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 . 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 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 -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 recognize ECM ligands. We have been also exploring Theme: GENETICS IN NEUROMUSCULAR DISEASES the interaction between the dissachardide repeat add- ed by LARGE (the LARGE-glycan) and its ECM li- gands. Using an inducible Large knockdown mouse PL 2.3 Congenital : The New model we determined that the avidity of α-dystroglycan Genetic Era and its ECM ligands correlates with LARGE-glycan Carsten G. BÖNNEMANN, Bethesda (Etats-Unis) length. Furthermore, muscle formation requires prop- Abstract not received er LARGE-glycan extension to prevent dystrophy.

PLENARY SESSION 02 PLENARY SESSION 02 Theme: GENETICS IN NEUROMUSCULAR DISEASES Theme: GENETICS IN NEUROMUSCULAR DISEASES PL 2.4 New genes in motor neuron PL 2.2 Single gene, gene panel and exome diseases sequencing approaches for neuromuscular diseases Wim ROBBERECHT, Leuven (Belgique) University of Leuven, Belgium Madhuri HEGDE, Atlanta (Etats-Unis) Department of Human Genetics, Emory University Our understanding of the hereditary causes of mo- School of Medicine tor neuron diseases is advancing rapidly. Here, the impressive progress in unraveling the genetics of Neuromuscular diseases (NMDs) are a group of amyotrophic lateral sclerosis (ALS) will be critically highly heterogeneous inherited genetic disorders that reviewed. Special attention will be devoted to the het- affect the peripheral nervous system and muscle re- erogeneity of the phenotype of ALS and to the con- sulting in gross motor disability. They comprise of tinuum between ALS and frontotemporal lobe over 200 Mendelian disorders all of which are rare degeneration. The role of aberrant RNA processing individually but have an approximate disease preva- and failure of proteostasis will be linked to the rele- lence of 1 in 3,000 altogether. Given the clinical and vant genetic changes in genes involved in these pro- genetic heterogeneity of NMDs, disease diagnosis is cesses, and novel ways to identify disease causing complicated and expensive through traditional bio- genes will be discussed. chemical and single gene testing. To improve and ex- pedite molecular diagnosis of NMDs we validated and launched several Next generation sequencing (NGS) based comprehensive panel and sub-panel tests. Comparing the clinical diagnostic yields of sin- gle gene (NMD associated) tests with that of the various NMD NGS panel and sub-panel tests NMD comprehensive panel testing has a 4-fold greater S6 Abstracts

PLENARY SESSION 03 molecules. The prospects of applying these biological Theme: IMMUNE-MEDIATED CONDITIONS IN NEUROMUS- agents will be discussed in the context of targeting the CULAR DISEASES main immune factors involved in the pathogenesis of each disorder. Their excessive cost and safety profi le, PL 3.1 Targets of immunotherapy in as well as the need to establish effi cacy with con- trolled studies, will be highlighted. autoimmune neuromuscular disorders and emerging new therapies Marinos DALAKAS, Athens / Philadephia (Grèce) PLENARY SESSION 03 Thomas Jefferson University, Philadelphia USA and Theme: IMMUNE-MEDIATED CONDITIONS IN NEUROMUS- University of Athens Medical School, Athens Greece CULAR DISEASES

The main autoimmune neuromuscular disorders PL 3.2 Emerging new concepts in (NMD) include the acquired demyelinating neuropa- thies such as GBS, CIDP and MMN; infl ammatory pathogenesis of infl ammatory myopathies myopathies (polymyositis, necrotizing autoimmune Olivier BENVENISTE, Paris (France) myositis, dermatomyositis and Inclusion Body Myo- Department of Internal Medicine and Clinical sitis), and neuromuscular transmission defects like Immunology - Reference Center for Neuromuscular and LEMS. These disorders are Disorders - AP-HP, UMRS 974, UPMC - INSERM - currently treated with the following non-specifi c im- Pitié-Salpêtrière Hospital, Paris, France munosuppressive or immunomodulating drugs and procedures, applied singly or in combination: Ste- Idiopathic infl ammatory myopathies include der- roids; Azathioprine; Mycophenolate Mofetil; Cyclo- matomyositis, polymyositis, immune-mediated nec- sporin; Methotrexate; Cyclophosphamide; rotizing and inclusion body myositis. The plasmapheresis; and IVIg. The judicious use of these myositis-specifi c antibodies lead to a serologic ap- agents, the means by which they exert their action and proach complementary to the clinico-pathological the pitfalls or limitations associated with their use will classifi cation, because strong associations of myosi- be discussed. Although these agents have had a sig- tis-specifi c antibodies with clinical features and sur- nifi cant impact in the treatment of autoimmune neu- vival have been documented, determining now ropathies and myopathies, they cause unacceptable myositis subclassifi cations. The role of these auto- side effects after long-term use and they are not al- antibodies remains elusive, particularly, it is not clear ways effective in inducing remission, necessitating if they are cause or consequence of the myositis. Nev- the need for exploring novel biological agents as fu- ertheless, the striking autoantibody profi les character- ture therapeutic options. Advances in biotechnology izing subsets of myositis patients provide valuable have promoted the development of a new class of clues to putative antigen triggers and clearly refl ect an products, mostly in the form of monoclonal antibod- underlying antigen driven process. Supporting this ies or fusion proteins that offer target-specifi c immu- contention, studies have shown that antibodies target- notherapy. Agents, currently on the market or in ing histidyltRNA synthetase (anti-Jo-1, encountered ongoing trials, appropriate for target-specifi c treat- during the antisynthetase syndrome) undergo affi nity ment of autoimmune neuromuscular disorders, are maturation, parallel disease activity, and do not co- directed against the following: 1) T cell Intracellular exist with other “myositis-specifi c” autoantibodies. Signaling Pathways associated with T cell activation, Previous work examining human T cell responses in such as monoclonal antibodies against CD52, Inter- Jo-1 antibody positive myositis patients provides ad- leukin 2-receptor (IL2 R), co-stimulatory molecules ditional evidence that this stereotypical antibody re- or compounds inhibiting Janus tyrosine Kinases sponse is driven by underlying antigen-specifi c T JAK1, JAK3; 2) B cells, against key B cell-surface cells. Perhaps most convincing, however, is the com- molecules or trophic factors of B cell activation; 3) bination of muscle and lung infl ammation resulting Complement, against C5, that intercepts the Membra- from subcutaneous immunization of various congenic nolytic attack complex formation; 4) Cytokines and mice with emulsifi ed murine histidyltRNA synthetase cytokine receptors, including IL-6, IL-17, the p40 that partially replicate the anti-synthetase syndrome subunit of IL12/1L-23; and 5) Lymphocyte migration in humans. Similarly, antibodies directed against the Abstracts S7 signal recognition particle (SRP) that guide polypep- PLENARY SESSION 03 tides into the endoplasmic reticulum are associated Theme: IMMUNE-MEDIATED CONDITIONS IN NEUROMUS- with severe forms of necrotizing myopathies. We CULAR DISEASES have previously shown that serum titers of anti-SRP auto-Abs closely correlate with CK levels and muscle PL 3.4 Emerging new concepts in weakness in patients. We have shown the presence of mouse SRP proteins reactive to human anti-SRP Ab pathogenesis of autoimmune by Western-Blot, immune-fl uorescence and immuno- gold electron microscopy on mouse muscle samples. Angela VINCENT, Oxford (Royaume Uni) Furthermore, mice injected with sera or purifi ed IgGs Emeritus Professor of Neuroimmunology, Nuffi eld from SRP+ patient showed a signifi cant decrease in Department of Clinical Neurosciences, University of muscular strength. This effect is dependant of the Oxford, 2012–2017 complement. These results also provide experimental evidence that injection of anti-SRP Abs may affect Over the last 40 years the concept of autoantibodies muscle function and therefore play a direct role in the affecting the number and function of both ligand-gat- pathogenesis of necrotizing autoimmune myopathies. ed and voltage-gated ion channels has widened con- siderably. In the 1970s, myasthenia gravis was shown to be caused by antibodies to acetylcholine receptors PLENARY SESSION 03 (AChRs), and these seminal fi ndings have helped lead Theme: IMMUNE-MEDIATED CONDITIONS IN NEUROMUS- the way to the recognition and treatment of other anti- CULAR DISEASES body-mediated diseases of the peripheral, autonomic and central nervous systems. PL 3.3 Emerging new concepts in In 2001, antibodies to MuSK were found in some of the myasthenia patients negative for AChR antibod- pathogenesis of peripheral neuropathy ies, and more recently antibodies to LRP4 in a small Hugh WILLISON, Glasgow (Royaume Uni) proportion. MuSK and LRP4 are postsynaptic mem- College of Medical, Veterinary and Life Sciences, brane proteins that are involved in AChR localisation University of Glasgow and function. These antibodies are now routinely de- tected in myasthenia and their pathogenicity is widely Infl ammatory changes are widely found in periph- accepted.MuSK antibodies are not common in many eral nerve in a range of acquired and heritable condi- populations but appear to be present in up to 50% of tions when they play important roles in mediating patients without AChR antibodies in myasthenia in glial and axonal injury, homeostasis and repair. The warmer countries. The incidence further south is not term autoimmune neuropathy is used to encompass yet clear. MuSK antibodies have been shown to pas- acute and chronic clinical phenotypes with a varying sively-transfer disease to mice, but it is still not en- underlying pathophysiological basis. Research in this tirely clear how binding of MuSK antibodies to MuSK area continues to progress rapidly, with the expecta- leads to neuromuscular transmission failure, although tion that syndrome biomarkers and new therapies will a reduction in AChR numbers at the neuromuscular emerge for use in clinical practice, alongside a deeper junction can be found. There may also be presynaptic understanding of pathophysiology. This presentation effects of MuSK antibodies which are not found with will focus on human and animal studies that are pro- AChR antibodies. LRP4 antibodies may act in a simi- viding major new insights into peripheral nerve as an lar manner since LRP4 is required for activation of immunologically active organ. Particular attention MuSK, but less work has been done on these rare an- will be paid to new antibody biomarkers, notably gly- tibodies. colipids and protein components of the nodal complex Other antibodies can affect the neuromuscular junc- and to downstream effector pathways, including com- tion. In the 1990s, antibodies to voltage-gated calcium plement activation. The fi ndings will be illustrated channels were identifi ed in the Lambert Eaton syn- through analysis of human biobanks and animal mod- drome, and antibodies to shaker type voltage-gated els. Gaps in knowledge, new concepts and directions potassium channels (VGKCs) in acquired neuromyo- for future research will be highlighted. tonia, a condition caused by peripheral nerve hyper- excitatibility that leads to muscle fasciculations, S8 Abstracts cramps and pain. Somewhat surprisingly, the VGKC PLENARY SESSION 04 antibodies were also identifi ed in central nervous sys- Theme: THERAPY IN NEUROMUSCULAR DISEASES tem disorders, particularly limbic encephalitis (mem- ory loss, sleep disorders and seizures) but this was explained by the fact that the antibodies turned out to PL 4.2 Antisense Oligonucleotide be directed at proteins that form part of VGKC com- therapies for Duchenne Muscular plexes in situ (2). The two principal proteins help lo- Dystrophy calise (CASPR2) and modify (LGI1) potassium Francesco MUNTONI, London (Royaume Uni) channel function. Dubowitz Neuromuscular Centre, These fi ndings helped to direct more attention to the UCL Institute of Child Health & Great Ormond central nervous system. Neuromyelitis optica is Street Hospital for Children caused by antibodies to the water channel, aquapo- London, UK rin-4 (AQP4) and is a chronic disease that leads to optic nerve and spinal cord infl ammation. Other anti- Antisense oligonucleotide (AON) induced exon bodies bind directly to CNS ligand-gated receptors. skipping, also known as splice switching AON, are Antibodies to NMDA receptors (NR1 principally) are increasingly being used in Duchenne muscular dys- found mainly in younger patients, often women and trophy (DMD), where several hundreds of children small children, who have a severe encephalopathy with deletions eligible to skipping of exons 44; or 45; with movement disorders. And antibodies to glycine or 51 or 53 have been studied in phase I, II and III receptors are associated with extreme rigidity and studies. brainstem disturbance which can be life threatening Two main chemistries are in use at the moment, the (Carvajal et al in press). Importantly, each of these 2’O Methyl (2’OMe) AON backbone; and the phos- antibodies bind to extracellular epitopes on the target phorodiamidate morpholino (PMO) AON backbone. proteins and there is evidence of their pathogenicity. Both backbones have been quite extensively stud- The conditions, although rare, can now be diagnosed ied before in relevant animal models such as the mdx regularly by serological tests and the patients treated mouse and, the PMO, also in the more severe dystro- with immunotherapies which lead to substantial im- phic dog models with encouraging results. provement. These preclinical promising results have been par- Intriguingly, although rather rare, neuromyelitis op- alleled by similarly encouraging results of the phase I tica can present in patients with previous or concur- and II studies of both chemistries. More recently how- rent myasthenia and myasthenia can also overlap with ever the failure of the only phase III study (in which a and Morvan’s syndrome. The ability 2’OMe AON targeting exon 51 was studied) raised of the immune system to recognise both peripheral concerns regarding both the rationale for exon skip- and central ion-channels and receptors or related pro- ping induced restoration of the reading frame in teins is proving a challenging but exciting area of DMD, and the possibility that current chemistries clinical . Disclosure: Angela Vincent and may achieve suffi cient correction to induce a clinical the University of Oxford hold patents and receive roy- benefi t to DMD patients. alties for antibody tests. In my presentation I will review both the data from the preclinical model; the effi ciency of the current AON to induce dystrophin expression; the biological PLENARY SESSION 04 signifi cance of different levels of dystrophin and the Theme: THERAPY IN NEUROMUSCULAR DISEASES available data from the clinical trials available to date. I will discuss why on the whole the positive data from PL 4.1 Loco regional gene therapy for the current approaches are both encouraging and not the result of chance improvement or “placebo” effect Duchenne Muscular Dystrophy patients in treated boys. I will also discuss possible reasons for Philippe MOULLIER, Nantes (France) the failed phase III study which will be important to treasure for future study design. Abstract not received Despite this recent set back, and provided the ex- pectations of what can be achieved with the current chemistries are understood, I remain optimistic that Abstracts S9 this approach will demonstrate clinical effi cacy in the PLENARY SESSION 04 future. While further chemistry development are like- Theme: THERAPY IN NEUROMUSCULAR DISEASES ly to be required to refi ne this approach, I consider the data so far encouraging as examples of addressing- for the fi rst time- the fundamental problem of dystro- PL 4.4 Therapy for GNE myopathy phin defi ciency in DMD boys. (DMRV/hIBM) Disclosure. The Author is involved in clinical trials Ichizo NISHINO, Tokyo (Japan) on antisense oligonucleotides in DMD with Prosensa (2’ O methyl antisense for skipping exon 45 and exon Abstract not received 53); and as the Chief Investigator, in collaboration with Sarepta Therapeutics, in a European Commis- sion funded study on a morpholino antisense to skip THEMATIC WORKSHOP 1.1 exon 53 (EU grant agreement No. 305370) Theme: DEVELOPMENT OF

PLENARY SESSION 04 TW 1.1.1 Regulation of muscle gene Theme: THERAPY IN NEUROMUSCULAR DISEASES expression by electrical activity: CtBP mediates repression PL 4.3 RNA targeted therapies for Laurent SCHAEFFER, Lyon (France) Abstract not received Charles A THORNTON, Rochester, NY (Etats-Unis) Department of Neurology, University of Rochester

Much of what we know about RNA toxicity as a THEMATIC WORKSHOP 1.1 mechanism for genetic has come from Theme: DEVELOPMENT OF NEUROMUSCULAR JUNCTION studies of myotonic dystrophy (DM). The underlying disease process is postulated to involve activation of TW 1.1.2 Making and Breaking signalling pathways, aberrant translation of repetitive RNAs, or sequestration of the binding proteins that Neuromuscular Synapses recognize RNA repeats. Once these disease mecha- Steven BURDEN, New-York (Etats-Unis) nisms were elucidated, it became possible to identify Molecular Neurobiology Program, Skirball Institute good targets for therapeutic intervention. Targeted of Biomolecular Medicine, NYU Medical School, therapies have been developed and tested in animal N.Y., N.Y. 10016 models, and robust effects have been reported using small molecules, gene therapy vectors, and antisense The formation and maintenance of neuromuscular drugs. Among these, it appears that antisense drugs synapses requires a complex exchange of signals be- will be the fi rst targeted treatments to reach clinical tween motor neurons and skeletal muscle fi bers lead- trials. This presentation will focus on RNA targeted ing to the formation of a highly specialized therapies for DM, in terms of how they were devel- postsynaptic membrane and a highly differentiated oped, how they can be tested in early phase trials, and nerve terminal. As a consequence, acetylcholine re- what steps may be necessary to optimize their impact ceptors (AChRs) become highly concentrated in the on clinical practice. postsynaptic membrane and arranged in perfect regis- ter with active zones in the presynaptic nerve termi- nal, insuring for rapid, robust and reliable synaptic transmission. During development, motor axons ap- proach and recognize muscle that is primed, or prepat- terned in the prospective synaptic region. Muscle prepatterning is established by MuSK, a receptor ty- rosine kinase, and Lrp4, a member of the LDLR fam- ily. Lrp4 associates with MuSK and stimulates MuSK kinase activity, increasing Lrp4 and MuSK expression S10 Abstracts and causing the clustering of Lrp4 and MuSK. Once infections, cancers, connective tissue diseases and clustered, Lrp4 functions as a direct retrograde signal other conditions. When vasculitis affects the PNS, for presynaptic differentiation, causing motor axons vasculitic neuropathy ensues. Vasculitis nomenclature to stop growing and develop specializations required is a set of names and defi nitions. Classifi cation sys- for neurotransmitter release. Nascent synapses are tems differentiate patients with vasculitis into cohorts stabilized by neuronal Agrin, which is released by for clinical research. Diagnostic criteria are used to motor nerve terminals and binds to Lrp4, stimulating diagnose vasculitis in individual patients. The 1994 further association between Lrp4 and MuSK and in- Chapel Hill Consensus Conference (CHCC) codifi ed creasing MuSK kinase activity. Lrp4 thus has a cen- nomenclature for 10 vasculitides. The 2012 CHCC tral role in coordinating synaptic differentiation, as updated these names/defi nitions and added new vas- Lrp4 not only binds Agrin and stimulates postsynaptic culitides. Large vessel vasculitides are giant cell arte- differentiation but also acts in turn as a direct retro- ritis (GCA) and Takayasu arteritis (TAK). Medium grade signal for presynaptic differentiation. Mutations vessel vasculitides are polyarteritis nodosa (PAN) and in Agrin, Lrp4 and MuSK, as well as additional genes Kawasaki disease (KD). Small vessel vasculitides that function in this signaling pathway, cause congen- (SVV) are divided by prevalence of immune deposits. ital myasthenia, and auto-antibodies to Lrp4, MuSK, Pauci-immune SVV are ANCA-associated: Granulo- or AChRs are responsible for myasthenia gravis. I matosis with polyangiitis (GPA; formerly Wegener will summarize experiments that have contributed to granulomatosis), microscopic polyangiitis (MPA) and this model of neuromuscular synapse formation, indi- eosinophilic granulomatosis with polyangiitis (EGPA; cate how this knowledge has provided insight into formerly Churg Strauss syndrome). Immune complex causes for , and describe a ther- SVV are anti-glomerular basement membrane dis- apeutic approach for preserving synapses and treating ease, cryoglobulinemic vasculitis, IgA vasculitis (for- neuromuscular diseases. merly Henoch Schönlein purpura), and hypocomplementemic urticarial vasculitis. Variable vessel vasculitides are Behçet disease and Cogan syn- THEMATIC WORKSHOP 1.1 drome. Secondary vasculitides are those “associated Theme: DEVELOPMENT OF NEUROMUSCULAR JUNCTION with systemic disease” or “associated with probable etiology.” CHCC2012 also defi ned single organ vas- culitides. Isolated PNS vasculitis is termed nonsys- TW 1.1.3 Signaling perturbations at the temic vasculitic neuropathy (NSVN). Primary NMJ systemic vasculitides commonly associated with neu- ropathy are EGPA, MPA, and MPA. Secondary vascu- David GLASS, Boston (Etats-Unis) litides featuring PNS involvement are HBV-PAN, HCV-cryoglobulinemia, and rheumatoid vasculitis. Abstract not received Neuropathies are infrequent in GCA and rare in TAK, KD, Behçet disease, Cogan syndrome and other im- mune complex SVV. In 1990, the American College THEMATIC WORKSHOP 1.2 of Rheumatology developed classifi cation criteria for Theme: VASCULITIC PERIPHERAL NEUROPATHY PAN, EGPA, GPA, hypersensitivity vasculitis, IgA vasculitis, GCA and TAK. In 2010, a Peripheral Nerve TW 1.2.1 Classifi cation and pathology of Society task force classifi ed vasculitic neuropathies the systemic and non systemic vasculitic into primary systemic, secondary systemic and non- systemic forms. The latter included NSVN and dia- neuropathies betic radiculoplexus neuropathy. There are no Michael P COLLINS, Milwaukee (Etats-Unis) validated diagnostic criteria for primary systemic vas- Medical College of Wisconsin, Milwaukee, WI, USA culitides. The Diagnostic and Classifi cation Criteria in Vasculitis study is designed to develop/validate di- Vasculitides are disorders defi ned by infl ammatory agnostic criteria and improve/validate classifi cation destruction of vessel walls. Some are caused by direct criteria for MPA, GPA, EGPA, PAN, GCA and TAK. infection, but most are autoimmune. Autoimmune The Peripheral Nerve Society task force derived diag- vasculitides can be primary or secondary to drugs, nostic criteria for pathologically defi nite, pathologi- Abstracts S11 cally probable, clinically probable and nonsystemic The gold standard for the diagnosis of vasculitic vasculitic neuropathy. neuropathy is demonstration of vasculitis in a nerve biopsy. Nerve and/or muscle biopsy sensitivity for vasculitis is between 60–70%. Combined nerve/mus- THEMATIC WORKSHOP 1.2 cle biopsy (usually sural nerve/gastrocnemius muscle, Theme: VASCULITIC PERIPHERAL NEUROPATHY superfi cial peroneal nerve/peroneus brevis muscle) can be considered for improved (~15%) yield com- pared to nerve biopsy alone. TW 1.2.2 Clinical approach to vasculitic neuropathies References: - Gwathmey KG, Burns TM, Collins MP, Dyck Alexander FJE VRANCKEN, Utrecht (Pays-Bas) PJB. Vasculitic neuropathies. Lancet Neurol University Medical Centre Utrecht, The Netherlands 2014;13:67–82. Vasculitic neuropathy presents in ~75% in the con- - Vrancken AFJE, Said G. Vasculitic neuropathy. text of primary systemic vasculitis or vasculitis sec- Handb Clin Neurol 2013;115:463–83. ondary to connective tissue disease, infectious disease, malignancy. In nonsystemic vasculitic neuropathy, vasculitis appears limited to the peripheral nervous THEMATIC WORKSHOP 1.2 system. Theme: VASCULITIC PERIPHERAL NEUROPATHY Except for multi-organ involvement, no clinical or pathological features distinguish systemic from non- TW 1.2.3 Treatment of vasculitic systemic vasculitic neuropathy. Women are more of- ten affected than men (ratio 3:2) and the average onset neuropathie age is 60. Typically there is a painful multifocal neu- Robert DM HADDEN, London (Royaume Uni) ropathy with (sub)acute onset of predominantly sen- King’s College Hospital, London, UK sory or sensorimotor defi cits. Over time there is progression to generalized polyneuropathy, usually Any non-immune cause (especially hepatitis B or C asymmetrical. Cranial nerves can be affected. Occa- infection) should be treated fi rst. sionally vasculitic neuropathy presents as pure sen- In systemic vasculitis, induction of remission is sory neuropathy, polyradiculoplexoneuropathy, or usually with cyclophosphamide for three months, mimicks Guillain-Barré syndrome. with intravenous pulses considered safer than daily The differential diagnosis includes diabetes, sys- oral dosing, together with high dose corticosteroids. temic disease (e.g., sarcoidosis, amyloidosis, malig- Cyclophosphamide dose is usually six pulses of 15 nancy), infection (e.g., leprosy, HIV, hepatitis B & C, mg/kg or 0.6 – 0.7 g/m2. In milder cases methotrexate Borrelia, CMV), AMSAN variant of Guillain-Barré is a less toxic alternative. Rituximab is at least as ef- syndrome. Features such as fever, weight loss, auto- fi cacious as cyclphosphamide and probably less toxic nomic symptoms, skin changes, musculoskeletal but more expensive. Plasma exchange may be used in symptoms provide important clues. Because an un- the most fulminant cases. Intravenous immunoglobu- derlying disease can manifest with the neuropathy, lin may be useful for short term benefi t in the presence ancillary investigations (blood & urine analysis, im- of severe infection. aging) are necessary to search for a cause and evaluate In non-systemic vasculitic neuropathy, there are no the presence of systemic infl ammation and multi- randomised trials of treatment so recommendations organ involvement. are based on case series and on extrapolation from Nerve conduction studies show axonal neuropathy, systemic vasculitis. Treatment may be stratifi ed by se- often multifocal and more extensive than the clinical verity. A few cases improve without treatment. Corti- defi cits. In up to 25% of cases transient ‘pseudo’ con- costeroids alone may suffi ce in many. Rapidly duction blocks are found, which refl ect ongoing Wal- progressive or steroid-refractory cases should addi- lerian degeneration and axonal damage from focal tionally receive methotrexate (15 – 25 mg/week) or ischemic nerve injury due to vasculitis. Electromyog- azathioprine (2.0 – 2.5 mg/kg/d) if of moderate sever- raphy demonstrates a neurogenic pattern, but a myo- ity, or cyclophosphamide if severe. Pulsed intrave- pathic pattern can be seen if there is muscle vasculitis nous methylprednisolone is optional for more severe or a disease predisposing to (infl ammatory) myopa- cases. thy. S12 Abstracts

