Animal models- #3046 P03- 45- Transcriptional control of muscle identity in Drosophila. A new type of muscles connecting internal organs. Laetitia Bataillé (1), Jean-Louis Frendo (1), Laurence Dubois (1), Alain Vincent (1) 1. Centre de Biologie du Développement, CNRS-Université Toulouse 3, Toulouse, France

Development of the musculature of the Drosophila larva is a classical model to decrypt Trancription Regulatory Networks (GRNs) controlling myogenesis and muscle identity. Each striated muscle is a single multinucleated fibre which forms by fusion of a Founder myoblast (FC) with fusion competent myoblasts. Muscle identity- orientation, shape, size, skeletal attachment sites and innervation specific to each skeletal muscle- reflects the expression, by each FC, of a specific combination of identity Transcription Factors (iTFs). Using a genetic screen in Drosophila, we recently identified several new Drosophila iTFs, including the Lim-Homeodomain protein Tup and the homeodomain protein Sine Oculis, the orthologs of mammalian Islet1 and Six1/2 proteins, respectively. It also revealed new functions of Eya , and D-MyoD in controlling the morphological diversity of Drosophila skeletal muscles. Along these studies, we found that Org-1, the Tbx1 ortholog, acts upstream of Tup/Islet-1 in controlling the development of both alary muscles (AMs) and previously undiscovered muscles in the thorax, which we named TARMs (Thoracic-Alary-Related Muscles). While AMs connect the exoskeleton to the heart, we found that TARMs connect to different elements of the digestive system, and that targeting of these internal organs is under Hox control.

Based on the morphology of TARMs and their connections to the digestive system and affixing to the trachea, we hypothesized that they play an architectural role. We are currently assessing the physiological consequences of genetically ablating them.

A core network of TFs, including Tbx1, Islet1 and MRFs, controls the development of cardiopharyngeal derivatives in mammalians. Our studies of a new type of Drosophila muscles provides a new, extended framework for studies of Islet1, Tbx1, Eya, Six functions and transcriptional regulatory interactions in the functional diversification of muscle lineages in humans and associated .

Myogenesis, alary muscle development, transcriptional networks

Animal models- #3047 P03- 46- Multiparametric longitudinal follow-up of HACD1-deficient CNM dogs Inès Barthélémy (1), Xavier Cauchois (1), Isabel Punzon (1), Stéphane Blot (1) 1. UMR U955 Equipe 10 BNMS Neurobiologie, Université Paris Est, Ecole nationale vétérinaire d'Alfort, Maisons-Alfort, France

Centronuclear myopathies (CNM) are congenital diseases with different inheritance patterns, originating from various genetic mutations, but sharing common features such as a generalized muscle weakness, centralized myofiber nuclei, myofiber atrophy, Page 45

 and triadopathy. Spontaneously affected dogs with centronuclear myopathies reproduce the human diseases from a clinical and histopathological point of view and are relevant to perform preclinical studies. Among them Labrador retriever dogs with HACD1-deficiency are a model of autosomal recessive centronuclear myopathy, and in addition, recently a family of patients with a mutation in the same gene has been identified. This study aimed to provide a multiparametric quantitative description of the disease evolution in this dog model.

Two CNM dogs and two healthy carrier littermates were iteratively followed-up during their first year using the following tests: gait analysis using accelerometry, respiratory inductance plethysmography, quantitative muscle ultrasound and muscle force measurement. Compared to their healthy carrier littermates, dogs affected with CNM exhibited a hypotonic gait attested by a decreased relative dorso-ventral power progressing over time. Respiratory inductance plethysmography revealed a decreased abdominal contribution to the inspiratory effort, attesting a diaphragmatic weakness. Quantitative muscle ultrasound showed a progressive increase of muscle echointensity and decrease of the CNM biceps femoris muscle section. Muscle force measurement showed a marked right-shift of the force-frequency relationship progressing with age. Interestingly, among the two CNM brothers, one showed a more severe phenotype, assessed by early (2 months of age) hypotonic gait and diaphragmatic weakness, associated with an early right-shifted force-frequency relationship, suggesting that EC-coupling defect could be a primary and key pathogenic feature consistent with triad morphologic alterations observed in these animals.

Despite the low number of dogs included in this study, the functional impairments are consistent with the observations made in other canine models of CNMs, such as X-linked myotubular myopathy. This study also confirms that the evaluation tools initially developed for dystrophin-deficient dogs can be easily used in other canine models of human myopathies, revealing specific functional impairments.

