ICNMD XIII 13th International congress on Neuromuscular Diseases Nice, France July 5-10, 2014 Plenaries Sessions Abstract Books Journal of Neuromuscular Diseases 1 (2014) S3–S79 S3 DOI 10.3233/JND-149001 IOS Press Abstracts PLENARY SESSION 01 branched phenotype has been reported in vivo in mdx Theme: BASIC SCIENCES, MUSCLE AND NERVE DEVELOP- animals or in Duchenne patients, it has been attributed MENT to fusion defects consequent to the cycles of regenera- tion occurring in dystrophic muscles. Our results rath- PL1.1 Modelling Duchenne Dystrophy er argue that the defect is intrinsic to the fi bers thus challenging current views on the origin of the pathol- with embryonic stem cells ogy of Duchenne Muscular Dystrophy. Olivier POURQUIE, Strasbourg (France) Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS (UMR 7104), Inserm PLENARY SESSION 01 U964, Université de Strasbourg, Illkirch. F-67400, Theme: BASIC SCIENCES, MUSCLE AND NERVE DEVELOP- France. MENT Whereas the in vitro differentiation of certain lin- eages such as cardiomyocytes or neurons from plu- PL1.2 Regulation of Muscle Satellite ripotent cells is now well mastered, the production of Cells other clinically relevant ones such as skeletal muscle Margaret Buckingham, Paris (France) remains notoriously diffi cult. During embryonic de- Department of Developmental Biology and Stem velopment, skeletal muscles arise from somites, Cells, CNRS URA 2578, Institut Pasteur, 25–28 Rue which derive from the presomitic mesoderm (PSM). du Dr Roux, Paris 75015, France. Based on our understanding of PSM development, we established conditions allowing effi cient differentia- Adult skeletal muscle homeostasis and regeneration tion of monolayer cultures of mouse embryonic stem relies on satellite cells. Unlike muscle progenitor cells (ES) cells into PSM-like cells without introduction of in the embryo, most adult satellite cells have activated exogenous genetic material or cell sorting. To opti- the myogenic determination gene, Myf5, and thus mize the differentiation of Embryonic Stem (ES) cells have acquired muscle identity. However, post-tran- toward the muscle lineage, we used a series of report- scriptional mechanisms prevent accumulation of er ES cell lines, expressing fl uorescent proteins under myogenic factors so that satellite cells constitute a re- the control of genes specifi c for key stages of myo- serve cell population which can be mobilized in re- genic development. Our optimized conditions were sponse to muscle damage. Quiescent satellite cells in inferred based on the development of the PSM in vivo their niche on the muscle fi bre employ protective and from a microarray series of early developmental strategies against toxins and stress. Once activated, stages of this tissue. We next established simple con- satellite cells proliferate and then begin to differenti- ditions to recapitulate primary and secondary/foetal ate into muscle fi bres. This process is accompanied by myogenesis in vitro from these PSM-like cells. Our major metabolic changes, as the differentiating cells strategy allowed for the production of contractile fi - progress from a glycolytic to an oxidative state with bers from pluripotent cells in vitro with an effi ciency extensive mitochondrial biogenesis. This leads to in- comparing well with current cardiomyocytes differ- creased production of reactive oxygen species (ROS). entiation protocols. The muscle fi bers produced are We show that Pitx transcription factors, also present striated and multinucleated and exhibit post-natal in the quiescent cell, regulate ROS levels by directly characteristics. They also provide a niche allowing activating genes in the antioxidant pathway. In the development of Pax7-positive satellite-like cells. Pitx2;Pitx3 double conditional mutant mice, ROS lev- We used these conditions to differentiate ES cells de- els are abnormally high leading to DNA damage, sat- rived from dystrophin-defi cient mdx mice. We show ellite cell senescence and impaired regeneration. In that these fi bers exhibit a strikingly abnormal organi- Pitx3 single mutants, on the other hand, premature zation of the myofi brils accompanied by a dramatic differentiation occurs. By manipulating ROS inhibi- increase in the number of branches. While such a tors in activated satellite cells we show that a moderate ISSN 2214-3599/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved This article is published online with Open Access and distributed under the terms of the Creative Commons Attribution Non-Commercial License. S4 Abstracts level of ROS, acting through the p38 kinase pathway, Therefore, the distinct transcriptional programs of is necessary for the correct timing of the onset of dif- MyoD and NeuroD2 