Monitoring of response is insensitive but usually temically or via intracerebral ventricular (ICV) relies on motor and sensory examination, disability injection remarkable rescue of SMA mice is obtained. scales, and infl ammatory markers; pain alone is an un- EMG recordings the compound muscle action poten- reliable marker of disease activity. tial (CMAP) and motor unit number estimates with To prevent relapse, corticosteroids, azathioprine, early delivery indicate an almost complete recovery. methotrexate or lefl unamide are generally continued We have also created a pig model of SMA by using for 18–24 months. Any relapse may be treated with scAAV9-sh RNA to pig SMN. The scAAV9-shRNA is corticosteroids usually with cyclophosphamide or injected intrathecaly into 5 day old piglets which re- rituximab. sults in effi cient transduction of motor neurons. Pigs Patients may also benefi t from treatment of neuro- developed marked weakness and have reduced pathic pain, optimisation of vascular risk factors, and MUNE and CMAP with clear signs of denervation de- rehabilitation therapies including orthoses. tected. Introduction of a second scAAV9 expressing Disclosure: RH received honoraria/expenses from human SMN( scAAV9-hSMN) results in rescue of CSL Behring, Baxter Healthcare, and Grifols. these pigs. If the rescue is performed when the pigs have symptoms then CMAP but not MUNE is in- creased and the pigs do show an improved phenotype. THEMATIC WORKSHOP 1.3 The scAAV9-SMN has now received IND and phase Theme: SPINAL MUSCULAR ATROPHY 1 trials are planned. In addition to AAV we have in- vestigated the use of antisense oligonucleotide of the morpholino chemistry for treatment of SMA. A series TW 1.3.1 Development of therapies for of different ASOs have been investigated strong ASOs SMA preclinical result in an extension of survival from 14 days to over 100 days with a single ICV injection. The SMN levels ARTHUR BURGHES, COLUMBUS (Etats-Unis) and incorporation of SMN exon 7 shows major in- 1Molecular and Cellular Biochemistry, Ohio State creases with ASO treatment. The ASO affect shows a University, Columbus, United States slow decay with time. We have examined CMAP and 2Department of Neurology, Ohio State University, MUNE with both early and late introduction of ASO Columbus, United States rescue therapy. In early rescue both CMAP and 3Department of Neuroscience, Ohio State University, MUNE showed marked rescue whereas latter only Columbus, United States CMAP showed an increase. We have further increased 4Department of Neurological Surgery, Ohio State survival by readministration of the ASO into the CNS University, Columbus, United States at 30 days. Thus SMN restoration with gene therapy, 5Gene Therapy, Nationwide Childrens Hospital, drug compounds or antisense oligonucleotides has a Columbus, United States major impact when delivered early in SMA. However 6Department of Biochemistry, Siriraj Hospital, it becomes important to consider how to improve Mahidol University, Bangkok, Thailand therapy latter in the course of the disease. 7Center for Comparative Genomics, Murdoch University, Perth, Australia 8Institute of Cell Biology, Univerity of Bern, Bern, Switzerland THEMATIC WORKSHOP 1.3 Theme: SPINAL MUSCULAR ATROPHY Spinal Muscular Atrophy (SMA) is caused by loss of the SMN1 gene and retention of SMN2. The SMN2 TW 1.3.2 Dysregulation of RNA gene differs from SMN1 in that it is ineffi cient at in- corporating exon7 due an alteration of a splice modu- processing in spinal muscular atrophy lator this results in less SMN being produced. We Livio PELLIZZONI, New York (Etats-Unis) have developed mice which model this situation. In 1Center for Motor Neuron Biology and Disease, SMA mice early induction of SMN results in rescue Columbia University, New York (NY) 10032, USA whereas late induction has a reduced impact. Using 2Department of Pathology and Cell Biology, Cre drivers and either reducing SMN or restoring high Columbia University, New York (NY) 10032, USA SMN levels, indicates that CNS is the key tissue to target. We have investigated various methods of resto- At the post-transcriptional level, expression of pro- ration of SMN. Using scAAV9-SMN delivered sys- tein-coding genes is controlled by a series of RNA Abstracts S13 regulatory events including nuclear processing of pri- THEMATIC WORKSHOP 1.4 mary transcripts, transport of mature mRNAs to spe- Theme: STEM CELLS AND MUSCLE REGENERATION cifi c cellular compartments, translation and ultimately, turnover. These processes are orchestrated through the dynamic association of mRNAs with RNA bind- TW 1.4.2 Eph/ephrin interactions ing proteins and ribonucleoprotein (RNP) complexes. generate fi ber type specifi city during Accurate formation of RNPs in vivo is fundamentally muscle development, regeneration and important to cellular development and function, and reinnervation its impairment often leads to human disease. The sur- vival motor neuron (SMN) protein is key to this bio- Dawn CORNELISON, Columbia (Etats-Unis) logical paradigm: SMN is essential for the biogenesis Associate Professor, Biological Sciences of various RNPs that function in mRNA processing, Investigator, Christopher S. Bond Life Sciences and genetic mutations leading to ubiquitous SMN de- Center fi ciency cause the neurodegenerative disease spinal University of Missouri - Columbia, MO 65211 - USA muscular atrophy (SMA). I will discuss the expanding role of SMN in the regulation of gene expression Each of the 200+ muscles found in adult mammals through its multiple functions in RNP assembly and is unique in its size, shape, and location as well as in advances in our understanding of how disruption of the functional connections it forms with other organ SMN-dependent RNA processing pathways can cause systems such as nerves and tendons. When the integ- motor neuron disease. rity of a muscle is disrupted, skeletal muscle is re- markable not only in its capacity to rapidly and effi ciently regenerate lost cell number and mass, but in the extent to which the pre-existing patterns intrin- THEMATIC WORKSHOP 1.3 sic to each muscle are recapitulated. One aspect of Theme: SPINAL MUSCULAR ATROPHY patterning that is critical for the specifi c function of any given muscle is its unique proportion and ar- TW 1.3.3 Clinical trials and rangement of fast and slow myofi bers to provide the necessary balance of force and endurance. Four dis- measures in SMA tinct muscle fi ber types are present in limb muscles, Eugenio MERCURI, Rome (Italie) which are generally divided into ‘slow’ and ‘fast’ on the basis of which sarcomeric heavy chain Abstract not received (MyHC) isoform they express: Type I/slow (small di- ameter, slow twitch, fatigue-resistant, oxidative fi bers innervated by slow, fatigue-resistant motor neurons) THEMATIC WORKSHOP 1.4 or Type II/fast (large diameter, fast twitch, fatigable, high force generating, glycolytic fi bers innervated by Theme: STEM CELLS AND MUSCLE REGENERATION fast motor neurons); fast myofi bers are further classi- fi ed as Type IIa, IIx/d, or IIb, in order of progressively TW 1.4.1 Regulation of satellite cell ‘faster’ phenotypes. migration manipulating the amoeboid Recent data from our group suggests that classical mechanisms for cell movement to repulsive guidance signaling through specifi c pairs of Eph receptors and their ephrin ligands could protect promote skeletal muscle regeneration slow myofi bers from innervation by fast motor neu- Ketan PATEL, Reading (Royaume Uni) rons during postnatal development or reinnervation after injury, and from fusion with muscle satellite Abstract not received cells originating on or specifi ed to form fast myofi - bers. These results not only present a mechanistic ex- planation for the fi ber type-specifi c innervation/ reinnervation and myoblast associations that have been noted for ober 50 years, but also have implica- tions for the understanding of the loss of specifi c mo- S14 Abstracts tor units in disease or aging and the design of intra-muscle-fi ber accumulations of ubiquitinated exogenous satellite cell-based therapies. multi-protein aggregates, several of which are confor- mationally-modifi ed and in congophilic β-pleated- sheet confi guration (therefore generically called THEMATIC WORKSHOP 1.4 “amyloid”). Conformationally-modifi ed proteins Theme: STEM CELLS AND MUSCLE REGENERATION present as congophilic amyloid within the s-IBM mul- tiprotein-aggregates include: amyloid-β (Aβ), mainly composed of the more toxic Aβ42; phosphorylated TW 1.4.3 Wnt signaling(s) and skeletal tau (including conformationally modifi ed tau); and muscle regeneration α-synuclein. Aβ42–oligomers, considered the most toxic form of Aβ, are also present in s-IBM muscle Fabien LE GRAND, Paris (France) fi bers. Because accumulation of these intra-muscle Institut Cochin, INSERM U1016, CNRS UMR 8104, fi ber proteinaceous aggregates is associated with and Université Paris Descartes, Sorbonne Paris Cité, presumably caused by protein unfolding/misfolding, Paris, France. s-IBM is considered a “conformational disorder”. In The author does not wish to be published s-IBM, abnormal accumulation of the multi-protein aggregates may result as documented by us a) in- creased transcription of several proteins, b) their ab- THEMATIC WORKSHOP 1.5 normal posttrantslational modifi cations and misfolding, and c) inadequate protein disposal. These Theme: INCLUSION BODY MYOSITIS phenomena indicate abnormal “myoproteostasis”, which, we postulate, is provoked or abetted by an ag- TW 1.5.1 Critical pathogenic mechanisms ing intracellular milieu. Very important pathogenical- associated with the muscle-fi ber ly are the impairments of protein disposal due to the degeneration specifi c to Sporadic inhibition of both the 26S proteasome and autophagy, endoplasmic reticulum stress, and impaired deacety- Inclusion-Body Myositis (s-IBM), and lation. Our most recent studies point to the important their treatment possibilities abnormalities associated with mitophagy, which Valerie ASKANAS, Los Angeles (Etats-Unis) might explain previously-demonstrated mitochondri- USC Neuromuscular Center, Department of al abnormalities. We have recently demonstrated in Neurology, University of Southern California Keck one of our in vitro models of s-IBM, based on the in- School of Medicine, Los Angeles, CA, USA hibition of autophagy, that treatment with sodium phenylbutyrate (NaPB) improved autophagy, elimi- The pathogenesis of s-IBM, the most common and nated vacuolization, and signifi cantly decreased Aβ42 severe muscle disease of older persons, is complex and its oligomers. Thus NaPB might be considered a and it involves multidimensional pathways most of potential treatment of s-IBM patients. which are still unresolved. Although both muscle- fi ber degeneration and mononuclear-cell infl amma- tion are characteristic components of the s-IBM THEMATIC WORKSHOP 1.5 pathology, but how each relates to the pathogenesis Theme: INCLUSION BODY MYOSITIS and which one plays a more important pathogenic role remain unsettled. In contrast to usually-treatable polymyositis and TW 1.5.2 How the impaired muscle dermatomyositis, both of which exhibit the same in- repair and regeneration contribute to the fl ammatory-cell pathology as s-IBM, anti-infl amma- sIBM pathogenesis, and their treatment tory treatments do not usually produce benefi t in possibilities s-IBM. We consider “aging” of the muscle-fi ber cel- lular milieu, and possibly genetic predispositions, the Massimiliano MIRABELLA, Rome (Italie) main risk factors leading to a multifactorial pathogen- Institute of Neurology, Catholic University, Roma, ic cascade that causes s-IBM muscle-fi ber degenera- Italy tion, atrophy and eventually death. s-IBM muscle Treatment of sIBM remains a challenge because of fi ber degeneration includes autophagic vacuoles and its complex pathogenesis and resistance to immuno- Abstracts S15 suppression due to an underlying muscle degenerative design strategies specifi cally targeting them in the dis- cascade. Satellite cells and fi bers presenting some re- ease treatment, alone or combined with stem cells generative features are often increased in sIBM and therapy. show activation of developmental pathways. Despite activation of repairing mechanisms, truly regenerat- ing muscle fi bers are scarce and regeneration is insuf- THEMATIC WORKSHOP 1.5 fi cient to counterbalance ongoing fi bers degeneration. Theme: INCLUSION BODY MYOSITIS We have shown age-related abnormalities account- ing for decline in muscle repair ability in sIBM myo- blasts that have constitutively impaired regenerative TW 1.5.3 How do immune-mediated capacity and intrinsic property, upon aging in vitro, to mechanisms contribute to sIBM accumulate Aβ. We also isolated and functionally pathogenesis, and why dysimmune characterized pericyte-derived mesoangioblasts treatments are largely not effective? (mAbs) from muscle biopsies and showed that mAbs, only in s-IBM muscle, display a myogenic differenti- Marinos DALAKAS, Athens (Grèce) / Philadephia ation defect rescued by overexpression of MyoD, en- (USA) visaging a possible cell-based therapeutic strategy. Because of a dual component of persisting infl am- Abstract not received mation and chronic muscle degeneration, in sIBM ex- ists a complex cross-talk between infl ammatory cells and skeletal muscle with activation of local progeni- THEMATIC WORKSHOP 2.1 tors and in situ recruitment of vessel-associated stem Theme: DEVELOPMENT OF MUSCLE LINEAGES (HEART/ cells. Macrophages may either adversely affect stem SKELETAL) cell differentiation or promote fi ber regeneration by releasing growth factors, neurotrophins and cytokines TW 2.1.1 Morphogenesis of muscles: a contributing to recruit local and circulating myogenic precursors. Consequently balance between fi ber de- bird’eye view generation and progenitors activation may be critical Christophe MARCELLE, Melbourne (Australie) for the clinical outcome. EMBL Australia; Australian Regenerative Medicine TWEAK is a multifunctional cytokine playing an Institute (ARMI), Monash University important role in tissue repair. We have shown dys- regulation of TWEAK-Fn14 axis in sIBM: this may In amniotes, all skeletal muscle is derived from an induce progressive muscle atrophy and reduce differ- embryonic structure known as the somite, with the ex- entiation of muscle precursor cells. TWEAK is a rep- ception of head muscles, that emerge from the parax- resentative molecule linking chronic infl ammation ial head mesoderm and prechordal mesoderm. Somite with defective regeneration and progressive atrophy. differentiation represents a exceptionally varied mi- Selective targeting of TWEAK might concurrently crocosm. In just a few hours, a wide spectrum of de- suppress sIBM degenerative and infl ammatory com- velopmental processes such as the mesenchymal to ponents counteracting muscle atrophy. Actually the epithelial transformation, and the converse epithelial aim to restore muscle mass is common to strategies to mesenchymal transition take place along with cel- currently investigated in sIBM patients (bimagrumab, lular differentiation, migration, morphogenesis and follistatin gene transfer, regular exercise programs). fusion. In the chick embryo, recent advances in elec- Blocking TWEAK activity may improve IBM mAbs troporation of expression vectors, siRNA constructs myogenic capacity allowing to use autologous cells in and tissue specifi c reporters in defi ned regions of so- regenerative cell therapy and to activate endogenous mites combined with recent advancements in live cell mAbs in vivo, inducing them to make new regenerat- confocal video microscopy, allow these biological ing fi bers. Identifi cation and modulation of factors phenomena to be readily observed and hypotheses selectively dysregulated in sIBM regulating myogen- tested extremely rapidly. This unique strength of the ic differentiation of mAbs, is a more handy approach avian system has opened the way to increasingly so- to enhance muscle regeneration compared to trans- phisticated experiments that address questions of in- plantation. Further insights into local cues fi nely regu- terest not only to the somite/muscle fi eld, but are also lating muscle regeneration in sIBM will be critical to fundamental in biology. Importantly, an ever-growing S16 Abstracts body of evidence indicates that morphogenesis and whether the spatial subcellular localization of ERK growth of skeletal muscles in birds is identical to that can act as a switch between proliferation (nuclear of mammals. ERK) and differentiation (cytoplasmic ERK) of mus- These technologies have allowed to characterize cle progenitors. A myristoylated peptide that blocks the morphogenic steps that lead to the formation of Importin7-mediated ERK nuclear translocation in- the primitive muscle in vertebrates and to identify the duced robust myogenic differentiation of muscle pro- embryonic origin of adult muscle stem cells, the satel- genitor/stem cells in both head and trunk. Our lite cells. Moreover, work we have recently performed fi ndings, corroborated by mathematical modeling, identifi ed the neural crest as a central player in the suggest that ERK shuttling between the nucleus and early steps of myogenesis, thus challenging textbook the cytoplasm provides a switch-like transition be- belief that this role is played by the neural tube. tween proliferation and differentiation of muscle pro- genitors.

THEMATIC WORKSHOP 2.1 Theme: DEVELOPMENT OF MUSCLE LINEAGES (HEART/ THEMATIC WORKSHOP 2.1 SKELETAL) Theme: DEVELOPMENT OF MUSCLE LINEAGES (HEART/ SKELETAL) TW 2.1.2 Head muscle origin, development and regeneration TW 2.1.3 Regulation of tissue-specifi c alternative splicing during development Eldad TZAHOR, Rehovot (Israël) 1Department of Biological Regulation, Weizmann of muscle lineage Institute of Science, Rehovot, Israel Thomas BRAUN, Bad Nauheien (Allemagne) 2Theoretical Systems Biology, Division of Molecular Biosciences, Imperial College London, London, UK Abstract not received 3Department of Orofacial Sciences and Program in Craniofacial and Mesenchymal Biology, University of California San Francisco, San Francisco, CA THEMATIC WORKSHOP 2.2 4Department of Pediatrics and Institute for Human Theme: DYSGLOBULINEMIC AND LYMPHOMA-RELATED Genetics, University of California San Francisco, PERIPHERAL NEUROPATHIES San Francisco, CA

The transition between the proliferation and differ- TW 2.2.1 Dysglobulinemic neuropathies entiation of progenitor cells is a key step in organo- Thierry KUNTZER, Lausanne (Suisse) genesis, and alterations in this process can lead to 1Nerve-muscle unit, Neurology service, Department developmental disorders. The fi broblast growth factor of Clinical Neurosciences, Lausanne University (FGF) - extracellular signal-regulated kinase 1/2 Hospital, Lausanne, and 2Department of Neurology, (ERK) signaling pathway is one of the most intensive- Basel University Hospital, Switzerland ly studied signaling mechanisms regulating both pro- liferation and differentiation. How a single molecule Coexistence of neuropathy and paraproteinemia (e.g., ERK) can regulate two opposing cellular out- (monoclonal gammopathy) is a common and complex comes is still a mystery. Using both chick and mouse problem seen in clinical practice with patients over 50 models we shed light on the mechanism responsible years of age and requires the distinction of specifi c for the switch from proliferation to differentiation im- syndromes. plicating ERK subcellular localization. Analysis of The different neurological groups include the anti- Sprouty mutant embryos in the mouse revealed that MAG neuropathy, those associated with Castelman’s increased FGF-ERK signaling suppressed head myo- disorder, POEMS (polyneuropathy, organomegaly, genesis. In chick embryos, manipulation of signaling endocrinopathy, monoclonal gammopathy, and skin molecules along the FGF-ERK signaling cascade in changes) syndrome, CANOMAD (Chronic ataxic vitro and in vivo demonstrated that blockage of this neuropathy with ophthalmoplegia, IgM paraprotein, pathway accelerated myogenic differentiation, where- cold agglutinins, and anti-disialyl antibodies), and the as its activation diminished it. We next examined Abstracts S17 neuropathies associated with MGUS, multiple my- distribution may resemble multifocal neuropathies, eloma, Waldenström macroglobulinemia and AL am- symmetric neuropathies or affect individual nerves or yloidosis with notable differences in the signs and plexus. Rarely also an intravascular spread of lym- symptoms among the different groups. The clinical phoma affect peripheral nerves. courses of these neuropathies are typically chronic Toxic: Treatment of patients with lymphoma often and progressive. A precise distinction of the type of involves neurotoxic drugs in particular vinka alka- haematologic disorder associated (benign or malig- loids, which are part of several chemotherapies. Other nant), investigation of other organs manifestations, drugs as nelarabine and potentially intrathecal agents and assessment of specifi c markers are mandatory. as MTX and cytosin arabinoside can be neurotoxic. There are guidelines to clarify when there is a patho- Brentuximab bedotin is an antibody drug conjugate genic link between the monoclonal gammopathy and which has been reported to cause neuropathies. Other the neuropathy (Joint Task Force of the EFNS and biological antibody based treatments as rituximab do PNS, 2010; Nobile-Orazio E. Handb Clin Neurol not seem to cause neuropathy. 2013).These steps are important to initiate an appro- As the treatment of lymphomas has been very suc- priate therapy that may include chemotherapy and/or cessful in the past years the knowledge of paraneo- immunosuppressive treatment targeting the neuropa- plastic , neoplastic and neurotoxic effects is important thy and the haematological dysfunction. for patient care. In addition also focal radiotherapeu- TK has an unrestricted educational grant from CSL- tic interventions have to be considered. Behring AG Switzerland for clinician-initiated re- search. AJS and TK perform consultancy work for References: Actelion Ltd Switzerland. Kelly JJ et al. Lymphoma and peripheral neuropa- thy: A clinical review. Muscle & Nerve, 2005; 31: 301–313. THEMATIC WORKSHOP 2.2 Giometto B, et al.. Paraneoplastic neurologic syn- Theme: DYSGLOBULINEMIC AND LYMPHOMA-RELATED drome in the PNS Euronetwork database: a European PERIPHERAL NEUROPATHIES study from 20 centers. Arch Neurol 2010;67:330–335. Grisariu Set al. Neurolymphomatosis: an Interna- tional Primary CNS Lymphoma Collaborative Group TW 2.2.2 Lymphoma associated report. Blood 2010;115:5005–5011. neuropathies Briani C, et al. Spectrum of paraneoplastic disease Wolfgang GRISOLD, Vienna (Autriche) associated with lymphoma. Neurology 2011;76:705– 1Department of Neurology, KFJ Hospital, Vienna, 710. Austria, 2 Department of Neurology, University Grisold W, et al. Peripheral neuropathies from che- Clinic Vienna, Austria motherapeutics and targeted agents: diagnosis, treat- ment, and prevention. Neuro-oncology 2012;14 Suppl Paraneoplastic: Paraneoplastic neuropathy in lym- 4:iv45-iv54. phoma is rare. Several reports describe uncharacteris- Grisold W , et al. Malignant cell infi ltration in the tic sensorimotor neuropathies, sensory neuronopathies peripheral nervous system. Handb Clin Neurol are rare. A subacute motor neuronopathy has been 2013;115:685–712. rarely observed. Demyelinating neuropathies as GBS and CIDP have been described to occur more frequently in non THEMATIC WORKSHOP 2.2 Hodgkin`s lymphoma than in Hodgkin`s disease. Theme: DYSGLOBULINEMIC AND LYMPHOMA-RELATED Associations with paraproteins have to be excluded. PERIPHERAL NEUROPATHIES Vasculitic and autonomic neuropathies are also de- scribed in individual cases. TW 2.2.3 Treatment options in Neoplastic: Neoplastic involvement in lymphoma can occur within the CSF space , by involvement of haemopathic-associated neuropathies the dura or outside of the CSF space, or both. Marinos DALAKAS, Athens / Philadephia (Grèce) The infi ltration of either cranial nerves or peripher- al nerves has been termed neurolymphomatosis . The Abstract not received S18 Abstracts

THEMATIC WORKSHOP 2.3 The exact mechanism by which the TTR amyloid Theme: PATHOMECHANISMS OF HEREDITARY NEUROPA- deposits cause the neuropathy are not fully under- THIES stood but the nerve damage probably at least partially arises from the toxicity of the TTR aggregates early in TW 2.3.1 Understanding the the disease while accumulation of amyloid in the nerves probably contributes to the pathogenesis of the pathomechanisms of peripheral nerve neuropathy in the late disease stages. amyloid The only specifi c treatment for TTR amyloidosis Mary M REILLY, London (Royaume Uni) for many years was orthotopic liver transplantation to MRC centre for Neuromuscular Diseases, UCL remove the source of abnormal TTR production. Pa- Institute of Neurology, Queen Square, London tients with the Val30Met mutation and in the early stages of their disease benefi t most from liver trans- The Familial Amyloid Polyneuropathies (FAP) are plantation. Understanding the pathomechanisms has a heterogeneous group of autosomal dominant disor- led to the development recently of other therapies in- ders originally described by Andrade in Portuguese cluding TTR stabilizers such as difl unisal and Ta- patients in 1952. FAP is one of a group of conditions famadis (Tafamadis is licensed in some countries), called the amyloidoses that are characterised by depo- TTR gene silencing strategies such as antisense oligo- sition of misfolded and aggregated fi brillar proteins nucleotide therapy and small interfering RNA therapy with abundant beta pleated structure in the extracel- (both undergoing clinical trials currently) and anti se- lular space. rum amyloid protein (SAP) monoclonal antibodies. In familial amyloid polyneuropathy (FAP), the commonest fi bril protein deposited as amyloid is a variant form of transthyretin (TTR) but FAP can also THEMATIC WORKSHOP 2.3 occur secondary to deposition of apolipoprotein A-1, Theme: PATHOMECHANISMS OF HEREDITARY NEUROPA- gelsolin and very rarely beta 2 microglobulin. Under- THIES standing the underlying pathomechanisms of FAP is the key to developing effective therapies. TW 2.3.2 Understanding the Over 100 mutations have been described in the TTR gene associated with TTR-related FAP. The most pathomechanisms of hereditary sensory common mutation, Val30Met, presents in the second/ and autonomic neuropathies third decade in Portuguese patients with a high pene- Ingo KURTH, Jena (Allemagne) trance. Patients typically present with a small-fi bre Jena University Hospital - Institute of Human neuropathic syndrome with prominent pain which Genetics, Jena, Germany progresses relatively rapidly. As the disease progress- es large fi bre involvement becomes apparent with a Axonopathies are neurodegenerative disorders that progressive sensory-motor neuropathy. Autonomic mainly affect the long processes of neurons [1, 2]. The dysfunction, gastrointestinal symptoms and cardiac longest axons can measure up to one meter posing a involvement is common. Other features include vitre- major challenge for crucial mechanisms such as traf- ous deposits and, less commonly, nephropathy and fi cking, energy utilization, signaling, and cytoskeletal leptomeningeal involvement. Cachexia is a common organization. Axonopathies of cortical motoneurons late feature of the disease. Mean survival in untreated comprise the group of hereditary spastic paraplegias patients is approximately 10 years from symptom (HSP/SPG), whereas lower motoneurons are typically onset. affected in distal hereditary motor neuropathies The neuropathy and other organ involvement in (dHMN). Both diseases are characterized by a length- FAP is due to the deposition of TTR as amyloid. Wild dependent axonal degeneration and are distinct from type TTR can be deposited as amyloid in senile car- motor neuron diseases where a combined degenera- diac amyloidosis and in FAP, the amyloid deposits tion of both upper and lower motoneuron cell bodies contain a mixture of both wild type and mutant TTR. occurs. Hereditary neuropathies can affect sensory The mutations in TTR appear to increase the tendency axons and include hereditary motor and sensory neu- of mutant TTR to dissociate from the stable tetrameric ropathies (CMT/HMSN) and hereditary sensory and form into the pro-amyloidogenic monomeric form. autonomic neuropathies (HSAN). Abstracts S19