Centronuclear myopathy, dog, HACD1, functional evaluation

Animal models- #3058 P03- 47- A feline model for congenital myasthenic syndrome with COLQ deficiency Marie Abitbol (1), Christophe Hitte (2), Philippe Bossé (1), Nicolas Blanchard-Gutton (1), Inès Barthélémy (1), Pablo Aguilar (1), Xavier Cauchois (1), Anne Thomas (3), Lionel Martignat (4), Stéphane Blot (1), Laurent Tiret (1) 1. Inserm IMRB U955-E10 BNMS Ecole nationale vétérinaire d'Alfort, Maisons-Alfort, France 2. Institut de Génétique et Développement de Rennes IGDR, UMR6290 CNRS?Université de Rennes 1, Rennes, France 3. Antagene, La Tour de Salvagny, France 4. ONIRIS-Ecole nationale vétérinaire de Nantes, Nantes, France

A congenital neuromuscular disorder characterized by skeletal muscle weakness, fatigability and variable electromyographic or muscular histopathological features has been described in the two related Sphynx and Devon Rex cat breeds. A four-month-old affected Sphynx cat was referred to our clinics and this proband allowed to collect clinical, histopathological and genetic data from a large family. Analysis of genome-wide SNP haplotypes specifically shared by a sib-pair of affected cats from this family, revealed six candidate regions. Using genotypes from five closely related cats, the disease was fine-mapped to a single region of feline C2. In that region, we further identified COLQ (collagen-like tail subunit of asymmetric acetylcholinesterase) as a good candidate. COLQ encodes the collagenic tail of acetylcholinesterase, which terminates signal transmission by hydrolysing acetylcholine at the neuromuscular junction. COLQ mutations were identified in human patients and in Labrador Retrievers affected by endplate acetylcholinesterase deficiency, a synaptic form of congenital myasthenic syndrome (CMS). A homozygous c.1190G>A missense variant located in exon 15 of COLQ was identified in the two affected Sphynx cats. This variant leads to a cysteine to tyrosine substitution at position 397 (C397Y), a highly-conserved residue within the C-terminal domain of the protein which mutation was previously shown to impair COLQ interaction with proteins from the synaptic network and to produce CMS in humans, and here we confirmed in an affected cat that it induces a loss of acetylcholinesterase clustering at the neuromuscular junction. Segregation of this variant was 100% consistent with the autosomal recessive mode of inheritance of the disorder; in addition, an affected, unrelated cat recruited thereafter, was also homozygous for the variant. Genotyping of a panel of 221 cats from 12 breeds failed to identify carriers in non-Sphynx and non-Devon Rex cats.

We propose that the c.[1190G>A] allele, which is associated with the neuromuscular disorder that was previously named spasticity, myopathy or muscular dystrophy of the Sphynx and Devon Rex, defines the first model of CMS in cats. Identifying this neuromuscular disorder as a CMS opens the opportunity to further evaluate in affected cats the efficiency of new treatments and to decipher the molecular mechanism of treatments currently used in human patients.

CMS, cat, neuromuscular junction, animal model

Animal models- #3252 P03- 48- Pre-clinical analyzes of motor activity and in mice: exemple of knock-out in the hemochromatosis HFE gene philippe noirez (1), Haidar Djemai (1), Damien Vitiello (1), Brice Fouque (1), Rémi Thomasson (1), Jean-François Toussaint (1) 1. IRMES EA7329, Université Paris Descartes, paris, France

Introduction: We recently showed that 80% of French sport winners in Olympic, World and Europeans competitions have mutations in the hemochromatosis HFE gene. Our objective was to estimate phenotypic and metabolic differences in performance between knockout (KO) and wild type (WT) mice for HFE gene.

Methods: 36 male sedentary mice of 6 months old (young, 10 KO and 12 WT) and 20 months old (old, 5 KO and 9 WT) were measured by magnetic resonance (Bruker, Germany) for their lean and fat mass and by echocardiography for their cardiac function.

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 Performance evaluation of each mouse proceeds on a one-way-treadmill equipped with calorimetric system (Phenomaster, TSE, Germany).

The following parameters were measured: oxygen consumption/carbon dioxide production (VO2, VCO2), respiratory exchange rate (RER = VCO2/VO2).