are established by a subset of ferentiation. Thus the physiological enhancement of factor-specifi c binding motifs and the lineage-deter- ROS production and mitochondrial content is an es- mined chromatin context of the cell. Chimeras be- sential regulator of muscle fi bre formation, as well as tween MyoD and NeuroD identify the specifi c a response to the rising energy demand of regenerat- molecular attributes that determine neurogenesis and ing muscle. myogenesis. PLENARY SESSION 01 PLENARY SESSION 02 Theme: BASIC SCIENCES, MUSCLE AND NERVE DEVELOP- Theme: GENETICS IN NEUROMUSCULAR DISEASES MENT PL 2.1 Genetics of dystroglycanopathies: PL1.3 Novel signalling pathways that new advances in dystroglycan post- control muscle mass translational processing Marco SANDRI, Padua (Italie) Kevin CAMPBELL, Iowa City (Etats-Unis) Howard Hughes Medical Institute, Department of Abstract not received Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, USA PLENARY SESSION 01 Theme: BASIC SCIENCES, MUSCLE AND NERVE DEVELOP- Dystroglycanopathies are muscular dystrophies in MENT which the aberrant post-translational modifi cation of dystroglycan results in loss of an essential link be- PL1.4 Genetic and epigenetic tween α-dystroglycan and its laminin-G domain-con- determinants of myogenesis taining extracullular matrix ligands. Recent genetic data has shown that mutations in at least sixteen genes Stephen TAPSCOTT, Seattle (Etats-Unis) encoding post-translational enzymes lead to a reduced Fred Hutchinson Cancer Research Center, Seattle, Xyl-GlcA dissacharide repeat on dystroglycan and WA USA cause congenital/limb-girdle muscular dystrophies, which can be accompanied by brain and eye abnor- MyoD and NeuroD2 are members of the basic-he- malities. Our previous efforts to understand the lix-loop-helix transcription factor family and regulate molecular mechanism underlying dystroglycan’s myogenesis and neurogenesis, respectively. ChIP-seq ability to bind the ECM led to identifi cation of a shows that MyoD and NeuroD2 share similar DNA novel phosphorylated O-mannosyl trisaccharide binding motifs. Both MyoD and NeuroD2 bind to a (N-acetylgalactosamine-β3-N-acetylglucosamine-β4- CAGCTG E-box, whereas the CAGATG E-box is mannose) on α-dystroglycan, and to the demonstra- preferred by NeuroD2 and the CAGGTG E-box is tion that addition of this phosphate residue is a preferred by MyoD. To some extent, the specifi c E- prerequisite for formation of the ligand-binding motif. box sequence determines the transcriptional program However, the biosynthetic pathway that leads to pro- of NeuroD2 and MyoD. The NeuroD2-specifi c CA- duction of the phosphorylated O-mannosyl glycan— GATG E-box binding site mediates is more highly as- the core saccharide that is extended by LARGE—had sociated with NeuroD2 regulated genes and has not been delineated. We now report that three of the stronger enhancer function in reporter constructs in newly identifi ed dystroglycanopathy genes (GTDC2, response to NeuroD2. Therefore, the DNA encoded B3GALNT2, and SGK196) are involved in synthesis sequence of the E-box is a genetic determinant of of the phosphorylated trisaccharide. We found that whether a gene will be activated by NeuroD2. How- GTDC2 is localized at the ER and has a protein ever, the specifi c E-boxes bound by each factor is O-mannose β1,4-N-acetylglucosaminyltransferase largely determined by the site accessibility in the activity, which led us to designate it as POMGNT2. chromatin, which varies depending on cell lineage. We also demonstrated that GTDC2 and B3GALNT2 Abstracts S5 can synthesize a GalNAc-β3-GlcNAc-β-terminus at diagnostic yield (63%) when compared to single gene the 4-position of protein O-mannose. The newly iden- testing (15–19%). This presentation will compare tifi ed CMD causative protein—SGK196—phosphor- clinical utility of targeted NGS panel test with that of ylates the 6-position of O-mannose using ATP, based whole exome sequencing (WES). Sanger fi ll-in of low on which we proposed to designate it as a protein coverage exons, copy number variation (CNV) analy- O-mannose kinase (POMK). SGK196 exhibits its sis by comparative genomic hybridization arrays phosphorylation activity only when the GalNAc-β3- (aCGH) and thorough in-house validation of the assay GlcNAc-β-terminus is linked to the 4-position of O- complements panel testing and allows detection of all mannose, indicating that this disaccharide serves as types of causative mutations, some of which (about the substrate recognition motif of SGK196. This strict 18%) may be missed by WES. specifi city of SGK196 explains why mutations in GTDC2 and B3GALNT2 cause congenital muscular dystrophy even though their product does not directly PLENARY SESSION 02