In HSAN sensory loss ranges from local numbness tion p.Tyr192Cys in ATL3 affects a residue in the over loss of proprioception to the complete inability GTPase-domain that is evolutionarily conserved to experience pain and affected individuals are highly throughout species and among all three human ATL exposed to injuries and mutilations. Loss of pain per- homologs, suggesting that Tyr192 is important for the ception also causes abnormal mechanical loading in proper function of the protein. Remarkably, the muta- the distal weight-bearing parts of the skeleton. This tion p.Tyr196Cys in ATL1 has been identifi ed as cause can fi nally result in neuropathic arthropathy and spon- of autosomal-dominant SPG3A with white matter taneous fractures. Fracture healing can be severely changes and seizures [18]. Tyr196 in ATL1, which is impaired leading to resorption of bones. The disorder homologous to p.Tyr192 in ATL3, is located at the in- is often complicated by osteomyelitis. Autosomal terface of the GTPase domains and its replacement by dominant and autosomal recessive forms of the disor- cysteine impaired dimerization in vitro. Mutation in der exist and known culprit genes encode proteins Tyr192 of the ATL3 protein results in a disruption of crucial for sphingolipid metabolism, neurotrophin ac- the cellular ER-network as demonstrated by heterolo- tion, axonal transport, DNA methylation, and mem- gous overexpression of mutant protein. brane shaping of organelles [3]. HSAN disorders show broad phenotypic overlap Despite remarkable non-allelic genetic heterogene- with the condition of “congenital inability to experi- ity of axonopathies, one of the common emerging dis- ence pain (CIP)”. Biallelic loss-of-function mutations ease pathways turns out to be an impairment of the in SCN9A, encoding sodium ion channel NaV1.7, structural organization of organelles. The architecture have been shown to cause an inability to experience and curvature of organelle membranes depends on pain by impairing the electrical signaling of morpho- different mechanisms including the insertion of inte- logically intact axons [19]. However, null alleles of gral membrane proteins into lipid bilayers [4–6]. Pro- SCN9A have been reported in individuals with loss of teins of the reticulon family are prototypes in bending large sensory fi bers, suggesting that neurodegenera- and stabilization of the membrane curvature of the ER tion and lack of ion channel activity are not mutually [7]. This class of molecules inserts a wedge-shaped exclusive. reticulon-domain composed of hydrophobic hairpins We recently showed that another type of “congeni- into the outer leafl et of the lipid-bilayers to induce tal inability to experience pain” results from a specifi c membrane bending. Mutations in reticulon-domain missense mutation in SCN11A, which encodes the proteins have been shown to cause spastic paraplegi- NaV1.9 voltage-gated sodium channel. This de novo as, i.e. mutations in RTN2 (reticulon 2) cause SPG12. mutation (p.Leu811Pro) was identifi ed in independent Moreover, nearly half of the HSP patients carry muta- individuals with the condition using whole-exome se- tions in SPAST (SPG4), REEP1 (SPG31), and ATL1 quencing. SCN11A / NaV1.9 is expressed in nocicep- (SPG3A), likewise encoding reticulon-domain con- tors, specialized sensory nerves that transmit pain taining ER-associated proteins [8, 9]. However, de- signals from the body periphery to the central nervous fects in membrane-shaping proteins also contribute to system. Mice that carry the missense mutation in Sc- HSAN as illustrated by mutations in FAM134B, en- n11a have reduced sensitivity to pain. Knockin-mice coding a reticulon-domain containing protein of the showed self-infl icted tissue lesions, likewise recapitu- ER and Golgi-compartment, which we found to be lating aspects of the human phenotype. Mutant causative for autosomal recessive HSAN2 [10]. Re- NaV1.9 channels in sensory neurons of knockin-mice cently, we linked mutations in the reticulon-domain are functional, but display excessive activity at resting containing GTPase ATL3 to sensory neurodegenera- voltages and cause sustained depolarization of pain- tion (HSAN1) [11]. Upon GTP binding the cytosolic sensing neurons. This gain-of-function in the basal GTPase domains of ATL-proteins dimerize. Subse- activity of NaV1.9 leads to progressive inactivation of quent GTP hydrolysis causes a major conformational other sodium and calcium channels, the principal change that pulls ATLs in opposing membranes to- components of the action potential in pain-sensing gether to facilitate membrane fusion [12–14]. By this neurons. A resultant conduction block prevents signal mechanism called homotypic fusion ATL-proteins transmission to the brain as supported by aberrant connect tubules of the ER to networks [15–17]. ATL3 synaptic transmission in the spinal cord of knockin- is particularly enriched in three-way junctions of the mice [20]. The fi ndings raise the possibility that ma- ER where it facilitates the composition of the polygo- nipulating NaV1.9 activity could be a new pathway nal ER network. The HSAN-related missense-muta- for treating pain. S20 Abstracts

Unraveling the genetic architecture of HSAN and the large G protein atlastin-1/SPG3A. Proceed- associated pain channelopathies will enlighten the ings of the National Academy of Sciences of mechanistic understanding of sensory function and the United States of America 108, 2216–2221 provide potential insights in translational concepts, (2011). which are urgently needed. [14] Liu, T. et al. Lipid interaction of the C terminus and association of the transmembrane segments [1] Suter, U. & Scherer, S.S. Disease mechanisms facilitate atlastin-mediated homotypic endo- in inherited neuropathies. Nat Rev Neurosci 4, plasmic reticulum fusion. Proceedings of the 714–26 (2003). National Academy of Sciences of the United [2] Zuchner, S. & Vance, J.M. Emerging pathways States of America (2012). for hereditary axonopathies. J Mol Med (Berl) [15] Hu, J. et al. A class of dynamin-like GTPases 83, 935–43 (2005). involved in the generation of the tubular ER [3] Rotthier, A., Baets, J., Timmerman, V. & Jans- network. Cell 138, 549–561 (2009). sens, K. Mechanisms of disease in hereditary [16] Muriel, M.-P. et al. Atlastin-1, the dynamin-like sensory and autonomic neuropathies. Nature GTPase responsible for spastic paraplegia SP- reviews Neurology 8, 73–85 (2012). G3A, remodels lipid membranes and may form [4] Shibata, Y., Hu, J., Kozlov, M. & Rapoport, T. tubules and vesicles in the endoplasmic reticu- Mechanisms shaping the membranes of cellular lum. Journal of neurochemistry 110, 1607– organelles. Annual review of cell and develop- 1616 (2009). mental biology 25, 329–354 (2009). [17] Orso, G. et al. Homotypic fusion of ER mem- [5] Park, S.H. & Blackstone, C. Further assembly branes requires the dynamin-like GTPase atlas- required: construction and dynamics of the en- tin. Nature 460, 978–983 (2009). doplasmic reticulum network. EMBO Rep 11, [18] McCorquodale, D. et al. Mutation screening of 515–21 (2010). spastin, atlastin, and REEP1 in hereditary spastic [6] Pendin, D., McNew, J. & Daga, A. Balancing paraplegia. Clinical genetics 79, 523–530 (2011). ER dynamics: shaping, bending, severing, and [19] Cox, J.J. et al. An SCN9A mending membranes. Current opinion in cell causes congenital inability to experience pain. biology 23, 435–442 (2011). Nature 444, 894–8 (2006). [7] Voeltz, G., Prinz, W., Shibata, Y., Rist, J. & [20] Leipold, E. et al. A de novo gain-of-function Rapoport, T. A class of membrane proteins mutation in SCN11A causes loss of pain per- shaping the tubular endoplasmic reticulum. ception. Nat Genet 45, 1399–404 (2013). Cell 124, 573–586 (2006). [8] Blackstone, C. Cellular pathways of hereditary spastic paraplegia. Annu Rev Neurosci 35, 25– THEMATIC WORKSHOP 2.3 47 (2012). Theme: PATHOMECHANISMS OF HEREDITARY NEUROPA- [9] Blackstone, C., O’Kane, C.J. & Reid, E. He- THIES reditary spastic paraplegias: membrane traffi c and the motor pathway. Nat Rev Neurosci 12, 31–42 (2011). TW 2.3.3 Understanding the [10] Kurth, I. et al. Mutations in FAM134B, encod- pathomechanisms of distal hereditary ing a newly identifi ed Golgi protein, cause se- motor neuropathies vere sensory and autonomic neuropathy. Nature Vincent TIMMERMAN, Antwerp (Belgique) genetics 41, 1179–1181 (2009). Peripheral Neuropathy Group, Molecular Genetics [11] Kornak, U. et al. Sensory neuropathy with bone Department, VIB and University of Antwerp, destruction due to a mutation in the membrane- Belgium shaping atlastin GTPase 3. Brain 137, 683–92 (2014). Distal hereditary motor neuropathies (HMN) are [12] Bian, X. et al. Structures of the atlastin GTPase characterized by predominant degeneration of motor provide insight into homotypic fusion of endo- neurons and their long axons, resulting in a length- plasmic reticulum membranes. Proceedings of dependent distal and atrophy. Hard- the National Academy of Sciences of the Unit- ing & Thomas (1980) have classifi ed distal HMN into ed States of America 108, 3976–3981 (2011). seven subtypes based upon age at onset, mode of in- [13] Byrnes, L. & Sondermann, H. Structural basis heritance and presence of additional clinical features. for the nucleotide-dependent dimerization of Genetic studies reported that dominant or recessive Abstracts S21 mutations in 17 genes may cause pure distal HMN or Periodic paralyses are genetic conditions of autoso- more complex forms: PLEKHG5, DCTN1, REEP1, mal inheritance with an onset in the second decade. SLC5A7, DNAJB2, FBXO38, GARS, BICD2, SETX, They manifest as acute and reversible attacks of muscle BSCL2, IGHMBP2, TRPV4, VAPB, ATP7A, HSPB1, weakness. Mutations in the gene encoding the muscu- HSPB3 and HSPB8. These genes code for proteins lar voltage-gated calcium channel are a major cause of with various functions ranging from more general to hypokalemic , a form of the disease motor neuron specifi c functions. The most remarkable with a concomitant decrease in blood potassium levels group of distal HMN genes are those coding for three during the attacks. If the causative gene has been known small heat shock proteins (HSPB1, B3 and B8). Al- for twenty years, patho-physiology has long remained though regulated by stress, they are constitutively ex- obscured. In contrast with other channelopathies, no pressed and responsible for quality control and protein major modifi cation of the biophysical properties was folding. The HSPBs are not only molecular chaper- observed in the mutated channel. Recent advances have ones but also involved in many essential cellular pro- helped to better understand the mechanism of the at- cesses such as apoptosis, autophagy, splicing, tacks with the discovery of a leaking current in the dynamics and neuronal survival. Tubulin channel. If attacks are better understood, there are still came out as the most striking differential interacting uncertainties in the way this leaking current modifi es protein to mutant HSPB1. This anomalous binding the properties of the muscle membrane and how these leads to stabilization of the (MT) network modifi cations can lead to the observed major ion fl uxes in a MAP-like manner. A transgenic mouse model for occurring across the muscle membrane during attacks. mutant HSPB1 confi rmed the enhanced interaction of The recent construction of functional models may help mutant HSPB1 with tubulin and can be treated by to unravel these questions as well as new possibilities HDAC inhibitors, demonstrating the potential for of observing abnormal ion fl uxes by MRI. All these ad- treating distal HMN. The wild type HSPB1 protein vances will be summarized and put in the context of seems to regulate the balance between centrosomal treatment of these incapacitating muscle disorders. and non-centrosomal MTs. Mutations in HSPB1 have also shown to disrupt the neurofi lament (NF) network and cause their aggregation. Neurofi laments are abun- THEMATIC WORKSHOP 2.4 dant structural proteins in neurons and play a key role Theme: CALCIUM FLUXES AND ION CHANNELS IN MUSCLE in neurodegeneration. Transduction of neuronal cells with mutant HSPB1 affect the NFs axonal transport and binding to the anterograde motor protein . TW 2.4.2 Genetic evidence that Ca2+ is These defi cits were also associated with an increased the key inducer of myofi ber necrosis in phosphorylation of NFs as well as an increased phos- muscular dystrophy phorylation of the cyclin dependent kinase Cdk5, which mediates the NF-phosphorylation. Inhibition of Jeffery D MOLKENTIN, Cincinnati (Etats-Unis) Cdk5 restored the NF-phosphorylation and binding to University of Cincinnati and Cincinnati Children’s kinesin. Altogether, specifi c mutations in HSPB1 af- Hospital Medical Center, Cincinnati, OH, USA fect the axonal transport via induced hyperphosphory- Muscular dystrophy (MD) refers to a clinically and lation of NFs and stabilization of the MT-network, and genetically heterogeneous group of degenerative mus- can be targeted by drugs in experimental models open- cle disorders characterized by progressive muscle ing possibilities for treatment of distal HMN. weakness and degeneration. Although the primary de- fect likely results from a loss of sarcolemmal integrity, the secondary molecular mechanisms leading to myo- THEMATIC WORKSHOP 2.4 fi ber necrosis is debated. One hypothesis suggests that Theme: CALCIUM FLUXES AND ION CHANNELS IN MUSCLE elevated cytosolic Ca2+ is an initiating event for many downstream sequelae resulting in myofi ber necrosis. TW 2.4.1 Periodic Paralysis related to Direct measurement of resting Ca2+ levels in myofi - calcium channelopathy bers from dystrophic animal models or humans has so far produced equivocal results, with some studies re- Bertrand FONTAINE, Paris (France) porting elevated Ca2+ while other studies report no National Reference Center for neuromuscular change. Here we undertook a genetic approach to ma- channelopathies and Institut Cerveau Moelle-ICM, nipulate Ca2+ directly in the mouse by transgenesis Hospital Pitié-Salpêtrière, Paris, France S22 Abstracts and gene-targeting. Our results uniformly support the tions in TM2 of RyR1 on EC coupling, bi-directional Ca2+ hypothesis of MD, such that mouse models with coupling between RyR1 and the dihydropyridine re- artifi cially elevated Ca2+ show fulminant disease, ceptor, and the conductance and gating of RyR1 chan- while mouse models with enhanced Ca2+ clearance nels incorporated into planar lipid bilayers. Following are resistant to MD when crossed with mouse models expression in RyR1-null myotubes, an uncoupling of this disease. Our collective results span transgenic phenotype was observed for a subset of CCD muta- mice that overexpress TRPC3, Stim1 and NCX1 as tions (L4647P, Delta4647–48, H4651P, and H4651R) ways of enhancing baseline Ca2+ levels in myofi bers located in the center and on one face of the TM2 (all induce MD-like disease), as well as mice express- α-helix. RyR1 function was essentially unaltered for ing dnTRPC3, dnTRPC6, dnOrai1, SERCA1 and CCD mutations located at either the N-terminal end NCX1 as ways of reducing pathologic Ca2+ infl ux (all (T4637A/I, G4638D/S/V, and M4640V) or on the op- of which protect from MD disease). We have also gen- posing face of the TM2 α-helix (L4650P). Immunocy- erated mice lacking the gene for cyclophilin D (Ppif) tochemistry and whole-cell patch clamp studies found and NCX1 (Slc8a1), which renders mitochondria less that all four of the uncoupling mutants in TM2 co-lo- sensitive to Ca2+-induced necrosis and hence, show calized with dihydropyridine receptor and enhanced less dystrophic disease when crossed with mouse L-type calcium currents. Additional mutations models of this disease. Our results suggest that eleva- (L4644P/R, L4647R, L4649P, A4653P, and tion in resting Ca2+ within the myofi ber in MD is the A4655P/R) were engineered to precisely defi ne the proximal event inducing muscle wasting downstream boundaries and helix-sidedness of the uncoupling of an unstable sarcolemma (due to loss of dystrophin module in TM2. Bilayer studies showed that while or other key structural genes that cause MD). potassium and calcium permeation were unaltered by the Delta4647–48, L4650P, and H4651P TM6 muta- tions, each of these mutants exhibited a markedly re- THEMATIC WORKSHOP 2.4 duced average channel open probability due to a Theme: CALCIUM FLUXES AND ION CHANNELS IN MUSCLE modest reduction in channel mean open time and larg- er increase in channel mean closed time. Together, these results demonstrate that one face of TM2 func- TW 2.4.3 Identifi cation of a Relay Gate in tions as an important RyR1 relay gate that couples the type I Ryanodine Receptor from dihydropyridine receptor activation and to channel functional analyses of a cluster of disease opening during excitation-contraction coupling and mutations in TM2 that mutations with this domain disrupt this relay gate function in the absence of change in ion permeation. Robert T DIRKSEN, Rochester, NY (Etats-Unis) 1Department of Pharmacology and Physiology, University of Rochester Medical Center, 601 THEMATIC WORKSHOP 2.5 Elmwood Avenue, Rochester. NY 14642, USA Theme: MYOFIBRILLAR AND GNE MYOPATHIES 2John Curtin School of Medical Research, Australian National University, PO Box 334, Canberra, ACT 2601, Australia TW 2.5.1 Myofi brillar myopathies - clinical characteristics Mutations in the type I ryanodine receptor (RyR1) that result in malignant hyperthermia and central core Bjarne UDD, Tampere (Finlande) disease can be found throughout the RyR1 sequence. Neuromuscular Research Center, Tampere Interestingly, a large number of the C-terminal disease University Hospital, Tampere & Folkhalsan Institute mutations are found in two clusters: one in the selec- of Genetics, University of Helsinki, Helsinki, Finland tivity/pore-lining region and a second cluster within the second conserved transmembrane domain (TM2) Myofi brillar myopathy is a diagnostic concept of the channel. We previously found that disease mu- based on muscle histopathology fi ndings indicating tations in the RyR1 selectivity fi lter lead to a form of disorganized myofi brils: dark and hyaline cytoplas- excitation-contraction “uncoupling” (i.e. reduced cal- mic changes on trichrome stain, abnormal sarcomeric cium release in the absence of store depletion), which protein aggregations, disintegrated myofi brillar struc- results from a disruption in calcium permeation. Here, tures on ultrastructure and rimmed vacuoles. Because we determined the effect of 11 different disease muta- many patients with a pathological diagnosis of myofi - Abstracts S23 brillar myopathy clinically show marked distal weak- the disease-causative gene. A precise diagnosis requires ness there is an overlap with the distal myopathies. identifi cation of a causative mutation in each patient. This was fi rst known for desminopathy, and later Muscle biopsies in patients carrying DES or CRY- shown for myotilinopathy and . Besides AB mutations show patches of -immunoreac- these three genetic disorders mutations in αB- tive inclusions that correspond to granulofi lamentous cristallin, BAG3 and -C cause marked myofi - material at the ultrastructural level. In patients with brillar myopathy. Myofi brillar myopathy changes can MYOT and ZASP mutations prominent desmin and also be observed in other diseases such as mutated myotilin immunoreactive inclusions, large numbers FHL-1, SEPN1, DNAJB6 and HMERF titinopathy, of vacuoles, inclusion bodies and fi lamentous bundles although other clinical or pathology features are usu- on EM are typically seen. A combination of the le- ally more characteristic for these disorders. In this sions mentioned above are normally seen in patients workshop we will highlight clinical, epidemiological, with mutations in fi lamin C rod domain. The presence muscle imaging and pathology features in this group of multiple cytoplasmic bodies in muscle fi bers is of muscle diseases with the aim of enhancing diag- typically observed in hereditary myopathy with early nostic accuracy for the patients which is of impor- respiratory failure (HMERF) resulting from muta- tance for the correct management of each of these tions in the FN3 domain. diseases. Some are associated with cardiomyopathy Muscle imaging is a powerful diagnostic tool or respiratory problems and others are not. Moreover, allowing differentiation between gene-dependent the molecular pathogenesis linked to defect turnover subtypes of MFM. Desminopathy and alphaB- and recycling of sarcomeric proteins as well as alter- crystallinopathy are characterized by early involve- natives regarding treatment options will be discussed. ment of semitendinosus, sartorius and gracilis at the mid-thigh, and anterior tibialis and peroneal group at mid-leg. A striking similar pattern of muscle THEMATIC WORKSHOP 2.5 involvement is observed in patients suffering from Theme: MYOFIBRILLAR AND GNE MYOPATHIES HMERF. By contrast myotilinopathy, ZASPopathy, and fi are characterized by early involve- ment of the semimembranosus, hip adductors and bi- TW 2.5.2 Diagnostic clues from muscle ceps femoris at the mid-thigh, and soleus and medial pathology and MRI in Myofi brillar gastrocnemius at the mid-leg level. Myopathies Montse OLIVÉ, Barcelona (Espagne) THEMATIC WORKSHOP 2.5 Institute of Neuropathology, Department of Pathology, and Neuromuscular Unit - IDIBELL- Theme: MYOFIBRILLAR AND GNE MYOPATHIES Hospital Universitari de Bellvitge, Barcelona, Spain TW 2.5.3 Protein aggregates and Rimmed Myofi brillar myopathies (MFM) are a group of Vacuoles: Possible therapeutic muscle disorders characterized morphologically by focal disintegration of myofi brils and accumulation of interventions in MFM degradation products into inclusions containing des- CONRAD WEILH, SAINT LOUIS (Etats-Unis) min and other proteins. These highly heterogeneous Department of Neurology, Washington University disorders are caused by mutations in desmin (DES), School of Medicine, Saint Louis, Missouri, 63119 USA. α(alpha)B-crystallin (CRYAB), myotilin (MYOT), Z- band alternatively spliced PDZ-containing protein The accumulation of proteinaceous inclusions (ZASP), fi lamin C (FLNC), Bcl-2-associated athano- underlies the pathogenesis of a growing list of protein gene-3 (BAG3) and FN3 titin (TTN) domain. Further aggregate myopathies or myofi brillar myopathies genetic heterogeneity is suspected since the causative (MFMs). The histopathologic spectrum of MFMs in- gene remains to be discovered in many patients suf- cludes, myofi brillar disorganization, protein inclu- fering from MFM. sions and occasionally rimmed vacuoles. Inherited Individual MFM subtypes have distinct clinical, mor- mutations in myofi brillar proteins such as desmin, phological and muscle imaging features depending on myotilin, and fi lamin-c cause these proteins to aggre- gate, become insoluble and accumulate in myofi bers S24 Abstracts leading to MFMs. Moreover, mutations in protein poorly equipped to administrate his life and live inde- chaperones, such as BCL2-associated athanogene 3 pendently with his helpers. (Bag3) and αB-crystallin, necessary for the proper The adult with DMD will also experience medical folding of myofi brillar proteins also leads to patho- complications that previously were not observed. In logically similar MFMs. Protein aggregates can be the Danish cohort, hospital admissions were most of- degraded via autophagy suggesting that enhancing au- ten attributable to pneumonia or abdominal pain, the tophagy in MFM may be therapeutic. We will discuss latter often caused by extreme constipation with ac- potential therapeutic interventions aimed at enhancing cumulated air. Surgical treatment of complications autophagy in skeletal muscle. In addition, we will substantially increases risk to the lives of the patients highlight our studies understanding and manipulating because it necessitates anaesthesia at a time where autophagy in skeletal muscle using MFM and protein they are medically vulnerable. In a Danish cohort fol- aggregate myopathy mouse models. These studies lowed for 30 years, the main cause of death was car- suggest that the manipulation of autophagic function diac insuffi ciency; in half of these cases, cardiac arrest in skeletal muscle holds promise in treating MFMs. was related to surgery procedures which would nor- mally be considered minor. The extended life expectancy of DMD patients ne- THEMATIC WORKSHOP 2.6 cessitates centralization of knowledge and follow-up Theme: ENMC SESSION: HIGHLIGHTS FROM RECENT ENMC in specialized centres in order to acquire experience WORKSHOPS with the medical complications and their manage- ment. The complexity of the medical care required and the transition from in-patient care to home care TW 2.6.1 Care for adults with DMD - with specialized home respiratory equipment and per- outcome of an ENMC workshop sonnel is another challenge in many countries. A spe- JES RAHBEK, Aarhus (Danemark) cially trained interdisciplinary team working in collaboration with the patient is needed to ensure that Over the past 30 years, the life expectancy of Duch- his everyday life will function well in his home, on his enne muscular dystrophy (DMD) patients has in- and his relatives’ own terms. creased thanks to advances in the care and treatment Important topics for further study and discussion of their respiratory and cardiac insuffi ciency, muscle include: strength deterioration, and . In a country like • Causes of death Denmark, the number of people with DMD aged 18 • Medical treatment years and older now exceeds the number younger than • Respiratory insuffi ciency, oral and upper airway 18. While the maximum age for survival was previ- problems ously approximately 20 years in the Western world, it • Endocrine aspects and nutrition currently extends to the fourth decade of life. • Motor function and activity A survey of the mainly steroid-naive Danish DMD • Organizing care for the adult with DMD population above the age of 20 (n = 74; age 20–46) • Quality of life revealed that from his mid twenties, a man with DMD is dependent on assisted ventilation 24 hours a day. He is able to sit all day in his powered THEMATIC WORKSHOP 2.6 with body and neck support. While he cannot move Theme: ENMC SESSION: HIGHLIGHTS FROM RECENT ENMC his limbs, he has a little residual muscle strength in WORKSHOPS one or more fi ngers that enables him to use a special joystick for his wheelchair and write on a computer. TW 2.6.2 Biomarkers in DMD: from the He is not able to feed himself and when fed, he takes discovery to the development toward a long time to chew and swallow his food. He cannot raise his voice and may have diffi culties in articula- clinical application and translation in tion, making his speech diffi cult to understand. He is other NMDs dependent on around-the-clock practical assistance. Alessandra FERLINI, FERRARA (Italie) Absence of social and cognitive skills, coupled with Department of Medical Sciences University of an insuffi cient educational level, may mean that he is Ferrara (Ferrara, Italy) Abstracts S25