We analyzed mice performances with 2 protocols: the first protocol consisted in increasing the speed by 1 cm.s-1 every 15s; VO2 peak was determined as the highest value of VO2 reached over 15s. The second protocol was carried out to 75% of their best speed reached during the previous test (Vmax).

Results: KO mice had a body weight (g) significantly higher (31±3.3 vs 28.5±3.3; p>0.05). Lean mass (g) was significantly more important both in young (22.3±0.4 vs 18.9±0.8; p>0.001) and in old (22.7±2.6 vs 20.2±0.8; p=0.01) KO mice. Their fat mass was also significantly reduced in both groups. The ejection fraction is significantly lower in KO mice than in WT at any age (in young: 72.3±3.4 vs 81.0±1.4 %, p>0.05; in old: 58.5±4.4 vs 72.1±4.5 %, p>0.01).

Results of the first protocol indicated that VO2 peak relative to lean mass (ml.h-1.g-1) was significantly reduced in KO mice. RER peak was lower in old (0.85±0.04) than in young (0.96±0.06) mice (p>0.001). The results of the second protocol were similar to those of the first one with regards to the RER peak and RER mean. During the second protocol, the time (sec) of race and the run distance (m) observed for WT were significantly higher than those of the KO mice (1634±1108 vs 978±667; p=0.05 and 504±316 vs 306±187; p>0.05 respectively).

Conclusions:

HFE-/- KO mice presented a decrease in oxygen uptake and a reduced capacity in endurance performance. This loss of performance seemed to be amplified with age. The cardiac function was greatly decreased in aged HFE-/- KO mice. In perspectives, in these mice, we will analyse the effect of endurance training on performance.

exercise, oxygen consumption, performance

Animal models- #3275 P03- 49- FGF/MAPK-dependent spatio-temporal patterning of the ascidian cardio-pharyngeal mesoderm FLORIAN RAZY-KRAJKA (1), Nicole KAPLAN (1), Wei WANG (1), Alberto STOLFI (2), Yelena BERNARDSKAYA (1), Lionel CHRISTIAEN (1) 1. NYU, NEW YORK, Etats-Unis 2. NYU, Array, France

FGF signals transduced by the Ras/MAPK pathway are crucial for the development of numerous tissues, from limbs to nervous system and teeth. Similarly, several FGF ligands are necessary for the correct development of the cardio-pharyngeal (CP) mesoderm, from which both the pharyngeal and the cardiac muscles arise. FGF signaling both promotes Tbx1 expression and pharyngeal muscle development and opposes early cardiac differentiation. Nonetheless, how sequential FGF/MAPK signals are integrated into the progressive deployment of the CP gene regulatory network remains elusive in vertebrates.

The tunicate Ciona intestinalis has emerged as a chordate model to study conserved aspects of CP development in a simple context with cellular resolution. Here, we built upon the exquisite knowledge of the cell lineage in Ciona to study the contribution of FGF/MAPK signals in the cell fate decisions of the multipotent CP progenitors called the trunk ventral cells (TVCs).

We show that MAPK signaling is first active in the TVCs and is then restricted to their lateral progeny as it is excluded from the medial heart precursors. Gain and loss-of-function assays show that FGF/MAPK signaling is both necessary and sufficient to promote the pharyngeal muscle identity at the expense of the heart fate. Remarkably, in the heart precursors, inhibition of FGF/MAPK transcriptional inputs occurs at the level of Ras activation and might require the Ets repressor Erf/Etv3, which is normally inhibited by MAPK-mediated phophorylation. Finally, positive feed-forward circuits involving FGF/MAPK signaling and the transcription factors Hand-related, Tbx1/10 and COE(Collier/Olf/Ebf) drive the progressive pharyngeal muscle specification and its restriction to a subset of the CPM

Altogether, our results describe how the progressive restriction of FGF/MAPK signaling impinges on the regulatory programs underlying heart vs. pharyngeal muscle specification and patterns the CP mesoderm in a simple chordate.

cardio-pharyngeal mesoderm, FGF signaling, cell patterning, gene regulatory network.

P04- Cardiac stem cells, cardiogenesis and cardiomyopathies- N° 50 to N° 65

Cardiac stem cells and cardiogenesis- #2396 P04- 50- Discovery and cardioprotective effects of the first non-peptide agonists of the G protein-coupled prokineticin receptor-1. ADELINE GASSER (1), Canan NEBIGIl (1), Laurent DESAUBRY (2) 1. UMR7242 CNRS, STRASBOURG, France 2. UMR7200 CNRS, STRASBOURG, France

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