Biomarkers have been defi ned as cellular, biochem- Disclosure of interest: AF is PI of Prosensa and ical, molecular alterations or biological characteris- GSK antisense-based clinical trials, participates to EU tics that are measurable in biological material as projects (BIO-NMD and Neuromics) and other re- indicator of normal biological or pathogenic process- search projects on rare diseases. es (Scotton C et al., 2013). Biomarkers may be used in differential and early diagnosis, and in monitoring of disease progression, regression, or therapeutic re- THEMATIC WORKSHOP 2.6 sponse. Duchenne Muscular Dystrophy (DMD) is a Theme: ENMC SESSION: HIGHLIGHTS FROM RECENT ENMC severe hereditary muscle disorder due to a variety of WORKSHOPS dystrophin gene mutations and presenting with vari- able clinical severity. Recently novel experimental drugs have been de- TW 2.6.3 Autophagy in Muscular veloped for DMD and several trials are ongoing, rais- Dystrophies: Translational approach ing the urgent need of having fi ne tools for measuring LUCIANO MERLINI, BOLOGNA (Italie) the trial outcomes as well as for optimizing the a Laboratory of Musculoskeletal Cell Biology, selection of eligible patients (Archevala-Gomez et al., Istituto Ortopedico Rizzoli, IOR-IRCCS, Bologna, 2012,; Mendell et al., 2012). The use of clinical Italy parameters measuring muscle strength and function is limited due to their dependence on motivation, large (Macro)autophagy is an evolutionarily conserved intra-individual variability and slow response time. intracellular system by which macromolecules and Conversely, molecular biomarkers may show earlier organelles are delivered to lysosomes for degradation response to treatment and refl ect the different patho- and recycling. This catabolic process is particularly physiological aspects of the disease. The use of bio- relevant for skeletal muscle that represents 40% of marker panels for the diagnosis, prognosis, and whole-body lean mass, thereby providing a tissue monitoring of DMD (and more in general of rare source for amino acids that can be used in times of chronic neuromuscular disorders) as well as to guide stress or starvation. Recent research has made it clear the choice of therapeutic regimens may signifi cantly that normal autophagy is fundamental for human improve the current clinical practice, by facilitating health, aging and life span, and malfunctioning or the evaluation of emerging therapies in drug trials and failure of autophagy is associated with a wide range regulatory approval. These biomarkers will also serve of human pathologies including muscular dystrophies for patients’ stratifi cation and for selection of appro- [1]. In particular alteration of autophagosome biogen- priate subjects for clinical trials. This will impact on esis has been found relevant for a number of muscular the economical load, patients’ care and novel thera- dystrophies including Ullrich congenital muscular pies. Biomarker discovery (proceeding by candidate dystrophy, DMD, Emery-Dreifuss muscular dystro- or via omics analysis) is vital in order to identify ex- phy and congenital muscular dystrophy 1A. Altera- ploratory markers to be further validated in larger pa- tion of autophagosome-lysosome fusion is instead tients’ cohorts and international collaborations are involved in Vici syndrome, Danon disease and Pompe mandatory in the validation stage, since rare diseases disease. Activation of autophagic fl ux has been proved patients are small in number. Enhancing cooperation benefi cial in mice models of Ullrich congenital mus- will allow to have validated clinical biomarkers trans- cular dystrophy, DMD, Emery-Dreifuss muscular latable in clinical practice. dystrophy, and inhibition of autophagic fl ux in a mouse model of congenital muscular dystrophy 1A. A References normocaloric low protein diet to reactivate autophagy -Arechavala-Gomeza V, et al., Antisense oligonu- was performed in patients with COL6-related myopa- cleotide-mediated exon skipping for Duchenne mus- thies. When compared to the baseline samples, cular dystrophy: progress and challenges. Curr Gene LC3II/I ratio and beclin1 appeared increased after Ther. 2012 12:152–60. Review. treatment both in leukocytes samples and in muscle -Mendell JR, et al., Gene therapy for muscular dys- biopsy of patients. As a group, our current recommen- trophy: lessons learned and path forward. Neurosci dations regarding the design of clinical trials modulat- Lett. 2012 Oct 11;527(2):90–9. ing autophagy are the following: 1) every effort -Scotton C, Biomarkers in rare neuromuscular dis- should be made to sample patient material under sim- eases. Exp Cell Res. 2013 Dec 31. S26 Abstracts ilar nutrient states, time of day and site of biopsy. 2) as Cachexia is a debilitating condition characterized no single autophagic biomarker is reliable, a panel of by extreme skeletal muscle atrophy that contributes several biomarkers should be assessed (i.e. LC3, p62, signifi cantly to morbidity and mortality. This wasting Lamp1/2) and include both morphologic/biochemical state of muscle largely derives from aberrant signal- markers as performed via electron microscopy, IHC ing of pathways that maintain a balance between the or immunoblotting along with gene expression stud- anabolism and catabolism of muscle protein. In ca- ies of these same biomarkers. 3) depending upon the chexia, this balance is tipped towards a catabolic state proposed mechanism of therapeutic intervention, resulting from the activation of the ubiquitin protea- measures of autophagy signaling pathways should be some and autophagy systems that regulate protein evaluated (i.e. mTOR, Akt, FOXO3, TFEB, beclin-1). breakdown, and reduced Akt and mTOR activities 4) In some cases, quantitating the changes in other au- that decrease protein synthesis. Whereas these events tophagic substrates may be useful (e.g. desmin, ubiq- are fi rmly established to reside within the myofi ber, uitinated proteins or TDP-43). Finally, 5) it is essential less regard has been given to potential contributory that new methods be developed to monitor autophagic factors that might act outside the myofi ber within the fl ux in vivo in human patients. muscle microenvironment. Recently, our laboratory described that muscles derived from tumor bearing References mice and pancreatic cancer patients with weight loss [1] Merlini L, Nishino I; On behalf of the Autopha- undergo a damage response. This damage was associ- gy in muscular dystrophy consortium. 201st ated with an expansion of both satellite and non-satel- ENMC International Workshop: Autophagy in lite progenitor cells that express Pax7 and commit to a Muscular Dystrophies – Translational Approach, differentiation program. However, we found that due 1–3rd November 2013, Bussum, The Nether- to persistent Pax7 expression, myoblasts are unable to lands. Neuromuscul Disord. 2014 (in press). complete their differentiation, and thus unable to fuse to damaged myofi bers. The ultimate fate of these cells is to undergo apoptosis, an event controlled by circu- THEMATIC WORKSHOP 3.1 lating microvesicles. These microvesicles contain Theme: STEM CELLS AND MUSCLE REGENERATION miR-21 that signal through TLR7 on myoblasts to mediate cell death via JNK signaling. Thus, we pro- pose a model of cachexia whereby Pax7 functions to TW 3.1.1 A cell-autonomous loss of impair the regenerative capacity of myogenic cells in muscle stem cell self renewal in aged the muscle microenvironment to drive muscle wast- muscle ing in cancer, a mechanism that might also be linked to the death of these myogenic cells mediated through Bradley OLWIN, Boulder (Etats-Unis) an extracellular vesicle signaling pathway involving miR-21 and TLR7. Abstract not received

THEMATIC WORKSHOP 3.1 THEMATIC WORKSHOP 3.1 Theme: STEM CELLS AND MUSCLE REGENERATION Theme: STEM CELLS AND MUSCLE REGENERATION TW 3.1.3 The role of the muscle stem cell TW 3.1.2 The Role of the Muscle niche in regulating regenerative Microenvironment in Cancer-Induced myogenesis Cachexia Michael RUDNICKI, Ottawa (Canada) Denis GUTTRIDGE, Columbus (Etats-Unis) Ottawa Hospital Research Institute, Regenerative 1 Department of Molecular Virology, Immunology, Medicine Program, Ottawa, Ontario, Canada and Medical Genetics, The Ohio State University, Columbus, OH, 43210, USA1, Satellite cells in adult skeletal muscle are a hetero- 2Pierre et Marie Curie University of Paris, Cedex 5, geneous population composed of stem cells and com- France mitted progenitors. Wnt7a signalling dramatically Abstracts S27 stimulates the symmetric expansion of satellite stem broad implications for the therapeutic use of Wnts as cells and this expansion requires Fzd7 and Vangl2, biologics. M.A.R. is a founding scientist in Fate Ther- both components of the planar cell polarity (PCP) sig- apeutics who have licensed the Wnt7a technology. naling pathway. Importantly, Wnt7a over-expression results in a large expansion of the satellite stem cell population, and Wnt7a defi ciency in impaired mainte- THEMATIC WORKSHOP 3.2 nance of the satellite cell compartment. Therefore, Theme: CONGENITAL MYASTHENIC SYNDROMES Wnt7a signaling through the planar cell polarity path- way controls the homeostatic level of satellite stem cells and hence regulates the regenerative potential of TW 3.2.1 An emerging subgroup of muscle. In differentiated myofi bers, Wnt7a binding to congenital myasthenic syndromes due to Fzd7 directly activates the Akt/mTOR growth path- mutations in genes associated with way thereby inducing myofi bre hypertrophy. Notably, N-linked glycosylation the Fzd7 receptor complex is associated with GNAS1 and PI3kinase in differentiated myofi bres but not in David BEESON, Oxford (Etats-Unis) myoblasts, and are required for Wnt7a to activate the Neurosciences Group, Weatherall Institute of Akt/mTOR growth pathway. Wnt7a/Fzd7 activation Molecular Medicine, Nuffi eld Department of Clinical of this pathway was completely independent of IGF- Neuroscience, University of Oxford, UK receptor activation. We found that Syndecan-4 (Sdc4) and Frizzled-7 (Fzd7) form a co-receptor complex in Congenital myasthenic syndromes (CMS) are he- satellite cells and that binding of the glycoprotein Fi- reditary disorders of neuromuscular transmission bronectin (FN) to Sdc4 stimulates the ability of Wn- characterised by fatigable muscle weakness. The t7a to induce the symmetric expansion of satellite number of cases recognised, at around 1:100,000 of stem cells. Newly activated satellite cells dynamically the UK population, is increasing with improved diag- remodel their niche by transient high-level expression nosis. The underlying genetic defects are diverse in- of FN. Knockdown of FN in prospectively isolated volving a series of different genes with a variety of satellite cells severely impairs their ability to repopu- different phenotypes. Mutations within the muscle late the satellite cell niche following transplantation acetylcholine receptor form the most common CMS into regenerating muscle. Conversely, in-vivo over- subtype. Here, we highlight a new emerging group of expression of FN with Wnt7a dramatically stimulates CMS, characterised by a limb-girdle pattern of mus- the expansion of satellite stem cells in regenerating cle weakness, which are caused by mutations in genes muscle. Therefore, activating satellite cells remodel that encode proteins in the initial steps of the N-linked their niche through autologous expression of FN that glycosylation pathway. We used whole exome se- provides feedback to stimulate Wnt7a signaling quencing to identify mutations in fi ve different genes through the Fzd7/Sdc4 co-receptor complex. Thus, associated with the N-linked glycosylation pathway FN and Wnt7a together regulate the homeostatic lev- that cause CMS. Expression studies in cultured myo- els of satellite stem cells and satellite myogenic cells tubes derived from patients with mutations in GFPT1 during regenerative myogenesis. We generated a trun- or DPAGT1 showed a signifi cant reduction of cell- cated Wnt7a variant, consisting of the C-terminal 137 surface AChR expression p < 0.0001. Inhibition of amino acids lacking the conserved palmitoylation GFPT1 or DPAGT1 enzymatic activity or siRNA sites, which retains full biological activity in skeletal silencing of GFPT1 or DPAGT1 expression both re- muscle. This includes binding to and signaling sulted in reduced AChR cell-surface expression. through its receptor Fzd7 to stimulate symmetric ex- Western blot and gene silencing experiments indicate pansion of satellite stem cells by activating the planar impaired assembly/maturation of the AChR pentamer cell polarity pathway, and inducing myofi bre hyper- is responsible for reduced expression at the endplate. trophy by signaling through the AKT/mTOR path- Mutations affecting N-linked glycosylation form part way. Furthermore, this truncated Wnt7a shows of the congenital disorders of glycosylation, and often enhanced secretion and dispersion compared to the lead to severe multisystem disorders. Surprisingly al- full-length protein. Together, these fi ndings open im- though N-linked glycosylation is ubiquitous, occur- portant new avenues for the development of a Wnt7a ring in all mammalian cells, symptoms in our patients as a treatment for muscle wasting diseases and have are often largely restricted to neuromuscular transmis- sion. We speculate that the neuromuscular junction, S28 Abstracts and in particular the acetylcholine receptor itself, is fects in the neuromuscular transmission leading to particularly sensitive to defects in biochemical path- muscle weakness accentuated by exertion, and are ways affecting glycosylation. heterogeneous for both their mode of inheritance and their pathophysiology. To date, even if the alteration of several genes have been shown to cause CMS, the THEMATIC WORKSHOP 3.2 molecular origin of about half of these pathologies Theme: CONGENITAL MYASTHENIC SYNDROMES remains still unknown. Using a candidate gene approach, the French CMS network has identifi ed mutations in the genes encod- TW 3.2.2 Diagnosis and therapy of ing MuSK and agrin. In order to demonstrate that congenital myasthenic syndromes - these variants were indeed responsible for the CMS clinical update phenotype, MuSK vectors or recombinant agrin pro- teins, either wild-type or bearing a mutation identifi ed Hanns LOCHMULLER, Newcastle (Royaume Uni) in patients, were tested for their ability to modify Institute of Genetic Medicine, Newcastle University MuSK phosphorylation or AChR aggregation in cell cultures or to alter the NMJ structures in rodent mus- Neuromuscular junction disorders, also called My- cles. Although the alterations of the agrin-MuSK- asthenic syndromes (MS), are a rare heterogeneous Dok7-AChR cascade in vitro were not always those group of acquired (Myasthenia Gravis, MG) and in- expected, the NMJ structural changes in rodent mus- herited (Congenital Myasthenic Syndromes, CMS) cle recapitulated those observed in the patient muscle neuromuscular disorders associated with distinctive biopsies. The involvement of the presynaptic com- clinical, electrophysiological, laboratory and ultra- partment was impressive as evidenced by the exuber- structural abnormalities. The genetic defects in CMS ant axonal growth and questions the role of mutant either impair neuromuscular transmission directly or MuSKs and agrins in these processes. Several other result in secondary impairments, which eventually mutations in the genes encoding MuSK or agrin have compromise the safety margin of neuromuscular been subsequently identifi ed, however not to the ex- transmission. More recently, we have identifi ed two tent of those in DOK7. In the case of the agrin gene, genes (DOK7, GFPT1) that cause fatigable weakness some mutations may be causing a misleading pheno- of muscles in a limb-girdle distribution, but rarely af- type of distal myopathy. fecting facial or eye muscles. We will cover the sig- This work on human mutations may reveal new nifi cant progress made in understanding the molecular functions of agrin and MuSK at the NMJ. In return, it pathogenesis of CMS, which is important for both pa- may allow a better understanding of the pathogenic tients and clinicians in terms of reaching a defi nite mechanisms underlying CMSs and the adaptive pro- diagnosis and selecting the most appropriate treat- cesses taking place at the NMJ in these patients. ment

THEMATIC WORKSHOP 3.2 THEMATIC WORKSHOP 3.2 Theme: CONGENITAL MYASTHENIC SYNDROMES Theme: CONGENITAL MYASTHENIC SYNDROMES

TW 3.2.3 Agrin and MuSK in congenital TW 3.2.4 Observations in recently myasthenia identifi ed congenital myasthenic syndromes (CMS) Daniel HANTAI, Paris (France) Inserm U1127, ICM, Groupe Hospitalier Pitié- ANDREW G ENGEL, ROCHESTER (Etats-Unis) Salpêtrière, Paris, France Department of Neurology, Mayo Clinic, Rochester, APHP, Centre de Référence des Maladies MN, USA Neuromusculaires, Groupe Hospitalier Pitié- To date 20 CMS disease genes have been identifi ed. Salpêtrière, Paris, France In some CMS the disease protein resides at pre-or Congenital myasthenic syndromes (CMS) repre- postsynaptic sites or in the synaptic space. More re- sent a group of genetic diseases characterized by de- cently genes involved in protein glycosylation or end- Abstracts S29 plate (EP) development and maintenance were THEMATIC WORKSHOP 3.3 identifi ed. The fi rst identifi ed gene affecting glycosyl- Theme: ELECTROPHYSIOLOGICAL APPROACHES IN DYSIM- ation was GFPT1. In our cohort of 11 pts 5 had inter- MUNE NEUROMUSCULAR DISEASES costal biopsies; all had hypoplastic EPs but only 3 had mild EP AChR defi ciency and a low miniature EP po- TW 3.3.1 Chronic infl ammatory tential (MEPP) amplitude. One pt with a mutation dis- rupting the muscle specifi c exon never moved in demyelinating polyradiculoneuropathy utero, remains quadriplegic at age 8 y, and has a se- (CIDP): electrodiagnostic evidence vere autophagic myopathy emphasizing importance Peter VAN DEN BERGH, Brussels (Belgique) of this exon in muscle function. The second identifi ed Neuromuscular Reference Centre, Dept. of gene affecting protein glycosylation was DPAGT1. Neurology, University Hospitals St-Luc, Brussels, We observed 3 pts who are intellectually disabled and Belgium have an autophagic myopathy. The observed missense mutations reduce protein expression, or decrease or Making the diagnosis of infl ammatory demyelinat- abolish enzyme activity, or cause many skipped and ing neuropathy, in particular when the disease course few nonskipped alleles. Immunoblots of muscle ex- is chronic, is often diffi cult. Diagnostic criteria are tracts indicate reduced to absent glycosylation of dif- very important but given the lack of a defi nitive diag- ferent proteins, including STIM1 which likely nostic marker and the limitations of laboratory stud- explains the tubular aggregates in this disease. EP de- ies, it appears impossible to reach a defi nitive velopment is affected by LRP4, a postsynaptic core- diagnosis in all patients. By defi nition, electrophysio- ceptor of neural agrin for activating MuSK. Acting in logical criteria for primary demyelination are de- concert with Dok7 and other proteins, LRP4 acts on signed to exclude abnormalities that can be explained rapsyn to concentrate AChR at the EP, enhance syn- by axonal degeneration. Therefore, lesser degrees of apse specifi c gene expression, and promote postsyn- demyelination can not be defi ned with complete cer- aptic differentiation. In a recent study with K. Ohno tainty electrophysiologically. Optimised electrophysi- and B. Ohkawara we identifi ed 2 mutations at the ological criteria are capable, however, to support the edge of the third beta-propeller domain of LRP4 in a diagnosis with different levels of probability (possi- 14-year-old girl with limb-girdle CMS. Both muta- ble, probable, defi nite). To reach the best possible sen- tions decrease binding affi nity of LRP4 for both sitivity levels, it is best to examine motor nerves MuSK and agrin. Intercostal muscle synaptic contacts bilaterally and to include proximal stimulation sites in are disarrayed and hypoplastic but other parameters the upper limbs. Electrophysiological criteria for on neuromuscular transmission are unaffected. Final- CIDP proposed by the EFNS/PNS Task Force are ly, in collaboration with L. Regal and J. Creemers, we highly sensitive and specifi c for primary demyelin- identifi ed a pt with isolated PREPL defi ciency with ation and lead to the correct diagnosis when clinical myasthenic symptoms since birth, a positive edropho- criteria are fufi lled in the very large majority of cases. nium test, and growth hormone defi ciency without cystinuria who responded transiently to pyridostig- mine during infancy. EP studies revealed decreased THEMATIC WORKSHOP 3.3 evoked quantal release and small MEPPs despite ro- Theme: ELECTROPHYSIOLOGICAL APPROACHES IN DYSIM- bust EP AChR expression. Because PREPL is an es- MUNE NEUROMUSCULAR DISEASES sential activator of the clathrin associated adaptor protein 1 (AP1), and AP1 is required by the vesicular ACh transporter, we attribute the small MEPPs to a TW 3.3.2 Guillan-Barrè syndrome: decreased ACh content of the synaptic vesicles. electrodiagnostic issues and challenges Antonino UNCINI, Chieti (Italie) Department of Neuroscience and Imaging, University “G. d’Annunzio”, Chieti-Pescara (IT)

Recent clinical and experimental studies indicated that axonal GBS subtypes with anti-ganglioside anti- bodies show, besides axonal degeneration, promptly S30 Abstracts reversible nerve conduction failure (RCF) affecting THEMATIC WORKSHOP 3.3 which can be recognized only by serial recordings. Theme: ELECTROPHYSIOLOGICAL APPROACHES IN DYSIM- This explain why Recovery in acute motor axonal MUNE NEUROMUSCULAR DISEASES neuropathy may be either very rapid and complete or prolonged with poor outcome according to the rela- TW 3.3.3 Neuromuscular junction tive amount of RCF and axonal degeneration in each patient. Not recognizing distal RCF may induce, on disorders: electrodiagnostic methods the basis of a single test, to diagnose axonal degenera- J GERT VAN DIJK, Leiden (Pays-Bas) tion and to formulate a bad prognosis. Moreover the lack of distinction among demyelinating conduction Abstract not received CB and RCF may fallaciously classify axonal GBS as acute demyelinating infl ammatory polyradiculoneu- ropathies. RCF has been recently demonstrated also THEMATIC WORKSHOP 3.4 in sensory fi bers and other clinical GBS variants and Theme: NEW APPROACHES OF IDENTIFICATION OF GENE: subtypes as pharingo-cervical brachial weakness, NEXT GENERATION SEQUENCING Fisher syndrome, and acute sensory ataxic neuropa- thy.These fi ndings suggests that GBS subtypes with antibodies anti-gangliosides belong to a continuous TW 3.4.1 New systems to interprete data clinical spectrum with a common pathophysiological from NGS analysis mechanism characterized by dysfunction/disruption Christophe BEROUD, MARSEILLE (France) of the node of Ranvier and could be more appropri- Aix Marseille Université, INSERM, GMGF UMR_S ately classifi ed as nodo-paranodopathies. 910, 13385, Marseille, France; Département de Génétique Médicale, Hôpital la Timone, Marseille, Selected references France Capasso M, Notturno F, Manzoli C, Yuki N, Uncini A. Reversible conduction failure in pharingeal-cervi- For many years, the sequencing of the human ge- cal-brachial variant of Guillain–Barré syndrome. nome was out of reach but recent advances in very Muscle Nerve 2010;42:608–6012. high throughput technologies have drastically reduced Capasso M, Notturno F, Manzoli C, Uncini A. In- costs leading to applications in clinical testing. Never- volvement of sensory fi bers in axonal subtypes of theless, if it is now technically possible to sequence Guillain–Barrè syndrome. J Neurol Neurosurg Psy- human genomes, we still have a long way to go to chiatry 2011;82:664–70. identify all genes responsible for human genetic dis- Uncini A, Kuwabara S. Electrodiagnostic criteria eases especially in the context of rare diseases. This for Guillain-Barrè syndrome: A critical revision and specifi c fi eld harbors today more than 8,000 diseases the need for an update. Clin Neurophysiol for which only 2,500 disease-causing genes have been 2012;123:1487–1495. identifi ed. To speed-up genes discoveries the Interna- Uncini A, Manzoli C, Notturno F, Capasso M. Pit- tional Rare Disease Research Consortium (IRDiRC) falls in electrodiagnosis of Guillain-Barrè syndrome (http://www.irdirc.org/) was created. Its goals are to subtypes. J Neurol Neurosurg Psychiatry facilitate the identifi cation of most genes involved in 2010;81:1157–1163. rare diseases as well as 200 new therapies by year Uncini A, Susuki K, Yuki N. Nodo-paranodopathy: 2020. To do so, many funders worldwide combine ef- beyond the demyelinating and axonal classifi cation in forts in a spirit of data sharing. Four projects have al- anti-ganglioside antibody mediated neuropathies. ready been funded at the European level including the Clin Neurophysiol 2013;124:1928–1934. RD-CONNECT project. It is a unique global infra- Uncini A, Notturno F, Capasso M. Natura non facit structure project that links up databases, registries, saltus in antiganglioside antibody-mediated neuropa- biobanks and clinical bioinformatics data used in rare thies. Muscle Nerve 2013;48:484–487. disease research into a central resource for researchers worldwide. One key element of this project is to pro- vide a central platform allowing data integration and analysis of various -omics data, fi rst and foremost ge- nomics. Abstracts S31

In this presentation, we will review various analysis engage a software developer at any stage. Our second pipelines, which are used to handle both Whole generation open source rare disease registry frame- Exome and Whole Genome Sequencing data as no work (RDRF) empowers registry curators to construct gold standard already exist. We will also describe the one or more patient registries without software devel- RD-CONNECT existing pipeline and tools as well as oper effort [1, 2, 3, 4]. New data elements for a di- future developments that will be progressively imple- verse range of phenotypic and genotypic features can mented in this global reference system that is tightly be defi ned at any time and can then be utilized and linked to the Neuromics EU-funded project. shared in any of the created registries. Fine grained, We will describe the three layers of data analysis: multi-level user and workgroup access can be applied raw data analysis leading to variant calling, data an- to each data element to ensure appropriate access and notation and variant prioritization in order to identify data privacy. Results from diverse bioinformatics bottlenecks and key systems that should be imple- analyses can then be incorporated into patient regis- mented in local pipelines to improve effi ciency, allow tries in a scalable fashion [5, 6]. data comparison and sharing. Finally, we will demonstrate how data sharing will References facilitate rare disease research through the example of [1] Bellgard, M., et al., A modular approach to dis- the Rare Disease Variant Database from the French ease registry design, Hum Mutat, 2012. 33(10): Rare Disease foundation. p. E2356–66. [2] Bellgard, M., et al., Dispelling myths about rare disease registry system development SCFBM THEMATIC WORKSHOP 3.4 2013. 8(1): p. 21. Theme: NEW APPROACHES OF IDENTIFICATION OF GENE: [3] Rodrigues M, Hammond-Tooke G, Kidd A, NEXT GENERATION SEQUENCING Love D, Patel R, Dawkins H, Bellgard M, Rox- burgh R. The New Zealand Neuromuscular Disease Registry. J Clin Neurosci. 2012 Sep TW 3.4.2 User-driven dynamic creation 18. of patient registries: a modular approach [4] Bellgard, M et al., Second Generation Registry to bioinformatics analysis for Framework, under revision, SCFBM, January neuromuscular diseases 2014. [5] Bellgard M, et al., Rare Disease Roadmap: Matthew BELLGARD, PERTH (Australie) Navigating the bioinformatics and translational Centre for Comparative Genomics - Murdoch challenges for improved patient health out- University, Murdoch 6150, Western Australia comes, submitted, HPT, Nov 2013. [6] Hunter, A., et al., Yabi: An online research en- Patient registries are essential, particularly for the vironment for Grid, High Performance, rare disease community. Their appropriate design, SCFBM, 2012, 7(1). implementation and deployment enables rapid deci- sion making and ongoing data mining, ultimately leading to improved patient diagnosis and treatment. Unfortunately, a major bottleneck encountered is the THEMATIC WORKSHOP 3.4 static nature of these implemented systems. Static in Theme: NEW APPROACHES OF IDENTIFICATION OF GENE: the way software developers work with stakeholders NEXT GENERATION SEQUENCING to determine requirements, design the system, imple- ment the required data fi elds and functionality for TW 3.4.3 FORGE Canada Consortium: each patient registry. Additionally, static in the way outcomes of a 2-year National Rare they are designed, making it diffi cult for disease reg- Disease Gene Discovey Project istries to effectively ‘communicate’ to share or inter- rogate patient phenotypic information. JACQUES MICHAUD, MONTREAL (Canada) Here we provide an overview of a registry frame- 1Centre Hospitalier Universitaire Sainte-Justine work that allows scientists and registry curators with 2Children’s Hospital of Eastern Ontario Research standard computing skills to dynamically construct a Institute complete patient registry from scratch, and customise 3McGill University it for their specifi c needs, with little or no need to 4Genome Quebec Innovation Centre S32 Abstracts

5Memorial University of Newfoundland Facioscapulohumeral dystrophy (FSHD) is associ- 6University of Calgary ated with a decondensation of the chromatin structure 7The Hospital for Sick Children of the D4Z4 repeat array on chromosome 4 and in- 8Centre of Genomics and Policy complete suppression of the D4Z4-encoded DUX4 9University of British Columbia retrogene in skeletal muscle. DUX4 is a germline 10British Columbia Cancer Agency transcription factor that is normally suppressed in so- 11Centre Hospitalier Universitaire de Québec matic tissue. Its expression in FSHD skeletal muscle activates germline and early developmental programs Inherited monogenic disease has an enormous im- as well as specifi c classes of retrotransposons. It also pact on the well-being of children and their families. suppresses the innate immune response to viral infec- Over half of the children living with one of these con- tion. ditions are without a molecular diagnosis due to the In the common form FSHD1, DUX4 expression in rarity of the disease, the marked clinical heterogeneity skeletal muscle is caused by contraction of the D4Z4 and the reality that there are thousands of rare disease array to a size of 1–10 units on chromosomes 4 that genes that are yet to be identifi ed. It is in this context contain a polymorphic DUX4 polyadenylation signal. that, in 2010, a Canadian consortium was formed to In the rare form FSHD2, D4Z4 chromatin deconden- rapidly identify a wide spectrum of pediatric-onset sation and DUX4 expression from chromosome 4 are rare disease genes using whole-exome sequencing. a consequence of mutations in the SMCHD1 gene on FORGE (Finding Of Rare disease GEnes in Canada) chromosome 18. SMCHD1 is a chromatin modifi er brought together clinicians and scientists from 21 Ge- that binds to the D4Z4 repeat to maintain a repressive netics Centres and three Science and Technology In- chromatin structure in somatic cells and in FSHD2 novation Centres from across Canada. From patients there is reduced SMCHD1 activity at D4Z4. nation-wide requests for proposals, 264 disorders FSHD2 is a heterogeneous condition since only 85% were selected for study from the 371 submitted; at of cases can be explained by SMCHD1 mutations. least one causative gene was identifi ed for 146 disor- SMCHD1 can also act as a modifi er of disease sever- ders (this includes 67 novel disease genes of which 41 ity in FSHD1 families as mutations in SMCHD1 ag- have been genetically or functionally validated and 26 gravate disease severity in FSHD1 families with are currently under study), yielding a success rate of borderline D4Z4 repeat sizes. 55% over a two-year period. Forty-fi ve of these disor- Collectively these fi ndings suggest specifi c mecha- ders had a neuromuscular component:14 had known nisms of FSHD pathology which will facilitate the genes identifi ed, 7 had potential novel genes, and 24 identifi cation of candidate biomarkers for disease di- remain unsolved. We will present our experience with agnosis and progression, as well as the development four strategies employed for gene discovery and dis- of therapeutic strategies for FSHD. cuss FORGE’s impact in a number of realms; from clinical diagnostics to the broadening of the pheno- typic spectrum of many diseases to the biological in- THEMATIC WORKSHOP 3.5 sight gained into both disease states and normal Theme: THERAPEUTIC APPROACHES IN FACIOSCAPULO- human development. Lastly, based on this experience, HUMERAL DYSTROPHY we will discuss the way forward for rare disease gene discovery both in Canada and internationally. TW 3.5.2 Aerobic exercise and cognitive behavior therapy reduce fatigue and slow THEMATIC WORKSHOP 3.5 progression of muscle MR fatty Theme: THERAPEUTIC APPROACHES IN FACIOSCAPULO- infi ltration in FSHD HUMERAL DYSTROPHY Baziel VAN ENGELEN, Nijmegen (Pays-Bas)

TW 3.5.1 Molecular mechanisms in Objective: The aim of this study was to investigate FSHD: identifi cation of druggable targets the effect of aerobic exercise training (AET) and cog- nitive behavior therapy (CBT) on chronic fatigue in SYLVERE VAN DER MAAREL, Leiden (Pays-Bas) patients with facioscapulohumeral muscular dystro- Leiden University Medical Center, Department of phy (FSHD). Human Genetics, The Netherlands Abstracts S33

Methods: We performed a multi-center, assessor- Under the genetic and epigenetic point of view, we blinded, randomized clinical trial (RCT). Fifty-seven can distinguish two forms of FSHD, identical in term patients with FSHD type 1 with severe chronic fatigue of clinical phenotype. Autosomal dominant FSHD1 is were randomly allocated to AET, CBT, or usual care associated to a pathogenic contraction of D4Z4 4Q (UC). Outcomes (including a 3 Tesla quantitative MR permissive allele, while FSHD2 is due to a mutation imaging of the upper leg muscles) were assessed at in SMCHD1 gene in presence of 4QA non-contracted baseline, immediately following 16 weeks of inter- permissive allele. vention, and after a 12-week follow-up. A linear The recent identifi cation of additive effect of mixed model for repeated measurements was used to FSHD1 and FSHD2 genetic abnormalities in some study the estimated group differences. families confi rmed the central role of toxic retrogene Results: Following treatment, both the AET and DUX4 in both these diseases. Hence, several thera- CBT intervention groups had signifi cantly less fatigue peutic strategies are being developed, aiming to avoid relative to the UC group, with a difference of -9.1 for DUX4 expression or its pathological consequences. AET (95%CI: -12.4 to -5.8) and -13.3 for CBT More recently, neuroimaging studies bring back the (95%CI: -16.5 to -10.2). These benefi cial effects last- attention to the role of infl ammation in FSHD clinical ed through the follow-up period, with a difference of progression and open the way for new therapeutic -8.2 for AET (95%CI: -12.4 to -5.8) and -10.2 for strategies development. CBT (95%CI -16.5 to -10.1). The patients who re- The future is full of promises for FSHD patients ceived CBT had an increase in registered and experi- and we need, more than ever, sensitive and specifi c enced physical activity, sleep quality, and social biomarker(s) able to characterize patients, quantify participation. The patients who received AET had an clinical progression and response to treatment. Some increase in registered physical activity only. The in- biomarkers are available and some are being devel- crease in registered physical activity in both groups oped. and the improvement in social participation following Biomarkers of interest in FSHD can be classifi ed as CBT were still present at follow-up. MRI determined clinical, epigenetic, genetic, immune and neuroimag- fat fractions showed decelerated progression of fatty ing biomarkers. Clinical biomarkers include manual infi ltration, most prominently in the adductor magnus muscular testing, quantifi ed muscular testing, age muscle. In this muscle fat percentage increased with corrected-Clinical Severity Score, Fatigue Severity 1.56% monthly (UC) with a difference of -1.21 for Score, pain assessment. Level of hypomethylation AET (95%CI: -1.99 to -0.34) and -1.73 for CBT correlates with the severity of clinical expression in (95%CI: - 2.56 to -0.87). FSHD2 patients and can be eventually used as a bio- Interpretation: This is the fi rst RCT showing that markers for therapy targeting SMCHD1 gene. If chronic fatigue can be ameliorated in patients with a DUX4 itself can hardly be used as genetic biomarker muscular dystrophy. AET and/or CBT reduce fatigue because of its limited expression, several genes asso- and slow progression of fatty infi ltration in patients ciated with germline and early stem cell development with FSHD. have been recently identifi ed as targets of the DUX4 transcription factor and are being tested as biomarkers for disease diagnosis and progression. Promising neu- THEMATIC WORKSHOP 3.5 roimaging biomarkers (SPECT, MRI), as well as im- Theme: THERAPEUTIC APPROACHES IN FACIOSCAPULO- mune biomarkers are also being tested. HUMERAL DYSTROPHY In conclusion, this presentation will describe the merits and limitations of known and recently discov- ered biomarkers for FSHD and the efforts that need to TW 3.5.3 Biomarkers and trial readiness be done by scientifi c community to be ready in view in FSHD1 and FSHD2; are we ready for of future clinical trials. the future? Sabrina SACCONI, NICE (France) 1Nice University Hospital, Neuromuscular Disease Specialized Center, Archet Hospital, Nice, France S34 Abstracts

THEMATIC WORKSHOP 4.1 transcription factor NFκB and its activation by acety- Theme: NEW INSIGHT INTO THE MECHANISM OF MUSCLE lation of p65 subunits. Overexpression of the SIRT1 ATROPHY deacetylase prevents this activation and activation of FoxO and blocks atrophy upon fasting or denervation. TW 4.1.1 New insight into the molecular Control of protein acetylation and degradation thus are attractive new drug targets. mechanism of muscle atrophy Alfred GOLDBERG, Boston (Etats-Unis) Dept Cell Biology, Harvard Medical School, Boston, THEMATIC WORKSHOP 4.1 MA 02115, USA Theme: NEW INSIGHT INTO THE MECHANISM OF MUSCLE ATROPHY Dramatic progress has been made recently in un- derstanding the cellular mechanisms for the atrophy of skeletal muscle that occurs with disuse, nerve in- TW 4.1.2 FoxO, autophagia and jury, fasting, glucocorticoid treatment, and many sys- mitochondria: the crossroad for the temic diseases, especially cancer. In these rapidly control of muscle atrophying muscles, there is a common program of Marco SANDRI, Padua (Italie) transcriptional changes, in which a set of atrophy-re- Department of Internal Medicine and Clinical lated genes (“atrogenes”) are induced or repressed Immunology coordinately. Among the most induced proteins are Reference Center for Neuromuscular Disorders components of the ubiquitin-proteasome pathway (UPS), especially the muscle-specifi c ubiquitin ligas- Idiopathic infl ammatory myopathies include der- es, atrogin-1 and MuRF1, which are induced by the matomyositis, polymyositis, immune-mediated nec- FoxO family of transcription factors. Overproduction rotizing myopathy and inclusion body myositis. of FoxO3 causes dramatic atrophy. MuRF1 is critical Clinical phenotype and histopathology examination in the ordered disassembly and degradation of the of muscle biopsies are determinant to distinguish myofi brillar apparatus, specifi cally components of the these myopathies from the others but detection of au- thick fi laments. However, a distinct E3, Trim32, cata- toantibody is now also crucial. The myositis-specifi c lyzes loss of thin () fi laments and the associated antibodies and myositis-associated antibodies lead to Z-band proteins and the cytoskeletal component, des- a serologic approach complementary to the clinico- min. Desmin is phosphorylated in atrophy and de- pathological classifi cation, because strong associa- graded in a Trim32-dependent manner. Its loss is tions of myositis-specifi c antibodies with clinical critical for the accelerated degradation of thin fi la- features and survival have been documented, deter- ments. The p97/VCP ATPase appears essential for this mining now myositis subclassifi cations. The presence process, and during atrophy, p97 appears to extract of anti-synthetase antibodies is associated with an ubiquitinated components from the myofi bril, since original histopathologic pattern between polymyositis p97DN mutants also block atrophy, and become tight- and dermatomyositis, and defi nes a syndrome where ly associated with myofi brils. In addition, FoxO3 interstitial lung disease drives the prognosis. Among stimulates the expression of genes for the autophagic/ the antisynthetase antibodies, some of them (anti-PL7 lysosomal pathway. Thus, during atrophy, the cell’s and PL12) have a worst prognosis than others (e.g. two main proteolytic systems are activated coordi- anti-Jo1) because of the severity of the interstitial nately to cause breakdown of different cellular com- lung disease. On the other hand, myositis may be ponents; the loss of contractile proteins via the UPS more common, more severe, and more resistant to and mitochondria via autophagy. This program for treatment for patients with anti-Jo1 than for those muscle wasting in disease states is also activated by with anti-PL12 and anti-PL7. Anti-MDA-5 antibody myostatin and its homolog, Activin A. Antagonists of is specifi cally associated with frequently amyopathic the myostatin/activin pathway inhibit FoxO-induced dermatomyositis, and defi nes a skin-lung syndrome proteolysis and are an attractive therapeutic approach. with sometimes a catastrophic disease course. Anti- These agents not only dramatically block muscle TIF1-γ is also associated with dermatomyositis but its wasting and cachexia, but also can prolong the viabil- presence in adults is highly predictive of cancer asso- ity of tumor-bearing animals. The induction of atro- ciation, which then drives the prognosis. On the other phy upon denervation of even fasting also requires the hand, anti-Mi2 antibody is associated with a classical Abstracts S35 form of dermatomyositis, with a better prognosis tions of myositis-specifi c antibodies with clinical since not particularly associated with interstitial lung features and survival have been documented, deter- disease, cancer, nor corticoresistance. Two other spe- mining now myositis subclassifi cations. The presence cifi c antibodies, anti-SRP and anti-HMGCR, are ob- of anti-synthetase antibodies is associated with an served in patients with immune-mediated necrotizing original histopathologic pattern between polymyositis myopathies and may be very useful to make the dif- and dermatomyositis, and defi nes a syndrome where ference with muscle dystrophies in case of slowly interstitial lung disease drives the prognosis. Among progressive onset and to allow the initiation of effec- the antisynthetase antibodies, some of them (anti-PL7 tive therapy (i.e. immunosuppressants). Interestingly, and PL12) have a worst prognosis than others (e.g. 40 to 60% of the patients presenting an immune-me- anti-Jo1) because of the severity of the interstitial diated necrotizing myopathies with anti-HMGCR re- lung disease. On the other hand, myositis may be ceived statins, outlining one of the roles of these drugs more common, more severe, and more resistant to in their muscle side effects. treatment for patients with anti-Jo1 than for those with anti-PL12 and anti-PL7. Anti-MDA-5 antibody is specifi cally associated with frequently amyopathic THEMATIC WORKSHOP 4.1 dermatomyositis, and defi nes a skin-lung syndrome Theme: NEW INSIGHT INTO THE MECHANISM OF MUSCLE with sometimes a catastrophic disease course. Anti- ATROPHY TIF1-γ is also associated with dermatomyositis but its presence in adults is highly predictive of cancer asso- ciation, which then drives the prognosis. On the other TW 4.1.3 Signalling mechanisms hand, anti-Mi2 antibody is associated with a classical mediating muscle homeostasis form of dermatomyositis, with a better prognosis David GLASS, Boston (Etats-Unis) since not particularly associated with interstitial lung disease, cancer, nor corticoresistance. Two other spe- Abstract not received cifi c antibodies, anti-SRP and anti-HMGCR, are ob- served in patients with immune-mediated necrotizing myopathies and may be very useful to make the dif- THEMATIC WORKSHOP 4.2 ference with muscle dystrophies in case of slowly progressive onset and to allow the initiation of effec- Theme: INFLAMMATORY MYOPATHIES tive therapy (i.e. immunosuppressants). Interestingly, 40 to 60% of the patients presenting an immune-me- TW 4.2.1 Specifi c auto-antibodies in diated necrotizing myopathies with anti-HMGCR re- infl ammatory myopathies: identifi cation ceived statins, outlining one of the roles of these drugs of new clinico pathological syndromes? in their muscle side effects. Olivier BENVENISTE, Paris (France) Department of Internal Medicine and Clinical THEMATIC WORKSHOP 4.2 Immunology Theme: INFLAMMATORY MYOPATHIES Reference Center for Neuromuscular Disorders , AP-HP, UMRS 974, UPMC - INSERM Pitié-Salpêtrière Hospital, Paris,france TW 4.2.2 Role of innate immunity in dermatomyositis Idiopathic infl ammatory myopathies include der- matomyositis, polymyositis, immune-mediated nec- Isabel ILLA, Barcelona (Espagne) rotizing myopathy and inclusion body myositis. Neuromuscular Diseases Unit, Neurology Clinical phenotype and histopathology examination Department, Hospital de la Santa of muscle biopsies are determinant to distinguish Creu i Sant Pau, Universitat Autònoma de Barcelona these myopathies from the others but detection of au- and Institut de Recerca Sant Pau, Barcelona. toantibody is now also crucial. The myositis-specifi c Centro Investigación Biomédica en Red de antibodies and myositis-associated antibodies lead to Enfermedades Neurodegenerativas a serologic approach complementary to the clinico- (CIBERNED), Madrid, Spain. pathological classifi cation, because strong associa- S36 Abstracts

This work has been supported by grant FIS 09/1964 According to these results, modulation of innate im- and FIS13/937 (ISCIII, Ministry of Health, Spain) mune mechanisms with specifi c drugs would be a and a research grant (XS-C) by The Myositis Associa- promising approach for the treatment of these diseases. tion. Infl ammatory myopathies (IM) are a heterogeneous The authors have no disclosures. group of myopathies that includes dermatomyositis [1] Engel AG F-AC. Myology: basic and clinical (DM), polymyositis (PM) and inclusion body myosi- 2004: 1321–1486. tis (IBM) among others. Although IM share the pres- [2] Rayavarapu S et al. Skelet Muscle 2013; ence of infl ammation, the clinical features and the 3(1):13. molecular mechanisms resulting in muscle injury [3] Greenberg SA et al. Ann Neurol 2005; 57: 664– probably differ in each of them [1]. MHC-I overex- 678. pression in muscle fi bers is a hallmark of IM. Previ- [4] Cappelletti C et al. Neurology 2011; 76: 2079– ous studies suggest that both immune and non-immune 2088. mechanisms play a role in muscle injury although the [5] Suárez-Calvet X et al. J Pathol 2014 Mar 6. In relative contributions of each one in each entity are press. not fully understood [2]. Type I interferons (IFN-I) play a central role in an- tiviral responses and IFN-I-inducible genes have been THEMATIC WORKSHOP 4.2 largely found in microarray studies in IM [3]. The IFN Theme: INFLAMMATORY MYOPATHIES induction is mainly mediated by the activation of in- nate immune receptors that recognize pathogens and endogenous danger signals. Several families of innate TW 4.2.3 Treatment of infl ammatory immune receptors are known, among them toll-like myopathies receptors (TLR) and Rig-like receptors (RLR). In PM Marinos DALAKAS, Athens / Philadephia (Grèce) and DM muscle biopsies, several TLR were found to University of Athens Medical School, Athens, Greece be increased [4]. Although its presence could be sec- and Thomas Jefferson University, Philadelphia, USA ondary to tissue damage, their activation by endoge- nous damage signals could have an important role in The infl ammatory myopathies include four distinct the perpetuation of the immune response within the entities: Polymyositis (PM), Dermatomyositis (DM), muscle. Inclusion Body Myositis (IBM), and Necrotizing Au- Using microarray technology from laser microdis- toimmune Myositis (NAM). A T-cell-mediated cyto- sected MHC-I positive pathological muscle fi bers we toxic process in PM and IBM, a complement-mediated have recently found a more prominent role of innate microangiopathy in DM, and a macrophage-mediated immunity in DM compared to PM and IBM. In par- antibody-fi xing process in NAM, are the hallmarks of ticular, RIG-I, a member of the RLR innate immune the underlying autoimmune processes. A common receptors, was overexpressed in the muscle fi bers of critical factor that affects therapeutic outcome is the DM but not in the others. In vitro studies have demon- distinction of PM from the diffi cult-to-treat mimics strated that RIG-I-activated myotubes induce MHC-I such as s-IBM and inlammatory dystrophies which overexpression, secretion of IFNβ and a positive feed- may have prominent secondary endomysial infl am- back of RIG-I. These results indicate that RIG-I in- mation. nate immunity contributes to initiating or perpetuating In uncontrolled studies, PM and DM respond to the immune response specifi cally in DM [5]. The per- prednisone, either alone or in combination with one petuation of the immune response mediated by RIG-I immunosuppressive drug (Azathioprine, Cyclospo- may occur through the recognition of viruses or dam- rine, Mycophenolate, Methotrexate), to some degree aged-cells derived particles containing, among other and for some period of time; in contrast, IBM is resis- molecules, RNAs. Similar innate immunity mecha- tant to most of these therapies, most times. Controlled nisms have been proposed in a dystrophic mouse studies have shown that IVIg is effective and safe for model. the treatment of DM. The clinical benefi t, which can be impressive in patients with early disease, is associ- ated with improvement in the muscle cytoarchitecture and resolution of the aberrant immunopathological Abstracts S37 parameters, including interception of complement ac- THEMATIC WORKSHOP 4.3 tivation and downregulation of ICAM-I, VCAM, Theme: THERAPEUTIC APPROACHES TO HEREDITARY MY- TGF-β, MHC-I and various immunoregulatory and OPATHIES structural genes. IVIg seems to be also effective in patients with PM but offers only minimal and tran- TW 4.3.2 Autologous myoblast cell sient help to a small number of patients with IBM. Rituximab seems helpful in some patients with PM, therapy for oculopharyngeal muscular DM or NAM, but a poorly designed controlled study dystrophy (OPMD): a phase I/IIa clinical did not show a difference in the outcome measures study between Rituximab and placebo. New biological Capucine TROLLET, Paris (France) agents currently on the market will be discussed as 1Sorbonne Universités, UPMC Univ Paris 06, promising new therapeutic options for the treatment Myology Research Center UM76, F-75013, Paris, of infl ammatory myopathies. They include various France monoclonal antibodies or fusion proteins against: a) 2INSERM UMRS974, F-75013, Paris, France molecules associated with T-cell-signaling pathways; 3CNRS FRE 3617, F-75013, Paris, France b) B cells antigens; c) Complement C5; and d) adhe- 4Institut de Myologie, F-75013, Paris, France sion or transmigration molecules. 5Service d’Oto-Rhino-Laryngologie et de Chirurgie Cervico-Faciale, Assistance publique-Hôpitaux de Paris (APHP), Hôpital Tenon, Paris, France THEMATIC WORKSHOP 4.3 6Unité de Thérapie Cellulaire et CIC-BT501, Hôpital Theme: THERAPEUTIC APPROACHES TO HEREDITARY Saint Louis, Assistance publique-Hôpitaux de Paris MYOPATHIES (APHP), Université Paris Diderot, Sorbonne Paris Cité, Paris, France TW 4.3.1 Advanced exon skipping 7Amiens Hospital, Hematology Department, Avenue antisense strategies for Duchenne René Laennec, 80054 Amiens Cedex 1 8 Muscular Dystrophy Service de Neurologie, Consultation de Pathologies Neuromusculaires, Institut de myologie, Hôpital Matthew WOOD, Oxford (Royaume Uni) Pitié Salpêtrière, Faculté de Médecine & Université Department of Physiology, Anatomy and Genetics, Paris VI Pierre et Marie Curie, Assistance publique- University of Oxford, OX1 3QX, United Kingdom Hôpitaux de Paris (APHP), Paris, France, 9CHU de Caen, Centre de Compétences des Oligonucleotides to modulate pre-mRNA splicing Pathologies Neuro-musculaires, Service de have therapeutic potential for a range of inherited Médecine Physique et Réadaptation, Avenue de la neuromuscular disorders. The classical example is Côte de Nacre, CS 30001, 14033 Caen Cedex 9, Duchenne muscular dystrophy (DMD), where modu- France et INSERM, U 1075 COMETE, France lation of pre-mRNA splicing of the DMD gene can restore a viable reading frame and the expression of Oculopharyngeal muscular dystrophy (OPMD) is an functional protein. This approach is currently being autosomal dominant inherited, slow progressing, late evaluated in clinical trials. A limitation of such meth- onset degenerative muscular disorder where a small ods is the poor ability to deliver oligonucleotides ef- group of specifi c muscles (pharyngeal and eyelid) are fectively to affected tissues including skeletal muscle, primarily affected, leading to dysphagia and ptosis. Its heart and brain. A possible solution to this problem is genetic basis is a trinucleotide repeat expansion rang- offered by the development of advanced oligonucle- ing from (GCG)8 to (GCG)13 in the N-terminus poly- otides utilising cell-targeting and cell-penetrating alanine domain of the the poly(A) binding protein peptides conjugated to or complexed with the oligo- nuclear 1 (PABPN1) gene. Mutated expanded PAB- nucleotide of choice. Such compounds provide great- PN1 protein accumulates as insoluble nuclear inclu- ly improved delivery and enhanced potency and are sions in muscles of OPMD patients. While the roles of being developed for future clinical studies in both PABPN1 in nuclear polyadenylation and in the regula- DMD and for other neuromuscular disorders, such as tion of alternative poly(A) site choice are established, spinal muscular atrophy. the molecular mechanisms behind OPMD including S38 Abstracts the specifi city of affected muscles and the role of these strongly effective treatments but new gene-based ther- inclusions remain undetermined. So far there is no apies are currently being developed. In the case of cure for OPMD patients. The dysphagia compromises DMD, a number of groups are testing gene therapy their life expectancy, and the classical myotomy is of- with adeno-associated virus vectors expressing engi- ten of transient benefi t. Since in a preclinical study, we neered micro- (AAV-MDs). In our hands, demonstrated good myogenic capacities of myoblasts highly sequence optimised AAV-MDs are available for expanded from clinically spared muscles, we launched use in mouse, dog and ultimately humans, expressed a phase I/IIa clinical study (ClinicalTrials.gov using a strong synthetic promoter specifi c for skeletal NCT00773227) using autologous myoblast transplan- and cardiac muscle cells have been tested in detail in tation following myotomy in adult OPMD patients. 12 mdx mice, and in the GRMD dog. Here we report the OPMD patients were included in the study, and were outcome of a detail study of AAV-canine MD delivery injected with a median of 178 million myoblasts fol- by isolated limb perfusion. No immunosuppression lowing myotomy. Feasibility, safety end points and was applied. In skeletal muscles of the treated limb we potential therapeutic benefi t of both autologous myo- report widespread vector biodistribution, and sustained blast transplantation as well as the surgical procedure high level MD expression. In addition the treated limb were assessed by radioimaging, quality of life ques- exhibited very signifi cantly improved parameters of tionnaire and drink test. Short and long-term (2 years) muscle turnover (regenerative fi bres), fi brosis (Colla- safety and tolerability were observed in all patients, gen type I), 1H-MRI and 31P-NMR spectroscopy, and with no adverse effects. There was an improvement in muscle strength. In the context of immune parameters, the quality of life score for all 12 patients, and no func- AAV administration elicited anti-AAV2/8 antibodies tional degradation in swallowing was observed for the and a transient elevation of serum cytokines, but no majority of patients. A cell dose dependant improve- evidence of cellular immunity to microdystrophin or to ment in swallowing was even observed in this safety the AAV vector was observed. The current optimised study. This trial supports the hypothesis that a local microdystrophin is thus highly functional, not only in injection of autologous myoblasts in the pharyngeal mice, but also in a large animal model, and that AAV2/8 muscles is a safe and effi cient procedure for OPMD vector system yields sustained microdystrophin ex- patients. Since the current protocol involves the autol- pression without adverse immune responses. Current ogous transplantation of unmodifi ed cells from spared studies are focussed on systemic delivery of AAV-ca- muscles - which still carry the genetic mutation - we nine MD vectors body-wide in the GRMD model. are now developing a new gene therapy strategy to ge- netically engineer patients cells prior to transplanta- tion in order to restore a normal PABPN1 allele. THEMATIC WORKSHOP 4.4 Theme: CONGENITAL MYOPATHIES

THEMATIC WORKSHOP 4.3 TW 4.4.1 and Theme: THERAPEUTIC APPROACHES TO HEREDITARY MY- OPATHIES related diseases: genetics, diagnosis and therapy TW 4.3.3 AAV microdystrophin gene Nigel G LAING, Perth (Australie) therapy for Duchenne Muscular Centre for Medical Research, University of Western Dystrophy Australia M519, Harry Perkins Institute of Medical Research, QQ Block, QEII Medical Centre, 6 Verdun George DICKSON, London (Royaume Uni) Street, Nedlands, Western Australia 6009, 1Royal Holloway - University of London, London, AUSTRALIA United Kingdom 2Atlantic Gene Therapy Institute, Nantes, France It is generally accepted that nemaline myopathy was 3Genethon Laboratories, Evry, France fi rst described in 1963 after electron microscopy be- 4AFM Institut de Myologie, Paris, France came available and the nemaline bodies, pathogno- monic for nemaline myopathy, could be clearly Muscular dystrophies refer to a group of inherited visualized. It then took 32 years, before slow alpha- disorders characterized by progressive muscle weak- (TPM3) was the fi rst gene found to be ness, wasting and degeneration. So far, there are no mutated in nemaline myopathy (1995). This fi nding Abstracts S39 refocused nemaline myopathy thinking from the Z- 2Institute for Stem Cell and Regenerative Medicine, disc, whose components most obviously accumulate in University of Washington, Campus Box 358056, nemaline bodies, to the thin fi lament and led relatively Seattle, WA 98109, USA. rapidly to mutations causing nemaline myopathy being 3Généthon, 1 bis rue de l’Internationale, 91002 Evry, identifi ed in genes coding for other thin fi lament pro- France. teins: (NEB)(1999), skeletal muscle alpha ac- 4Department of Human Nutrition, Foods and tin (ACTA1)(1999), slow T (TNNT1)(2000), Exercise, Virginia Polytechnic Institute and State beta-tropomyosin (TPM2)(2002), and muscle-specifi c University, Blacksburg, VA 24061, USA. cofi lin (CFL2)(2007). More recently, several BTB 5The Manton Center for Orphan Disease Research, Kelch proteins have been associated with nemaline Children’s Hospital Boston, Harvard Medical myopathies: (KBTBD13)(2010) with rod-core disease, School, 300 Longwood Ave, Boston, MA 02115, then, with the advent of next generation sequencing USA. (NGS) gene discovery, KLHL40 and KLHL41 (2013) 6Department of Pathology and Laboratory Medicine, with severe early, including foetal, onset nemaline my- Children’s Hospital and Medical College of opathy. More genes remain to be found. Whether the Wisconsin, Milwaukee, WI, USA Kelch proteins associated with nemaline myopathy are 7Wake Forest Institute for Regenerative Medicine, also related to the thin fi lament remains to be deter- 391 Technology Way, Winston-Salem, NC 27101, mined. Diseases related to nemaline myopathy include USA. actin aggregate myopathy, cap disease and zebra-body 8Comprehensive Cancer Center, Wake Forest myopathy and these have been associated with muta- University Health Sciences, School of Medicine, tions in some of the genes associated with nemaline Medical Center Boulevard, Winston-Salem, NC myopathy (ACTA1, CFL2, TPM2 and TPM3). Mo- 27157, USA. lecular diagnosis of nemaline myopathy is best per- 9INSERM U649, Atlantic Gene Therapies, CHU formed by NGS, because of the high levels of genetic Hôtel Dieu, 44300 Nantes, France. heterogeneity and the major role of the giant protein 10Molecular Genetics and Microbiology Department, nebulin. The identifi cation of the proteins affected in University of Florida, Gainesville, FL 32611, USA. nemaline myopathy has allowed investigation of the pathobiology of the disease. This has identifi ed defects Loss-of-function mutations in the myotubularin in excitation contraction coupling and thin fi lament gene (MTM1) cause X-linked myotubular myopathy length in the pathobiology of nemaline myopathy. This (XLMTM), a fatal pediatric disease of skeletal muscle included the identifi cation of increased thin fi lament characterized by small centrally nucleated myofi bers sensitivity to calcium associated with ACTA1 muta- containing abnormal mitochondrial accumulations. tion and a clinical phenotype of episodic hypercontrac- By previous local studies in Mtm1-mutant mice we tion and hypertonicity rather than the universally demonstrated potential effi cacy of gene therapy to expected and weakness of nemaline myopa- treat the disease. We will present results showing that thy. This hypercontraction has altered the clinical de- systemic administration of a single dose of a recombi- scription of nemaline myopathy to muscle dysfunction nant serotype-8 adeno-associated virus (AAV8) vec- rather than muscle weakness. The different pathobiolo- tor expressing the murine myotubularin to gies indicate that different routes to therapy will be Mtm1-defi cient knockout mice at the onset or at late needed for mutations associated with nemaline myopa- stages of the disease led to robust improvement in thy, even different mutations within one protein. contractile force, corrected the muscle pathology and prolonged survival throughout a 6-month study. Simi- larly, single-dose intravascular delivery of a canine THEMATIC WORKSHOP 4.4 AAV8-MTM1 vector in XLMTM dogs rescued al- most completely motor and respiratory functions, and Theme: CONGENITAL MYOPATHIES prolonged lifespan in the absence of toxicity and im- mune response. These results demonstrate the thera- TW 4.4.2 Gene replacement therapy for peutic effi cacy of AAV-mediated gene therapy for myotubular myopathy myotubular myopathy in small and large animal mod- els, and provide proof of concept for future clinical Anna BUJ-BELLO, Evry (France) trials in XLMTM patients. 1Department of Rehabilitation Medicine, School of Medicine, University of Washington; S40 Abstracts

THEMATIC WORKSHOP 4.4 we demonstrate molecular and physiological amelio- Theme: CONGENITAL MYOPATHIES ration of the toxic effects of mutant RNA in the HSA(LR) mouse model of DM1 by systemic admin- istration of peptide-linked morpholino (PPMO) anti- TW 4.4.3 Understanding - and improving sense oligonucleotides bearing a CAG repeat - sarcomeric function in Nemaline sequence. Intravenous administration of PPMO con- Myopathy jugates to HSA(LR) mice led to redistribution of Mbnl1 protein in myonuclei and corrections in abnor- Coen OTTENHEIJM, Amsterdam (Pays-Bas) mal RNA splicing. Additionally, was com- Dept. of Physiology, VU University Medical Center, pletely eliminated in PPMO-treated HSA(LR) mice. Amsterdam, The Netherlands These studies provide proof of concept that neutral- Nemaline myopathy - the most common non-dys- ization of RNA toxicity by systemic delivery of anti- trophic - is caused by mutations sense oligonucleotides that target the CUG repeat is in genes encoding proteins of the thin fi lament, a ma- an effective therapeutic approach for treating the skel- jor constituent of the muscle’s sarcomere. A hallmark etal muscle aspects of DM1 pathology. feature of nemaline myopathy is muscle weakness, Disclosures: A.J.L., L.M.M., P.A.P., S.H.C., and the cause of which remains only partly understood. B.M.W own stock in Sanofi . This presentations aims to provide an overview of the current knowledge regarding the pathophysiology of muscle weakness in nemaline myopathy and to high- THEMATIC WORKSHOP 4.5 light recent developments in targeting thin fi lament Theme: THERAPEUTIC APPROACH IN MYOTONIC DYSTRO- function using calcium sensitizers. PHY

TW 4.5.2 Systemic delivery of an THEMATIC WORKSHOP 4.5 antisense oligonucleotide (ASO) targeting Theme: THERAPEUTIC APPROACH IN MYOTONIC DYSTRO- DMPK RNA improves the phenotype of PHY DMSXL mice TW 4.5.1 Systemic delivery of a peptide- Jack PUYMIRAT, Quebec (Canada) 1 PMO of CAG sequence neutralizes Unit of Human Genetics, CHUQ/CHUL, 2705 Bd Laurier, Qué bec, G1V4G2, Canada. mutant RNA toxicity in a mouse model of 2Isis Pharmaceuticals Inc, 2855 Gazelle Court, DM1 Carlsbad, CA 92010, USA. 3 Andrew LEGER, Boston (Etats-Unis) Neuromuscular Reference Center, Henri Mondor 1Genzyme, a Sanofi Company, Cambridge, MA Hospital, INSERM U955, Cré teil, France. 4 02142, USA INSERM U781, Institut Imagine, 75015 Paris, France. Expansions of CUG trinucleotide sequences in 5Massachusetts General Hospital, 55 Fruit St, RNA transcripts provide the basis for toxic RNA gain- Boston MA 02114, USA of-function that leads to detrimental changes in RNA 6University of Rochester Medical Center, 601 metabolism. A CTG repeat element normally resides Elmwood Ave, Rochester, NY 14642, USA in the 3’ untranslated region of the dystrophia myo- tonica-protein kinase (DMPK) gene, but when ex- Antisense oligonucleotides (ASO) provide thera- panded it is the genetic lesion of myotonic dystrophy peutic opportunities for a wide variety of human dis- type 1 (DM1), a hereditary neuromuscular disease. eases. Improvements in ASO chemistry and design The pathogenic DMPK transcript containing the CUG have advanced the technology such that systemic ad- expansion is retained in ribonuclear foci as part of a ministration of therapeutic ASOs has produced ro- complex with RNA-binding proteins such as muscle- bust, antisense-mediated target inhibition and blind-like 1 (MBNL1), resulting in aberrant splicing therapeutic benefi t in multiple species, including in of numerous RNA transcripts and consequent physi- humans. ASOs are clearly an effective therapeutic ological abnormalities including myotonia. Herein, modality for liver-expressed genes, as exemplifi ed by Abstracts S41 the recent FDA approval of the ASO drug, Kynamaro. THEMATIC WORKSHOP 4.5 Extending the power of ASO drugs to extra-heptatic Theme: THERAPEUTIC APPROACH IN MYOTONIC DYSTRO- tissues represents one of the next key steps for the PHY technology. Here we describe the identifi cation and characterization of ASOs containing both 2’-me- TW 4.5.3 Targeting nuclear expanded thoxyethyl (MOE) and the next generation con- strained ethyl (cEt) chemistry, that were designed to repeats to correct RNA gain-of-function target the expression of the DMPK gene in skeletal effects in DM1 muscle. This target was selected because in myotonic Denis FURLING, Paris (France) dystrophy type I (DM1), a CUG expansion within the 1Center of Research in Myology UMRS974, UPMC/ DMPK gene results in RNA transcripts that are aber- Inserm/CNRS, Institut de Myologie, F-75013, Paris, rantly retained in the nucleus producing a toxic gain- France of-function effect in skeletal muscle and other tissues. 2Inserm UMR837–1 and Univ. Lille Nord de France, . Previous study demonstrated that ASOs targeting Jean-Pierre Aubert Research Center, Alzheimer & transcripts with expanded CUG repeats reverse mo- , F-59045 Lille, France lecular changes induced by the transgene (Wheeler et al., Nature 488:111–115). Myotonic Dystrophy type 1 (DM1) is one of the A screen of >3,000 candidate ASOs targeting dif- most common form of inherited neuromuscular disor- ferent regions of the human DMPK gene produced ders in adults characterized by myotonia, progressive numerous potent DMPK lead ASOs. Two of these muscle weakness and wasting, cardiac conduction ab- leads, one with MOE chemistry that was selective for normalities, cognitive as well as other multisystemic human DMPK and one with cEt modifi cations which defects. This autosomal dominant disease is caused was active against both human and mouse DMPK. by an expanded trinucleotide (CTG) n > 50 repeats These ASOs completely abolished nuclear foci, pref- located in the 3’ non-coding region of the DMPK erentially decreased the levels of mutant RNAs gene. The size of the expansion is generally correlated (mtRNAs) up to 88%, induced MBNL1 redistribution with the clinical severity and the age of onset of the and corrected the splicing of a subset of mis- spliced disease. Expression of pathogenic DMPK transcripts pre-RNAs, in DM1 cells in vitro. In DMSXL mice, containing expanded CUG repeats (CUGexp-RNAs) subcutaneous injection of ASOs reduced the levels of results in a toxic RNA gain-of-function mechanism. mtRNAs between 30–40% (MOE) and 67–75% (cET) The CUGexp-RNAs are retained into the nuclei as ri- in 6 skeletal muscles and 30% in the heart (cEt-ASO). boprotein aggregates or foci that sequester MBNL The cET-lead decreased the number of foci up to 70% RNA splicing factors leading to functional loss of in the quadriceps. DMSXL mice treated with cEt- MBNL and alternative splicing misregulations of a ASO also showed a signifi cant increase in body subset of pre-mRNAs. Thus, mis-splicing of CLCN1 weight from 17,9 ± 1,9 g to 18,7 ± 1,7 g (P = 0,016), and BIN1 pre-mRNAs contributes respectively to whereas no signifi cant effect was observed in mice myotonia and muscle weakness in DM1. Several treated with the MOE-ASO. cEt-ASO also increased strategies are currently under development to reverse forelimb force determined by the grip test (+3,4 ±7 g, toxic CUGexp-RNAs dominant effects. P = 0.008), motor performances determined by the In this study, we propose to restore MBNL function coat hanger test and, induced a complete (cEt) or par- and correct alternative splicing misregulations in tial (MOE) disappearance of immature muscle fi bers DM1 cells by interfering with abnormal CUGexp- at the histological levels. Both ASOs had no toxic ef- RNA:MBNL sequestration in order to release endog- fects. enous MBNL factors. To this purpose, we have This study demonstrates: 1) An effi cient strategy to engineered a modifi ed MBNL∆ that keeps its RNA select ASOs active in skeletal muscle; 2) The in vivo binding property but shows reduced splicing activi- proof of the principle of a therapeutic antisense strat- ties. To evaluate its ability to inhibit CUGexp-RNA egy for the treatment of DM1. toxicity, we fi rst expressed a GFP-MBNL∆ construct in DM1 muscle cells by using lentiviral vectors. GFP- MBNL∆ colocalized with the CUGexp-RNA foci and splicing misregulations as well as differentiation de- fects were corrected in DM1-GFP-MBNL∆ muscle S42 Abstracts cells. To further assess this approach in vivo, intra- failure. This may be interpreted, by a single electro- muscular injections of AAV-GFP-MBNL∆ vectors physiological study, as demyelinating conduction were performed in HSA-LR mice expressing 220CTG block or distal axonal degeneration leading to errors in skeletal muscles. As observed in DM1 cells in cul- in classifi cation and in establishing prognosis. More- tures, colocalization of GFP-MBNL∆ with nuclear over the term axonal may be misleading as it is com- CUGexp-RNA foci in myofi bers indicates that monly associated to axonal degeneration and not to a MBNL∆ will compete and release endogenous MBNL transitory, promptly reversible, dysfunction of the ex- from these aggregates. Splicing alterations of several citable axolemma. To focus on the site of nerve injury transcripts were normalized or nearly corrected in and overcome the classifi cation diffi culties, we pro- HSA-LR injected muscles, and the myotonia was pose the new category of nodo-paranodopathy which abolished. In conclusion, we propose that a modifi ed seems appropriate to various acute and chronic neu- MBNL∆ gene therapy approach could represent an ropathies associated with anti-ganglioside antibodies alternate or complementary therapeutic approach for and we think better systematizes the neuropathies DM1. characterized by an autoimmune attack targeting the nodal region.

THEMATIC WORKSHOP 5.1 Theme: PNS SESSION 1: PATHOGENESIS OF IMMUNE MEDI- THEMATIC WORKSHOP 5.1 ATED NEUROPATHIES Theme: PNS SESSION 1: PATHOGENESIS OF IMMUNE MEDI- ATED NEUROPATHIES TW 5.1.1 Immunology and the nerve TW 5.1.3 Conduction block: Schwann Hans-Peter HARTUNG, Dusseldorf (Allemagne) cell or Axon pathology? Abstract not received Satoshi KUWABARA, Chiba (Japon) Department of Neurology, Graduate School of Medicine, Chiba University THEMATIC WORKSHOP 5.1 Theme: PNS SESSION 1: PATHOGENESIS OF IMMUNE MEDI- Conduction block can be produced by either demy- ATED NEUROPATHIES elination or axonal dysfunction, if the safety factor for impulse transmission is critically impaired. Guillain- Barré syndrome (GBS) is classifi ed into the two major TW 5.1.2 Nodo-paranodopathy: beyond categories; acute infl ammatory demyelinating poly- the classifi cation of myelinopathy and neuropathy (AIDP) and acute motor axonal neuropa- axonopathy thy (AMAN), presenting demyelinating and axonal Nobuhiro YUKI, Singapore (Singapour) conduction block respectively. Over the past 20 years, Department of Medicine, National University of major advances have been made in understanding the Singapore immunopathogenesis and pathophysiology of AMAN. AMAN is characterized by pure motor involvement, In some anti-ganglioside antibody-mediated neu- antecedent Campylobacter jejuni enteritis, and serum ropathies, human and experimental data suggest a anti-ganglioside antibodies. Compared with AIDP, common pathogenic mechanism of dysfunction/dis- AMAN has more rapid progression and earlier nadir, ruption at the node of Ranvier resulting in a patho- and two patterns of recovery (rapid improvement by physiologic continuum from transitory nerve resolution of conduction blocks, and slow recovery by conduction failure to axonal degeneration. The tradi- axonal degeneration). Electrophysiological studies tional classifi cation of polyneuropathies into demye- frequently reveal, as well as axonal degeneration, rap- linating or axonal may generate some confusion in the idly reversible nerve conduction block/slowing (re- electrophysiological diagnosis of Guillain-Barré syn- versible conduction failure); the time-course suggests drome subtypes associated with anti-ganglioside anti- functional or microstructural changes at the nodes and bodies. The axonal forms show, besides axonal paranodes. It is now established that AMAN is caused degeneration, promptly reversible nerve conduction by molecular mimicry of human gangliosides by the Abstracts S43 bacterial lipo-oligosaccharide. An animal model of nal neuropathy associated with subtle signs of AMAN immunized by GM1 was developed, and its demyelination, CIDP should be envisaged. If the clin- histological studies show disruption of nodal sodium ical and electrophysiological European Federation of channel clusters, and paranodal myelin detachment; Neurological Societies / Peripheral Nerve Society these changes are likely to account for conduction (Joint task force)) criteria are not present, we usually block in human AMAN. Conventional electrodiag- opt for a NB to confi rm the diagnosis of CIDP, a treat- nostic criteria for AMAN were based on assumption able disorder. The diagnostic strategy of CIDP, in- of simple axonal loss, and therefore should be recon- cluding the decision to perform a NB has been sidered. The mechanisms for axonal conduction block discussed recently by a group of French neurologists due to microstructural nodal/paranodal pathology are (The French study CIDP group). discussed. Sometimes a few perivascular T cells may be seen Kuwabara S, Yuki N. Axonal Guillain-Barré syn- on paraffi n-embedded or frozen sections, while a few drome: concepts and controversies. Lancet Neurol macrophages are diffusely scattered in the endoneu- 2013;12:1180–8. rium. According to the histopathological diagnostic Hiraga A, et al. Patterns and serial changes in elec- criteria for CIDP of the AAN (ref), which are still use- trodiagnostic abnormalities of axonal Guillain-Barré ful, more than fi ve demyelinated fi bers must be de- syndrome. Neurology 2005;64:856–60. tected. On semi-thin sections at low magnifi cation, the heterogeneous distribution of these de- and remy- elinating lesions between different fascicles and in- THEMATIC WORKSHOP 5.1 side some fascicles may be readily visualized in some Theme: PNS SESSION 1: PATHOGENESIS OF IMMUNE MEDI- cases. By EM, various extents of onion bulb prolifera- ATED NEUROPATHIES tions composed of concentric Schwann cell processes may be seen around more or less completely demye- linated axons. Such axons may be devoid of a myelin TW 5.1.4 Nerve pathology: still helpful in sheath or wrapped only by a few myelin lamellae in- the diagnosis of immune neuropathies? dicating a remyelinating process; some macrophages Jean-Michel VALLAT, Limoges (France) may destroy the myelin sheaths or myelin debris may National reference center for rare peripheral be visible inside a macrophage well demarcated from neuropathies, department of neurology, university the Schwann cell cytoplasm. A loss of nerve axons is hospital, Limoges, France. a consistent fi nding; these secondary axonal lesions develop at a variable rate. In almost all cases, there are In this presentation, we will speak mainly about clusters of regeneration, which generally refl ect abor- Chronic Infl ammatory Demyelinating Polyneuropa- tive attempts at repair. thies (CIDP) and peripheral neuropathies associated As mentioned below, these morphological lesions are to a monoclonal dysglobulinemia which are treatable common to CIDP associated with a monoclonal dys- conditions. In such patients, the diagnosis is made globulinemia and also to some other general disorders. with suffi cient certainty in many cases based on clini- A NB has to be discussed on a case-by-case basis and so cal, electrophysiological and CSF fi ndings. Neverthe- will be reserved to clinically and/or electrophysiologi- less, CIDP could and should be more often diagnosed cally atypical cases including patients with unexcitable by nerve biopsy (NB), especially in atypical cases nerves (Vallat et al 2003). Without this evidence, a sig- (Vallat et al., 2003). nifi cant number of unrecognized CIDP patients may be Otherwise, a polyneuropathy (PN) can be observed classifi ed as having chronic idiopathic axonal neuropa- in any type of benign or malignant monoclonal gam- thy and will not be treated appropriately. mopathy and is a frequent complication of plasma- POLYNEUROPATHIES ASSOCAITED TO A cell dyscrasias. Sometimes aggressive treatments may MONOCLONAL DYSGLOBULINEMIA: be indicated if the nerve biopsy demonstrates lesions The prevalence of neuropathies in patients present- that are responsible for the polyneuropathy (PN). ing a monoclonal gammopathy (MG) has not been CHRONIC INFLAMMATORY DEMYELINAT- evaluated with precision in large prospective studies; ING PERIPHERAL POLYNEUROPATHIES: nevertheless it has been shown that such PN can be When dealing with a chronic isolated polyradiculo- observed in any type of benign or malignant MG and neuropathy with electrophysiological pattern of axo- are a frequent complication of plasma-cell dyscrasias S44 Abstracts such as “monoclonal gammopathy of undetermined . IgG ; IgA : signifi cance” (MGUS), multiple myeloma, Walden- ström’s disease, POEMS (polyneuropathy, organo- In these patients the antigens are unknown, there is megaly, endocrinopathy, monoclonal gammopathy, no anti-MAG activity. In a few cases, immunofl uores- and skin changes) syndrome, Castleman’s disease, and cence of frozen nerve sections with specifi c antibod- light-chain amyloidosis. These cases of clinically di- ies demonstrate annular bindings to numerous verse peripheral neuropathies either occur before the myelinated sheaths which correspond to widenings of discovery of the gammopathy or as complications of it. the myelin lamellae comparable to those described in In any case, a coincidental association has to be dis- IgM-anti MAG neuropathies. Sometimes these le- cussed. sions may be associated to deposits of immunoglobu- A NB may be of particular importance in patients lins in the endoneural interstitial tissue (see next under potentially neurotoxic chemotherapy. Actually, paragraph). Such fi ndings argue in favor of the MG in such cases, the clinical and electrophysiological causing the neuropathy. (Vallat et al 2011). fi ndings only, cannot discriminate between a PN in- duced by a therapeutic agent and other mechanisms. - Deposits in the interstitial tissue: Aggressive treatments such as a specifi c chemothera- . Immunoglobulins py and/or a bone marrow transplant may be indicated if the nerve biopsy demonstrates that the lesions that Immunoglobulin deposits of any kind: IgG, IgA or we will discuss, are responsible for the PN. IgM or light chains can only be evidenced by nerve biopsy. They must not be confused with amyloidosis . Endoneural immunoglobulin deposits: on routine stainings and are better identifi ed by immu- - Intramyelin infi ltration: nocytochemistry on frozen sections and by EM. Actu- . IgM: ally, according to their size, they can be detected either by immunofl uorescence on frozen sections or only by At the present time if anti-MAG antibodies are sig- EM. Ultrastructurally, they may have a quite charac- nifi cantly elevated in the serum of a patient, NB is teristic aspect as digitiform, fi brillar or tubular struc- usually not done. Nevertheless, in a few reported cas- tures etc…(Moorhouse et al 1992); if not, immunoEM es, this procedure has revealed other lesions associat- using anti-immunoglobulin antibodies has to be done ed to the characteristic widenings of the myelin to confi rm their specifi city of such. In any case, a cryo- lamellae such as malignant cells and amyloidosis de- protein should be looked for carefully; if present, we posits.These widenings consist of a 23-nm spacing recommend to fi x it to compare its ultrastructure with between the separated leafl ets of the intermediate line, the ultrastructure of the endoneural deposits; their ul- which contains an electrolucent material. The ultra- trastructural similarity would confi rm the presence of structural features of the dense lines remain un- the abnormal immunoglobulin inside the nerve. changed. These widened lamellae are associated with a dilatation of the outer mesaxon and are mainly lo- . Amyloid deposits: cated on the outer part of the sheath. In some fi bers, the widening is restricted to the outermost lamellae, Amyloid deposits are also multifocal, so that they and careful examination is required to notice it. Such are not seen on all the sections of the NB and can be features are indicative of a dysglobulinemia, and in a missed. A negative examination does not exclude an patient with PN of unknown origin, should prompt the amyloid neuropathy. The sensibility of nerve biopsy search for an M component using appropriate immu- for the diagnosis of systemic amyloidosis is said to be nologic investigations. Widenings of myelin lamellae around 85%. are observed in Waldenström’s macroglobulinemia On paraffi n-embedded sections, amyloid deposits and in monoclonal gammopathy of undetermined sig- appear as round deposits, scattered in the endoneuri- nifi cance (MGUS). Such lesions have also been de- um and sometimes joined to thickened capillary walls. scribed in a few cases in the absence of a AL amyloidosis results from the transformation of an dysglobulinemic neuropathy. They should not be con- immunoglobulin light chain, which may be identifi ed fused with other types of uncompacted myelin lamel- on frozen specimens with a specifi c antibody. These lae which may be encountered in other neuropathies deposits are stained by Congo red and thiofl avine. By such as P0 mutations or in POEMS syndrome. EM examination, amyloid deposits are extracellular Abstracts S45 and composed of characteristic bundles of un- tected on routine paraffi n embedded sections and their branched, 7–10 nm-wide fi brils in an irregular matted specifi c types are confi rmed by immunopathological form; they are sometimes seen in intimate contact examination of deep-frozen NB. Cellular infi ltrates with the basement membranes of endothelial cells and may disappear after multiple sections, which are re- of Schwann cells. The severity of the lesions of the quired for the immunopathological studies. myelinated and unmyelinated fi bers is common in In some cases, the NB may also help to fi nd out amyloidosis, whether of genetic or dysimmune origin. whether the neuropathy is due to an associated mono- Even in an association of a monoclonal IgG, which clonal gammopathy or to the presence of the lympho- may be coincidental, it is recommended to look sys- matous cells or a combination of both (Kelly and tematically for a transthyretin (TTR) mutation. Immu- Karcher 2005; Vallat et al 1995). In such a context, no-EM is of value for differentiating amyloidosis due detection of a clonal immunoglobulin or T-cell recep- to light chain from TTR amyloidosis which may in- tor gene recombination may be very useful to confi rm duce important genetic consequences. the lymphomatous infi ltration. This is commonly per- . POEMS syndrome (Crow Fukase syndrome): formed by using standardized PCR techniques de- Usually, the diagnosis is clinical and is confi rmed signed to assess the diversity of the junctional regions by increased levels of vascular endothelial growth in immunoglobulin or T-cell receptor genes. PCR factor in the serum. As the clinical signs are often in- techniques require low amounts of DNA and have a complete, the diagnosis may be missed; in a few cases good sensitivity (1–5%) to detect clonality (Langerak VEGF may be normal. So, in such cases a NB is still et al 2007). realized. In rare cases of angiotropic lymphoma, there are Specifi c immunocytochemistry studies using im- lymphomatous cells visible in the lumen of arterioles munoglobulin antibodies are negative. EM may dis- in nerve and muscle specimens (Vital et al, 1989). close uncompacted myelin lamellae (UML) which are . CIDP : characteristic features of this condition and may be If electrophysiological studies are in favor of a de- visible in 1–7% of the myelinated fi bers (Vital et al, myelinating polyneuropathy which appears to have 2003). Such a myelin modifi cation corresponds to no direct link with the MG, CIDP is probable and may portions of the mesaxon that are neither fl attened nor result from a common immunological disorder whose compacted and that are visible at least around one primary target antigen remains unknown. To diagnose semi-circumference of the axon, on three or more this type of PN is important as CIDP is a treatable consecutive lamellae; so, there is abnormally widened disorder (Vallat et al 2010). The most common asso- space between major dense lines. Fragments of cyto- ciation is with IgG. As for the “primitive” CIDP cases, plasm are often seen between these lamellae. These nerve biopsy is not done in typical cases, but may be unusual lesions are commonly observed in inner and quite useful to diagnose atypical ones (Vallat et al middle layers of the myelin sheaths, but may also con- 2003); the pathological lesions have been described in cern the outer layer and must be differentiated from the chapter about CIDP. widened myelin lamellae, which are observed in pa- So, in these hematologic disorders it is important to tients with an IgM-antiMAG positive activity. So, the discuss a NB because the demonstration of such spe- presence of numerous UML on a nerve biopsy may cifi c lesions, as early as possible in the course of the serve to distinguish POEMS from nerve CIDP and PN, will determine treatment options. It seems that other demyelinating peripheral nerve disorders, main- usually, this microscopic study is done not early ly in the early phase of the disorder. The signifi cance enough, so that a treatment is instaured too late and of UML with regard to the pathogenesis of POEMS is the evolution unsurprisingly poor. The frequency of still unknown. these lesions is surely under-estimated. NB may also Otherwise, the nerve fi ber lesions are usually char- reveal several processes in the same case; some au- acterized by a mixture of segmental demyelination thors have shown that that fi nding serum anti-MAG and axonal degeneration. However, in some cases antibodies does not exclude the diagnosis of amyloi- pure axonal lesions are present . Infl ammatory infi l- dosis. tration of the endoneurium and occasionally of the If therapy of the blood disorder is in progress, its epineurium, though sparse, may be seen. possible role needs also to be evaluated so that other . Malignant cells : strategies or drugs can be instaured. The presence of lymphoma B or T cells in the pe- ripheral nerve parenchyma, usually in the epineurium, No disclosure. again can only be shown by NB; these cells are de- S46 Abstracts

References: Vallat, J.M., Funalot, B., and Magy, L. (2011). AAN (1991) Research criteria for diagnosis of Nerve biopsy: requirements for diagnosis and clinical chronic infl ammatory demyelinating polyneuropathy value. Acta Neuropathol 121, 313–326. (cidp). Report from an ad hoc subcommittee of the Vallat, J.M., Tabaraud, F., Magy, L., Torny, F., Ber- american academy of neurology aids task force. Neu- net-Bernady, P., Macian, F., and Couratier, P. (2003). rology 41:617–618. Diagnostic value of nerve biopsy for atypical chronic Joint Task Force of the EFNS and the PNS. (2010) infl ammatory demyelinating polyneuropathy: evalua- European Federation of Neurological Societies/Pe- tion of eight cases. Muscle Nerve 27, 478–485. ripheral Nerve Society. Guideline on management of Vital C, Héraud A, Vital A.(1989) Acute mononeu- chronic infl ammatory demyelinating polyradiculo- ropathy with angiotropic lymphoma. Acta Neuro- neuropathy: report of a joint task force of the Euro- pathol 78:105–7. pean Federation of Neurological Societies and the Vital C, Vital A, Bouillot S (2003). Uncompacted Peripheral Nerve Society--First Revision. J Peripher myelin lamellae in peripheral nerve biopsy. Ultra- Nerv Syst 2010; 15:1–9. struct Pathol 27:1–5. Kawagashira Y, Koike H, Tomita M et al. (2010) Vital C, Héraud A, Vital A. (1989) Acute mononeu- Morphological progression of myelin abnormalities ropathy with angiotropic lymphoma. Acta Neuro- in IgM-monoclonal gammopathy of undetermined pathol 78:105–7. signifi cance anti-myelin-associated glycoprotein neu- ropathy. J Neuropathol Exp Neurol 69, 1143–1157. Kelly JJ, Karcher DS (2005) Lymphoma and pe- THEMATIC WORKSHOP 5.2 ripheral neuropathy: A clinical review. Muscle Nerve Theme: RECENT ADVANCES IN UNDERSTANDING ANTI- 31:301–313. GENIC TARGETS IN MYASTHENIA GRAVIS Langerak AW, Molina TJ, Lavender FL, et al. (2007) Polymerase chain reaction-based clonality testing in tissue samples with reactive lymphoprolif- TW 5.2.1 Defects of immune regulation in erations: Usefulness and pitfalls. A report of the Myasthenia Gravis patients with anti- biomed-2 concerted action bmh4-ct98–3936. Leuke- acetylcholine receptor antibodies mia 21:222–229. Sonia BERRIH-AKNIN, Paris (France) Moorhouse DF, Fox RI, Powel HC. (1992). Immu- Sorbonne Universités, UMRS 974 UPMC – INSERM, notactoid-like endoneurial deposits in a patient with CNRS FRE 3617 and Institut de Myologie, Paris, monoclonal gammopathy of undetermined signifi - France cance and neuropathy. Acta Neuropathol;84:484–94. The French CIDP Study Group (2008). Recommen- Myasthenia Gravis (MG) is an autoimmune disease dations on diagnostic strategies for chronic infl amma- in which the thymus frequently presents signs of in- tory demyelinating polyradiculoneuropathy (CIDP). J fl ammation and T cells display defects in suppressive Neurol Neurosurg Psychiatry 2008;79:115–8. regulation. These immune defects can be due to either Vallat, J.M., Tabaraud, F., Magy, L., Torny, F., Ber- the defective function of Treg cells or the resistance of net-Bernady, P., Macian, F., and Couratier, P. (2003). Tconv cells to suppression. Diagnostic value of nerve biopsy for atypical chronic In order to determine which cells were responsible infl ammatory demyelinating polyneuropathy: evalua- for this defect and to address the mechanisms in- tion of eight cases. Muscle Nerve 27, 478–485. volved, we performed cross-experiment studies using Vallat JM, Sommer C, Magy L (2010) Chronic in- purifi ed thymic Treg cells and Tconv cells from con- fl ammatory demyelinating polyradiculoneuropathy: trols and MG patients. We fi rst confi rmed that MG Diagnostic and therapeutic challenges for a treatable Treg cells were defective in suppressing control Tconv condition. Lancet Neurol 9:402–412. proliferation, and we demonstrated for the fi rst time Vallat, J.M., Tabaraud, F., Magy, L., Torny, F., Ber- that MG Tconv cells were resistant to Treg cell sup- net-Bernady, P., Macian, F., and Couratier, P. (2003). pression. The activation of MG Tconv cells triggered Diagnostic value of nerve biopsy for atypical chronic a lower upregulation of FoxP3 and a higher upregula- infl ammatory demyelinating polyneuropathy: evalua- tion of CD4 and CD25 than control cells. To investi- tion of eight cases. Muscle Nerve 27, 478–485. gate the factors that could explain these differences, we compared the transcriptomes of purifi ed thymic Treg and Tconv cells from MG patients and controls. Abstracts S47

Many of the pathways revealed by this analysis are leading to muscle weakness. Antigenic modulation is involved in other autoimmune diseases, and T cells a temperature dependent mechanism requiring clat- from MG patients exhibit a Th1/Th17/Tfollicular sig- rhin and . nature. An increase in IL-17-related genes was only In MUSK MG are mainly of the IgG4 subclass. observed in Treg cells, while increases in the pro-in- They interfere with the interaction of LRP4 with fl ammatory cytokines, IFN-γ, and TNF-α were ob- MUSK. This process does not require the presence of served in both Treg and Tconv cells. In addition, the complement nor crosslinking of MUSK at the cell role of TNF-α in the defect in Tconv cells from MG surface. Anti MUSK antibodies bind to the fi rst Ig- patients was confi rmed by functional studies (Grad- Like domain of MUSK preventing the binding of olatto et al, J. Autoim, 2014). Finally, we showed that MUSK to LRP4 (I. Koneczny PLOS one 2013 and thymic epithelial cells from MG patients contribute to M.Huijbers PNAS 2013). The disruption of the the regulation defects of T cells, and normal thymic MUSK-LRP4 complex inhibits the MUSK phosphor- epithelial cells can partially restore the immunosup- ylation by neural agrin. Similarly to AChR MG, pressive features of MG T cells. MUSK MG endplates are severely damaged resulting Altogether, our results indicate that the immuno- in severe clinical symptoms. regulatory defects observed in MG patients are caused In contrast LRP4 MG patients have less severe clin- by both Treg cell and Tconv cell impairment and in- ical symptoms.( Zisimopoulou J 2013) volve thymic epithelial cells, as well as several pro- Lrp4 is a member of the low-density lipoprotein infl ammatory cytokines, with TNF-α playing a key related receptor family and the receptor for neuronal role in this process. The chronic infl ammation present agrin and activates MuSK Antibodies against LRP 4 in the thymus of MG patients could provide an expla- are of the IgG1 subclass and may therefore fi x com- nation for the escape of thymic T cells from regulation plement when and possibly also disrupt the interac- in the MG thymus. tion between LRP4 and MUSK. In all types of autoimmune MG it is crucial to lower the antibody titers rapidly which is best achieved by THEMATIC WORKSHOP 5.2 plasmapheresis in addition it is to immunosuppressive Theme: RECENT ADVANCES IN UNDERSTANDING ANTI- therapy. Targeting plasma cells with proteasome in- GENIC TARGETS IN MYASTHENIA GRAVIS hibitors opens new perspectives for rapid improve- ment of MG. TW 5.2.2 Effector mechanisms of autoantibodies in Myasthenia Gravis THEMATIC WORKSHOP 5.2 Marc DE BAETS, Maastricht (Pays-Bas) Theme: RECENT ADVANCES IN UNDERSTANDING ANTI- GENIC TARGETS IN MYASTHENIA GRAVIS Myasthenia gravis is a prototype autoimmune dis- ease with circulating antibodies against the nicotinic TW 5.2.3 New autoantigens in MG acetylcholine receptor (AChR) or muscle specifi c ki- nase (MUSK) or Low density lipoprotein receptor- Socrates TZARTOS, Athens (Grèce) related protein 4 (LRP4). Although the autoimmune aHellenic Pasteur Institute, Athens, Greece process is initiated in the thymus the bulk of the auto- bTzartos Neurodiagnostics, Athens, Greece antibodies are produced in the bone marrow by plas- cNeurology Department, Aeginition Hospital, Athens, ma cells. Greece In AChR MG antibodies against extracellular do- dNeurological Institute “C. Besta”, Milano, Italy mains of the AChR mainly a main immunogenic re- eInstitute of Neurology, Catholic University, Rome, gion (MIR) increase the degradation rate of AChRs at Italy the postsynaptic membrane in the presence of com- fIstanbul University, Istanbul, Turkey plement. The postsynaptic folding is decreased leav- gHadassah Hebrew University Medical Center, ing less space for the reduced numbers of AChRs and Jerusalem, Israel sodium channels. These phenomena are called anti- hUPMC and INSERM, Paris, France genic modulation and complement mediated focal ly- iSchool for Mental Health and Neuroscience, sis, result in a neuromuscular transmission defect Maastricht University, The Netherlands S48 Abstracts jThe Cyprus Institute of Neurology and Genetics, VERONIQUE PAQUIS, Nice (France) Nicosia, Cyprus 1IRCAN, UMR CNRS 7284/INSERM U1081/UNS, kDepartment of Neurology, Medical University of School of Medicine, Nice Sophia-Antipolis Warsaw, Poland University, France lSerbia, Clinic of Neurology ,Clinical Center of 2Department of Medical Genetics, National Centre Serbia, Serbia for Mitochondrial Diseases, Nice Teaching Hospital, mNorway Department of Neurology, Ullevaal France University Hospital, Oslo, Norway nBarcelona University, Spain Mitochondrial fusion and fi ssion are fundamental oCharles University Prague Czech Republic processes that govern mitochondrial function from yeast to humans. In mammalian cells, thre large GT- Double-seronegative myasthenia gravis (dSN-MG, Pases are important for mitochondrial fusion which without detectable AChR and MuSK antibodies, mea- requires the coordinated fusion of the outer and inner sured with the classical assays), occurring in about membranes. The mitofusins Mfn1 and Mfn2 are lo- 20% of MG patient, presents a serious gap in MG di- cated on the mitochondrial outer membrane and are agnosis and understanding. Recently, autoantibodies involved in early steps of membrane fusion. The dy- against LRP4 have been identifi ed in several dSN- namin-related protein OPA1 is associated with the in- MG sera. Our aim was the development of sensitive ner membrane and is essential for membrane fusion. assays to be used routinely in the diagnosis of MG for Mitochondrial fi ssion is mediated by a single dyna- the reliable and effi cient detection of antibodies in min-related protein, DRP1. Loss of either fusion or dSN-MG. We have applied cell based assays (CBA) fi ssion activity results in dysfunctional mitochondria. based on LRP4, MuSK and AChR clusters expressed In addition to its well recognized function of control- in HEK293 cells. With these CBAs we have found ling mitochondrial morphology, mitochondrial fusion 18.7% (119/635), 14.3% (100/700) and 3.5% (7/200) clearly protects mitochondrial function. The need for in the sera of dSN-MG patients from 12 countries be- exchange of matrix content between mitochondria, ing positive for LRP4, MuSK and AChR clusters, re- including mtDNA copies, explains probably in part spectively. Interestingly, we found that double positive the importance of this process. sera, namely LRP4+/AChR+(7.5%) or LRP4+/ Mutations in MFN2 and OPA1 genes have been as- MuSK+ (18.2%) are more frequent, compared to sociated with neurodegenerative disorders. MFN2 AChR+/MuSK+ double positives as described in the mutations are a major cause of primary axonal Char- literature. Moreover, we found that ocular MG was cot-Marie-Tooth disease type 2A (CMT2A), an auto- frequent among the LRP4+ patients. The clinical data somal dominant neuropathy that impairs motor and of the LRP4-MG patients showed that at disease onset sensory neurons. OPA1 mutations are responsible for symptoms were much milder than in AChR and Dominant Optic Atrophy (DOA), that is characterized MuSK MG. However, although LRP4 antibodies by retinal ganglion cell degeneration leading to optic were not detected in any of 90 tested sera from healthy neuropathy. Recently, it has been shown that both controls and were rare in other neuroimmune diseas- MFN2 and OPA1 mutations can be responsible for es, they were detected in 24/104 (23.4%) patients with syndromic forms of DOA, including mitochondrial amyotrophic lateral sclerosis (ALS), suggesting a myopathy with COX-negative and ragged red fi bres, broader involvement of the LRP4 antibodies. Overall called DOA “plus” phenotypes. These phenotypes are the three CBAs detected autoantibodies in the 36.5% related to mtDNA instability with multiple mtDNA of the dSN-MG patients. deletions, suggesting that mitochondrial fusion con- trols mtDNA integrity. These novel clinical presenta- tions and the link with mtDNA maintenance will be THEMATIC WORKSHOP 5.3 discussed. Theme: NEUROMUSCULAR DISORDERS OF MITOCHONDRI- AL FUSION AND FISSION THEMATIC WORKSHOP 5.3 TW 5.3.1 Novel phenotypes associated Theme: NEUROMUSCULAR DISORDERS OF MITOCHON- DRIAL FUSION AND FISSION with mitochondrial dynamics defi ciency Abstracts S49

TW 5.3.2 Neuromuscular complications mtDNA deletions in these COX-negative muscle fi - in patients with autosomal dominant bres. optic atrophy Our experimental fi ndings have fi rmly established OPA1 as a novel disorder of mtDNA maintenance. Patrick YU-WAI-MAN, Newcastle (Royaume Uni) Dissecting the disease mechanisms leading to optic 1Wellcome Trust Centre for Mitochondrial Research, atrophy and neuromuscular involvement in DOA will Institute of Genetic Medicine, Newcastle University, have important therapeutic implications. Newcastle upon Tyne, UK. 2Departments of Neurology and Ophthalmology, References Royal Victoria Infi rmary, Newcastle upon Tyne, UK. 1. Yu-Wai-Man P et al. Multi-system neurological disease is common in patients with OPA1 mutations. Autosomal dominant optic atrophy (DOA) is the Brain. 2010;133:771–86. most common inherited optic nerve disorder in the 2. Yu-Wai-Man P et al. OPA1 mutations cause cyto- general population. It affects at least 1 in 25,000 indi- chrome c oxidase defi ciency due to loss of wild-type viduals and OPA1 is the major causative gene in about mtDNA molecules. Human Molecular Genetics. 60% of families. Visual loss starts in early childhood 2010;19:3043–52. secondary to the highly tissue-specifi c loss of one cell type – the retinal ganglion cell (RGC). Although pro- gressive visual failure remains the defi ning feature of THEMATIC WORKSHOP 5.3 DOA, we recently described the expanding pheno- Theme: NEUROMUSCULAR DISORDERS OF MITOCHON- typic spectrum associated with OPA1 disease. Up to DRIAL FUSION AND FISSION 20% of mutational carriers developed a more severe “DOA+” phenotype characterised by prominent neu- romuscular features such as myopathy, peripheral TW 5.3.3 Mitochondrial division and neuropathy, ataxia, and chronic progressive external mitophagy in the brain and heart ophthalmoplegia. Histochemical and molecular char- Hiromi SESAKI, Baltimore (Etats-Unis) acterisation of skeletal muscle biopsies revealed the Department of Cell Biology, Johns Hopkins presence of cytochrome c oxidase (COX) defi cient fi - University School of MedicineBaltimore, MD, USA bres and multiple mitochondrial DNA (mtDNA) dele- tions in the majority of patients harbouring Mitochondria divide and fuse at different rates in pathogenic OPA1 mutations. different cell types. While mitochondria are highly By investigating these tissue samples further, we dynamic in the brain, mitochondria are very static in have uncovered some key pathological consequences the heart. Nonetheless, altered mitochondrial division of OPA1 mutations, namely: and fusion in these two organs are associated with 1. A three-fold increased risk of developing the many diseases such as neurological disorders and more severe DOA+ phenotype with missense OPA1 heart failure. In this talk, I will discuss our recent fi nd- mutations involving the GTPase domain compared ings on the physiological functions of mitochondrial with other mutational subgroups. These patients also division in neurons and cardiomyocytes using tissue- had signifi cantly worse visual acuities with more pro- specifi c mouse knockout for the mitochondrial divi- nounced thinning of the RGC layer. The greater dele- sion protein Drp1. When Drp1 was deleted in terious consequences of this specifi c group of OPA1 Purkinje cells in the cerebellum, mitochondrial tu- mutations on RGC survival is therefore clearly linked bules elongated due to excess fusion, became large to the development of multisystem organ involvement spheres due to oxidative damage, and accumulated in DOA+, suggesting a dominant-negative effect. ubiquitin and mitophagy markers, leading to respira- 2. The induction of a signifi cant in vivo mitochon- tion defects and neurodegeneration. Treatment with drial oxidative phosphorylative defect, with the fre- antioxidants rescued cell death in Drp1KO Purkinje quency of COX-defi cient muscle fi bres being over cells. When Drp1 was lost in cardiomyocytes, mito- four times higher in the DOA+ group compared with chondria also became defective in mitophagy and ac- the pure DOA group. cumulated oxidative damage, resulting in lethal 3. A marked mitochondrial proliferative response cardiac failure. Our fi ndings suggest that mitochon- and the accumulation of clonally-expanded, somatic drial division serves as a universal mechanism to pro- S50 Abstracts tect the organelle from oxidative stress and thus ally, the MS is closely associated with insulin resis- ensures tissue maintenance in mammalian organs. tance in metabolically active tissues like fat and muscle. Recently insulin resistance was shown to be present in the peripheral nervous system, with disrup- THEMATIC WORKSHOP 5.4 tion of intracellular signaling leading to Mt and endo- Theme: METABOLIC NEUROPATHY plasmic reticulum stress, ROS accumulation, DNA damage and the development of DN. In summary, the newly evolving concept that all components of the TW 5.4.1 Underlying Metabolic metabolic syndrome, and not just hyperglycemia, un- Mechanisms in the Pathogenesis of derlie the onset and progression of DN, has broad im- Diabetic Neuropathy plications on not only the development of new therapies but also the clinical care of patients. Eva L FELDMAN, Ann Arbor (Etats-Unis) University of Michigan, Department of Neurology, Ann Arbor, Michigan, USA. THEMATIC WORKSHOP 5.4 382 million people have diabetes in 2013 and by Theme: METABOLIC NEUROPATHY 2035 this will rise to 592 million. Type 2 diabetes ac- counts for 90% of all cases with the greatest number TW 5.4.2 Metabolic neuropathy: Clinical of affected people between 40 to 59 years of age. manifestations and current treatment Eighty percent of people with diabetes live in low- and middle-income countries. At least half of all peo- Brian CALLAGHAN, Ann Arbor (Etats-Unis) ple with diabetes develop diabetic neuropathy (DN), a Department of Neurology, University of Michigam condition with signifi cant morbidity including foot ulcers and amputations. The underlying pathogenesis Of the metabolic syndrome (MetS) components, of DN is a source of active investigation. A decade diabetes and pre-diabetes have the strongest evidence ago, the “glucocentric” hypothesis stated that DN was supporting a pathogenic link with neuropathy, but due in large part to impaired glucose fl ux and metabo- each of the other components also have evidence sup- lism. Excess neuronal glycolysis overloads the mito- porting their association with neuropathy in diabetic chondrial electron transport chain, leading to the populations. Specifi cally, obesity has been shown by generation of reactive oxygen species (ROS). This multiple investigators to be associated with neuropa- process occurs in parallel with increased fl ux through thy in diabetic patients.(1–3) Similarly, in a study of the polyol and hexosamine pathways as well as the 427 diabetic patients with mild to moderate diabetic generation of advanced glycation endproducts, with neuropathy, elevated triglycerides correlated with loss all 3 pathways further promoting ROS accumulation. of sural nerve myelinated fi ber density, a direct ana- A vicious feed forward cycle occurs of continuous im- tomical measurement of neuropathy.(4) Furthermore, paired glucose fl ux, disruption of downstream path- patients with normoglycemia and neuropathy have the ways, ROS accumulation and cellular injury. same prevalence of MetS components as those with In the last decade, this hypothesis has been expand- IGT and neuropathy, and an even higher prevalence of ed, with two ideas. First, nervous system injury in pa- MetS components than those with diabetes and no tients and experimental models of DN are not limited neuropathy.(5) These results indicate that MetS and its to neurons, axons and nerve terminals, but also in- components are likely to be important in non-diabetic clude glial cells and endothelial cells of the microvas- populations as well. Given the clustering of MetS culture. Secondly, DN is secondary to not only components, hypertension, hypertriglyceridemia, glucose-mediated injury but also nervous system in- dyslipidemia, and particularly obesity are prime can- jury secondary to the metabolic syndrome (MS). The didates to be the essential factors underlying the neu- MS is a cluster of risk factors that include abdominal ropathy present in patients with type 2 diabetes. obesity, dyslipidemia with high triglycerides and low The only component of MetS with an established HDL cholesterol, hypertension and hyperglycemia. treatment for the prevention of neuropathy is diabetes. Dyslipidemia promotes nervous system infl amma- Enhanced glucose control has been shown to decrease tion, ROS and apoptosis and in human clinical studies the incidence of neuropathy in patients with type 1 of type 2 diabetes and DN, is more closely related to diabetes, with little effect in those with type 2 diabe- nervous system injury than hyperglycemia. Addition- tes.(6–9) In type 1 diabetes, enhanced glucose control Abstracts S51 can be achieved through diet and exercise and insulin. 5. Smith AG, Rose K, Singleton JR. Idiopathic neu- Similar diet and exercise regimens with the addition ropathy patients are at high risk for metabolic syn- of metformin, sulfonylureas, and other less common drome. J Neurol Sci. 2008 Oct 15;273(1–2):25–8. drugs, provides improved glycemic control but little PubMed PMID: 18606421. Pubmed Central PMCID: protection against neuropathy in type 2 diabetes. Diet 2576295. Epub 2008/07/09. eng. and exercise in those with pre-diabetes and neuropa- 6. Effect of intensive diabetes treatment on nerve thy has been shown to increase nerve fi ber density, but conduction in the Diabetes Control and Complica- no controlled clinical trial has been performed to con- tions Trial. Ann Neurol. 1995 Dec;38(6):869–80. fi rm this fi nding.(10) Furthermore, diet, exercise, and PubMed PMID: 8526459. Epub 1995/12/01. eng. metformin reduce the incidence of diabetes in those 7. Intensive blood-glucose control with sulphonyl- with pre-diabetes, but the effect on the prevention of ureas or insulin compared with conventional treat- neuropathy is unclear.(11) While effective pharma- ment and risk of complications in patients with type 2 ceutical treatments exist for hypertension, hypertri- diabetes (UKPDS 33). UK Prospective Diabetes glyceridemia, and dyslipidemia, no studies have Study (UKPDS) Group. Lancet. 1998 Sep investigated the effect of these interventions on the 12;352(9131):837–53. PubMed PMID: 9742976. prevention or improvement of neuropathy. Similarly, Epub 1998/09/22. eng. while diet and exercise programs and medications can 8. Duckworth W, Abraira C, Moritz T, Reda D, be effective in the treatment of obesity, no current data Emanuele N, Reaven PD, et al. Glucose control and exist on the effect of these interventions on peripheral vascular complications in veterans with type 2 diabe- neuropathy in this population. Importantly, diet and tes. The New England journal of medicine. 2009 Jan exercise regimens have the potential to treat MetS as a 8;360(2):129–39. PubMed PMID: 19092145. Epub whole; however, compliance and long term mainte- 2008/12/19. eng. nance on these regimens are notoriously diffi cult. 9. Ismail-Beigi F, Craven T, Banerji MA, Basile J, Confl icts of interest: Dr. Callaghan receives re- Calles J, Cohen RM, et al. Effect of intensive treat- search support from Impeto Medical and performs ment of hyperglycaemia on microvascular outcomes center certifi cations for the ALS Association. in type 2 diabetes: an analysis of the ACCORD ran- domised trial. Lancet. 2010 Aug 7;376(9739):419–30. 1. Straub RH, Elbracht R, Kramer BK, Roth M, PubMed PMID: 20594588. Epub 2010/07/03. eng. Palitzsch KD, Scholmerich J. Infl uence of digoxin- 10. Smith AG, Russell J, Feldman EL, Goldstein J, like immunoreactive factor on late complications in Peltier A, Smith S, et al. Lifestyle intervention for pre- patients with diabetes mellitus. Eur J Clin Invest. diabetic neuropathy. Diabetes Care. 2006 1994 Jul;24(7):482–7. PubMed PMID: 7957506. Jun;29(6):1294–9. PubMed PMID: 16732011. Epub Epub 1994/07/01. eng. 2006/05/30. eng. 2. Tesfaye S, Chaturvedi N, Eaton SE, Ward JD, 11. Knowler WC, Barrett-Connor E, Fowler SE, Manes C, Ionescu-Tirgoviste C, et al. Vascular risk Hamman RF, Lachin JM, Walker EA, et al. Reduction factors and diabetic neuropathy. N Engl J Med. 2005 in the incidence of type 2 diabetes with lifestyle inter- Jan 27;352(4):341–50. PubMed PMID: 15673800. vention or metformin. N Engl J Med. 2002 Feb Epub 2005/01/28. eng. 7;346(6):393–403. PubMed PMID: 11832527. Pubmed 3. Van Acker K, Bouhassira D, De Bacquer D, Central PMCID: 1370926. Epub 2002/02/08. eng. Weiss S, Matthys K, Raemen H, et al. Prevalence and impact on quality of life of peripheral neuropathy with or without neuropathic pain in type 1 and type 2 THEMATIC WORKSHOP 5.4 diabetic patients attending hospital outpatients clin- Theme: METABOLIC NEUROPATHY ics. Diabetes Metab. 2009 Jun;35(3):206–13. PubMed PMID: 19297223. Epub 2009/03/20. eng. 4. Wiggin TD, Sullivan KA, Pop-Busui R, Amato TW 5.4.3 Advances in the diagnosis and A, Sima AA, Feldman EL. Elevated triglycerides cor- clinical interventions for metabolic relate with progression of diabetic neuropathy. Diabe- neuropathy tes. 2009 Jul;58(7):1634–40. PubMed PMID: 19411614. Pubmed Central PMCID: 2699859. Epub Gordon SMITH, Salt Lake City (Etats-Unis) 2009/05/05. eng. University of Utah School of Medicine, Department of Neurology S52 Abstracts

Current guidelines recommend that diagnosis of betic Peripheral Neuropathy. Journal of Diabetes and distal symmetric polyneuropathy (DSP) rely on the Its Complications. 2013 (26):424–429. demonstration of a combination of symptoms, signs 5. Smith AG. Impaired glucose tolerance and meta- and confi rmatory neurophysiological or pathological bolic syndrome in idiopathic neuropathy. Journal of testing1. Reliable recognition of clinical features of the Peripheral nervous system : JPNS. 2012 May;17 DSP is challenging, even for experts, resulting in reli- Suppl 2:15–21. ance on nerve conduction studies (NCS)2. However, NCS are frequently normal in early DSP, particularly Disclosure: Dr. Smith receives grant support from among patients with preferential small fi ber involve- the NIH (DK064814), ADA (ADA7–111-AEC23). ment. Skin biopsy with measurement of intraepider- mal nerve fi ber density (IENFD) has demonstrated excellent diagnostic performance for patients with THEMATIC WORKSHOP 5.5 suspected small fi ber neuropathy. Several other tech- Theme: NUCLEAR ENVELOPE / NUCLEAR MATRIX niques, such as corneal confocal microscopy (CCM) hold promise as diagnostic tools. While there are no pharmacological interventions shown to alter the nat- TW 5.5.1 Emerin-LAP1 Interaction and ural history of diabetic or idiopathic DSP, data from a X-linked Emery-Dreifuss Muscular growing number of studies suggest lifestyle interven- Dystrophy tions, particularly mixed aerobic exercise and strength training, may be effective in enhancing peripheral Howard WORMAN, New York (Etats-Unis) 1 nerve regenerative capacity, resulting in slowed neu- Department of Medicine 2 ropathy progression and improved patient symp- Department of Pathology and Cell Biology, College toms.3,4 These strategies likely work by addressing of Physicians & Surgeons, Columbia University, NY the detrimental effects of obesity and its downstream 10032 USA 3 complications of prediabetic insulin resistance, dys- Department of Neurology 4 lipidemia and other aspect of the “Metabolic Syn- Department of Cell and Developmental Biology drome”5. There are also a number of symptomatic University of Michigan Medical School, Ann Arbor, approaches to DSP with demonstrated effi cacy. Cur- MI 48109 USA rent approaches to diagnosis of DSP will be reviewed. X-linked Emery-Dreifuss muscular dystrophy is Current treatment approaches, including a brief re- caused by loss of function of emerin, an integral pro- view of symptomatic management will be discussed. tein of the inner nuclear membrane. Mice lacking New models for therapeutic development, including emerin, however, are essentially normal, suggesting innovative clinical trial designs for DSP will be the existence of compensating factors. We show that discussed. lamina-associated polypeptide1 (LAP1) interacts with emerin and that conditional deletion of LAP1 from References: striated muscle causes muscular dystrophy. Striated 1. Tesfaye S, Boulton AJ, Dyck PJ, et al. Diabetic muscle-selective LAP1 knockout mice also display neuropathies: update on defi nitions, diagnostic crite- ventricular systolic dysfunction with abnormal induc- ria, estimation of severity, and treatments. Diabetes tion of genes encoding cardiomyopathy-related pro- Care. 2010 ed. 2010 Oct;33(10):2285–2293. teins. Skeletal muscle pathology in mice with LAP1 2. Dyck PJ, Overland CJ, Low PA, et al. Signs and deleted from this tissue is worsened in the absence of symptoms versus nerve conduction studies to diag- emerin. LAP1 levels are signifi cantly higher in mouse nose diabetic sensorimotor polyneuropathy: Cl vs. than human skeletal muscle, potentially explaining NPhys trial. Muscle Nerve. 2010 ed. 2010 why mice lacking emerin are essentially normal Aug;42(2):157–164. whereas humans develop muscular dystrophy. Reduc- 3. Smith AG, Russell J, Feldman EL, et al. Lifestyle ing LAP1 by approximately half also induces skeletal intervention for pre-diabetic neuropathy. Diabetes muscle abnormalities in emerin null mice. Condition- Care. 2006 Jun;29(6):1294–1299. al deletion of LAP1 from hepatocytes yields mice that 4. Kludig P, Pasnoor M, Singh R, et al. The Effect exhibit normal liver function and are indistinguish- of Exercise on Neuropathic Symptoms, Nerve Func- able from littermate controls, demonstrating a selec- tion, and Cutaneous Innervation inPeople with Dia- tive role for LAP1 in striated muscle. These results Abstracts S53 establish that LAP1 interacts physically and function- 2012). This led us to hypothesize a model of how ab- ally with emerin. They are also consistent with emerg- normalities of A-type lamins may lead to cardiomy- ing case reports of mutations in the gene encoding opathy. We proposed a mechanism for the LAP1 causing muscular dystrophy in human patients. pathogenesis of LMNA-cardiomyopathy, for which abnormal cardiac activation of MAP kinases lead to an increase in AKT/mTOR signaling, which reduces THEMATIC WORKSHOP 5.5 tolerance to energy defi cit (Choi et al. 2012). Theme: NUCLEAR ENVELOPE / NUCLEAR MATRIX These results provide proof of principle for small molecule inhibition as a therapeutic option to delay the onset of heart failure in LMNA-cardiomyopathy. TW 5.5.2 Lamin A/C and striated muscle Pharmacological blockade of MAP kinase (Wu et al. 2011; Muchir et al., 2011) or AKT/mTOR (Choi et al. 2012) cascade in Lmna H222P knock-in mice blunts Antoine MUCHIR, Paris (France) left ventricular dilatation and deterioration in cardiac 1Inserm, U974, Paris, F-75013, France contractility. While it remains unclear how alterations 2Universite Pierre et Marie Curie-Paris 6, UM 76; in A-type lamins lead to activation of these signaling CNRS, UMR7215; Institut de Myologie, IFR14, in the heart, these studies clearly show that the abnor- Paris F-75013, France mal activations are involved in the pathophysiology 3Department of Medicine of LMNA-cardiomyopathy. 4Department of Pathology and Cell Biology, College Muchir et al. Activation of MAPK Pathway Links of Physicians and Surgeons, Columbia University, LMNA Mutations to Cardiomyopathy in Emery-Dre- 630 West 168th Street, New York, NY 10032, USA. ifuss Muscular Dystrophy. J. Clin. Invest. 2007. The nuclear lamina underlies the nuclear face of the Wu et al. Mitogen activated protein kinase inhibi- inner nuclear membrane of eukaryotic cells. Because tors improve heart function and prevent fi brosis in of its well-described roles in maintaining nuclear en- cardiomyopathy caused by lamin A/C gene mutation. velope integrity and providing anchoring sites for Circulation 2011. chromatin domains, the nuclear lamina plays a sig- Choi et al. Temsirolimus activates autophagy and nifi cant role in both the nuclear architecture and func- ameliorates cardiomyopathy caused by lamin A/C tion. The main components of the nuclear lamina are gene mutation. Sci. Transl. Med. 2012. intermediate fi lament proteins, the nuclear lamins. LMNA encodes the A-type lamins. The discovery that can be caused by LMNA mu- THEMATIC WORKSHOP 5.5 tations has resulted in a re-evaluation of the function Theme: NUCLEAR ENVELOPE / NUCLEAR MATRIX of the nuclear lamina. An emerging body of work sup- ports a new view of the nuclear lamina as a node that TW 5.5.3 Abnormal nuclear mechanics integrates and transduces a range of signals. Hence, beyond its classical barrier function, studies of the and mechanotransduction in muscular nuclear lamina are increasingly providing insights laminopathies into basic aspects of cellular organization and func- Jan LAMMERDING, New-York (Etats-Unis) tion and providing novel insights into the pathogene- Department of Biomedical Engineering & Weill sis of human disease. Institute for Cell and Molecular Biology To explore the pathogenesis of LMNA-cardiomy- Cornell University Ithaca, NY USA opathy, we carried out a genome-wide RNA expres- sion analysis of hearts from a mouse model of the Unlike the static pictures found in many text books, disease (Lmna H222P knock-in). This analysis re- cells live in a highly dynamic, mechanically active en- vealed signifi cant differences in expression of genes vironment. Cells exert physical forces on intracellular encoding proteins in stress-activated MAP kinases structures and their environment to facilitate move- and AKT/mTOR signaling pathways in the mutated ment and migration; at the same time, cells and organ- mice (Muchir et al. 2007). We confi rmed an aberrant elles are subjected to forces from their surroundings, increase in both signaling in hearts from Lmna H222P resulting in often substantial deformations. Here, I knock-in mouse hearts (Muchir et al. 2007, Choi et al. will focus on intracellular biomechanics, particularly the and its connection to the cytoskeleton. S54 Abstracts

Recent discoveries provide compelling evidence that Introduction: Randomized controlled trials showed the physical properties of the nucleus and its struc- that intravenous immunoglobulin (IVIg) and plasma tural organization are critical for a multitude of cellu- exchange (PE) are effective in patients with Guillain- lar functions. For example, cell migration requires Barré syndrome (GBS)1,2,3. Based upon trial results, coordinated movement of the nucleus with the cell these treatments need to be started early in the course body, and neutrophils have evolved highly lobulated of disease, within the fi rst two weeks (or within four nuclei that increase their physical plasticity, enabling weeks for PE) after onset of weakness, presumably passage through narrow spaces. Not surprisingly then, before partial irreversible nerve damage has been oc- disturbed nuclear structure and mechanics, often curred. Despite standard IVIg or PE treatment, about caused by inherited mutations in nuclear envelope 25% of patients require artifi cial ventilation and 20% proteins such as lamins, are responsible for many dis- is still unable to walk after half a year. It is clear that eases, including muscular dystrophies, dilated cardio- treatment needs to be improved, especially in the myopathy, familial partial lipodystrophy, and group of patients being severely involved and having Hutchinson-Gilford progeria syndrome. My labora- a slow recovery phase. tory has developed novel experimental techniques to Predict the course of disease in GBS by using pre- study the effect of mutations on nuclear structure and diction models: Since the course and severity of dis- mechanics and the physical coupling between the nu- ease can vary widely, it is a challenge to predict the cleus and the cytoskeleton, as well as to examine how course of disease in individual patients. I will discuss changes in these properties can modulate cellular two simple prognostic tools that easily can be used at functions. Using these techniques, we have demon- the patient’s bedside to predict the prognosis. The strated that lamin mutations associated with muscular Erasmus GBS Respiratory Insuffi ciency Scale dystrophies can reduce nuclear stability, thereby ren- (EGRIS) enables to calculate the chance to need arti- dering cells more susceptible to damage in mechani- fi cial ventilation within two weeks after admission4. cally active tissues such as muscle and providing a It can help to decide if a patient may need direct ICU potential explanation for the muscle-specifi c pheno- admission or otherwise very frequent neurological types. Interestingly, we also found that many of these and pulmonary controls at a ward. This scale can be mutations impair cellular mechanotransduction sig- used already at hospital admission and only requires naling, i.e., the ability of cells to respond to mechani- three variables: duration between onset of weakness cal stimulation, which could further exacerbate the and admission, presence of facial or bulbar weakness, muscular phenotypes. In addition, mutations in nucle- and the MRC sumscore of 6 pairs of limb muscles. ar envelope proteins that cause muscular dystrophies Another helpful scale is the modifi ed GBS Out- can also disrupt nucleo-cytoskeletal coupling, which come Scale (mEGOS)5. This scale can be used al- is critical for nuclear positioning, intracellular force ready one week after hospital admission to predict the transmission, cellular polarization, and migration. I chance to walk unaided after 6 months. To determine will present these and other recent fi ndings from our this chance, it requires the MRC sumscore (range laboratory and discuss the close relationship between 0–60) at 7 days after admission, the age of the patient nuclear mechanics and cellular organization and its and the presence of diarrhea before onset of GBS. role in muscular disease caused by mutations in nu- There is a website available (www.gbstools.org) that clear envelope proteins. enables to use EGRIS and mEGOS and to do the cal- culations in a very easy way. Usage of prediction models in clinical trials: THEMATIC WORKSHOP 6.1 mEGOS is being used in the ongoing Second IVIg Theme: PNS SESSION 2: NEW OPTIONS IN THE TREATMENT Dose (SID)-GBS randomized placebo controlled trial OF IMMUNE MEDIATED NEUROPATHIES that is running in the Netherlands, and in the interna- tional prospective (ISID)-GBS study as part of the international GBS outcome study (IGOS). Both stud- TW 6.1.1 Guillain-Barré syndrome: how ies investigate the effect of a second IVIg course in to assess and to treat individuals with GBS patients with a poor prognosis. poor prognosis GBS patients may have a secondary deterioration or turn out to have acute-onset CIDP: Approximately Peter A. VAN DOORN, Rotterdam (Pays-Bas) 10% of GBS patients have a secondary deterioration Erasmus MC University Hospital Rotterdam, The often named ‘treatment-related fl uctuation’ (TRF). Netherlands Neurologists should take care for these fl uctuations, Abstracts S55 since it requires re-treatment with IVIg of PE. improves with immune therapies. A few variants of Additionally, about 5 percent of patients initially di- CIDP have been described including Distal Acquired agnosed as GBS turn out to have acute onset CIDP Demyelinating Symmetric (DADS), multifocal ac- (A-CIDP). These patients should be treated like CIDP, quired demyelinating sensorimotor neuropathy also which indicates repeated IVIg infusions, or even a named Lewis Sumner syndrome, purely sensory, switch to treatment with corticosteroids. It can be dif- purely motor and focal demyelinating neuropathy. If fi cult to make a distinction between GBS-TRF and their inclusion as atypical CIDP is useful in prescrib- A-CIDP. From a prospective study it turned out that ing expensive therapy such as intravenous immuno- patients initially diagnosed as GBS, who have 3 or globulins (IVIg) that are effective in CIDP, it does not more deteriorations, of those having deteriorations af- help to clarify whether these are phenotypic variants ter 8 weeks from onset of weakness very likely have of CIDP or separate diseases. This has also therapeu- A-CIDP6. tic implications since motor CIDP may worsen with steroids that is often effective in CIDP. Several im- References: mune therapies have been reported to be effective in 1. van Doorn PA, Ruts L, Jacobs BC. Clinical fea- CIDP including steroids, plasma exchange and IVIg. tures, pathogenesis, and treatment of Guillain-Barre It is therefore diffi cult for the clinician to decide when syndrome. Lancet Neurol 2008;7:939–50. to start treatment in CIDP and what therapy to fi rst 2. Hughes RA, Swan AV, van Doorn PA. Intrave- use. This decision should consider the effi cacy, cost nous immunoglobulin for Guillain-Barre syndrome. and side effects of these therapies. This also explain Cochrane Database Syst Rev 2012;7:CD002063. why patients with minimal symptoms not interfering 3. Raphael JC, Chevret S, Hughes RA, Annane D. with their daily life are usually treated with symptom- Plasma exchange for Guillain-Barre syndrome. Co- atic therapy and periodic assessment. A few random- chrane Database Syst Rev 2012;7:CD001798. ized trial in CIDP have shown a comparable short-term 4. Walgaard C, Lingsma HF, Ruts L, Drenthen J, effi cacy of IVIg and oral corticosteroids and of IVIg van Koningsveld R, Garssen MJ, van Doorn PA, Stey- and plasma exchange in and more recent trials have erberg EW, Jacobs BC. Prediction of respiratory in- shown that both IVIg and steroids have prolonged ef- suffi ciency in Guillain-Barre syndrome. Ann Neurol fi cacy in CIDP. A recent randomized controlled trial 2010;67:781–7. comparing the six-month effi cacy of IVIg and intrave- 5. Walgaard C, Lingsma HF, Ruts L, van Doorn PA, nous methylprednisolone showed that IVIg were Steyerberg EW, Jacobs BC. Early recognition of poor more frequently effective and tolerated than steroids prognosis in Guillain-Barre syndrome. Neurology during the fi rst six month of treatment, although, 2011;76:968–75. when effective, steroids were less frequently associ- 6. Ruts L, Drenthen J, Jacobs BC, van Doorn PA, ated with deterioration than IVIg after therapy discon- Dutch GBSSG. Distinguishing acute-onset CIDP tinuation. There were not signifi cant differences in the from fl uctuating Guillain-Barre syndrome: a prospec- proportion of patients experiencing adverse events tive study. Neurology 2010;74:1680–6. even if slightly more adverse events were observed after steroids. It remains unclear whether these advan- tages are suffi cient to balance the much higher cost of THEMATIC WORKSHOP 6.1 IVIg compared to steroids. Even if similarly effective Theme: PNS SESSION 2: NEW OPTIONS IN THE TREATMENT to IVIg, plasma exchange is usually considered the OF IMMUNE MEDIATED NEUROPATHIES third choice since it is more invasive for the patients and has an higher prevalence of side effects. A num- ber of immunosuppressive agents have been also re- TW 6.1.2 When and how to treat CIDP? ported to be useful in CIDP even if their effi cacy was Eduardo NOBILE-ORAZIO, Milan (Italie) not confi rmed in randomized trials. Dept. of Medical Biotechnology and Translational The author received personal compensation for Medicine, Milan University, 2nd Neurology, IRCCS serving in the Steering or Medical Advisory Board of Humanitas Clinical Institute, Rozzano, Milan, Italy Baxter, Italy, CSL Behring, Italy, Kedrion, Italy, and Novartis, Switzerland. He received honoraria for lec- Chronic infl ammatory demyelinating polyradiculo- turing from Baxter, Italy, CSL Behring, Italy, Grifols, neuropathy (CIDP), is a chronic acquired demyelinat- Spain, and Kedrion, Italy and travel supports for Sci- ing neuropathy that in over 80% of the patients entifi c Meetings from Baxter and Kedrion. S56 Abstracts

THEMATIC WORKSHOP 6.1 routine electrophysiological examination. Some may Theme: PNS SESSION 2: NEW OPTIONS IN THE TREATMENT have “MMN without PMCB.” OF IMMUNE MEDIATED NEUROPATHIES Randomized controlled trials show that intravenous immunoglobulin has a benefi cial effect on strength TW 6.1.3 Multifocal motor neuropathy: and ability. (van Schaik et al. Intravenous immuno- globulin for multifocal motor neuropathy. Cochrane IVIg and ... what else? Database of Systematic Reviews 2009 DOI: David R CORNBLATH, Baltimore (Etats-Unis) 10.1002/14651858.CD004429.pub2) Department of Neurology, Johns Hopkins University Other immunosuppressants have been trialled: School of Medicine, Baltimore, MD, USA. • In a randomized placebo-controlled trial of my- cophenolate mofetil, there was no signifi cant Multifocal Motor Neuropathy (MMN) is a rare, benefi t. male predominant, young onset syndrome character- • CTX has been used successfully in small num- ized by progressive asymmetric distal weakness with bers of patients but the risks likely outweigh the partial motor conduction block on nerve conduction benefi ts. studies, and clinical improvement following IVIg ad- • Rituximab has been trialled in open studies with- ministration. out success. MMN should enter the differential diagnosis of any • Recent open trial of eculizumab suggested a full patient with a slowly or stepwise progressive asym- trial. metrical limb weakness without objective sensory ab- • Trials of other immunosuppressants should be normalities, upper motor neuron or bulbar signs or undertaken. (Umapathi et al. Immunosuppressant symptoms. and immunomodulatory treatments for multifocal The Core Criteria are Slowly progressive or step- motor neuropathy. Cochrane Database of Sys- wise progressive, focal, asymmetrical limb weakness tematic Reviews 2012 DOI: 10.1002/14651858. AND Lack of objective sensory abnormalities except CD003217.pub4). for minor vibration sense abnormalities in lower limbs. Disclosures: Consultant: Bristol-Myers Squibb Supportive clinical criteria include predominant up- Company, Cigna Health Management, Inc., CSL Beh- per limb involvement, decreased or absent tendon re- ring GmbH, DP Clinical, Inc., InVivo, Merck and Co., fl exes in the affected limbs, absence of cranial nerve Seattle Genetics, Inc., Shire Pharma PLC, Sun Phar- involvement, cramps and fasciculations in the affect- maceuticals. Data Safety Monitoring Board: Acorda ed limb, and response in terms of disability or muscle Therapeutics, Inc., Pfi zer Inc., Johnson & Johnson, strength to immunomodulatory treatment. GlaxoSmithKline plc., ISIS Pharmaceuticals, Novar- Supportive criteria include elevated IgM anti-gan- tis Corp. Technology Licensing: Johnson & Johnson, glioside GM1 antibodies, increased CSF protein, Shire Pharma PLC, Seattle Genetics, Inc., Genentech. magnetic resonance imaging showing increased sig- Board of Directors: GBS-CIDP Foundation Interna- nal intensity on T2-weighted imaging associated with tional, Foundation for Peripheral Neuropathy, The Pe- a diffuse nerve swelling of the brachial plexus, and ripheral Nerve Society. objective clinical improvement following IVIg treat- ment. Exclusion criteria include upper motor neuron THEMATIC WORKSHOP 6.1 signs, marked bulbar involvement, sensory impair- Theme: PNS SESSION 2: NEW OPTIONS IN THE TREATMENT ment other than minor vibration loss in the lower OF IMMUNE MEDIATED NEUROPATHIES limbs, and diffuse symmetric weakness during the ini- tial weeks. The presence of partial motor conduction block TW 6.1.4 Rituximab in anti-MAG (PMCB) in motor nerve fi bers is the electrodiagnostic neuropathies: Yes or No? hallmark of the disease. Some patients with otherwise Jean-Marc LEGER, PARIS CEDEX 13 (France) typical MMN have no detectable PMCB, probably National Referral Center for rare Neuromuscular because these blocks are activity-dependent or are lo- Diseases, University Hospital Pitié-Salpêtrière and cated in nerve segments which cannot be assessed by Abstracts S57

University Pierre et Marie Curie (Paris VI) Paris, elinating neuropathy, but the limitations of these 2 France RCT should be considered.

Within the spectrum of chronic immune-mediated neuropathies, demyelinating neuropathy associated THEMATIC WORKSHOP 6.2 with IgM monoclonal gammopathy and antibodies Theme: NEW THERAPIES IN MYASTHENIA GRAVIS against myelin-associated glycoprotein (MAG) is a distinct entity that typically presents with progressive sensory ataxia and painful paresthesias in the 4 limbs. TW 6.2.1 New therapies for myastenia The disease may progress slowly over many years in gravis: just for refractory disease some patients, whereas others develop signifi cant dis- Amelia EVOLI, Rome (Italie) ability mostly due to dysesthesia and ataxia, thus there Institute of Neurology, Catholic University, Roma, is a need to develop effective treatments. Despite sev- Italy eral arguments in favor of a highly probable autoim- mune mechanism underlying this disease, there is Myasthenia Gravis (MG) comprises several disease insuffi cient evidence from most pilot studies or ran- subtypes with distinct pathogenic and clinical charac- domized controlled trials (RCT) on IgM anti-MAG teristics (1). Current treatment is based on a combina- demyelinating neuropathy to recommend any particu- tion of symptomatic medication, thymectomy, lar immunotherapy (Lunn and Nobile-Orazio, Co- long-term and short-term immune therapies. chrane Review 2012). Rituximab is a genetically The development of new therapeutic agents for MG engineered chimeric murine/human monoclonal anti- has been relatively marginal when compared to the body directed against CD20, a protein present on the remarkable expansion in other autoimmune diseases. surface of normal and malignant pre-B and mature B A possible reason for such disparity is that conven- cells until differentiation into plasma cells. Its effi ca- tional treatment is effective, as it can control symp- cy has been supported by several uncontrolled stud- toms in most patients. However, this outcome often ies, and the results of 2 recent RCT versus placebo are requires prolonged and even life-long immunosup- now available. In the fi rst one (Dalakas et al. 2009), pression. Moreover, a proportion of patients have re- after 8 months, by intention-to-treat (ITT), 4 of 13 fractory disease as they complain of disabling rituximab-treated patients improved by ≥ 1 leg IN- weakness or frequent relapses and/or require too high CAT score compared with 0 of 13 patients taking pla- doses of steroids or other immunosuppressive agents cebo (p=0.096). Excluding one rituximab included with serious side effects (2). Refractoriness to con- patient who had normal INCAT score at entry, and ventional treatment appears to be more common in thus could not improve, the results were signifi cant patients with thymoma and with muscle-specifi c ki- (p=0.036). In addition, the Time to 10m walk was sig- nase (MuSK) antibodies (3–4). nifi cantly reduced in the rituximab group (ITT; The experience with biological agents in MG is still p=0.042). Based on clinical assessments and patient limited and mostly based on small patient series. Tu- questionnaires, among the 13 placebo-treated pa- mor necrosis factor-inhibitors should be avoided as tients, 6 had worsened, 7 remained unchanged, and they can induce MG worsening (1). Complement in- none improved at month 8; in contrast among the 13 hibition with eculizumab proved effective in a phase rituximab-treated patients, 1 worsened, 5 remained II trial in patients with acetylcholine receptor (AChR) unchanged, and 7 improved. In the second one (Léger antibodies (5). Many uncontrolled studies on B-cell et al. 2013), ITT analysis, with imputation of missing depletion with rituximab have collectively reported a ISS values by the last observation carried forward high rate of positive results, with differences between method, showed a lack of mean change in ISS at 12 MuSK-MG and AChR-MG. Recent studies have months (p=0.92). However, changes were observed, shown multiple effects of rituximab on the immune in per protocol analysis at 12 months, for the number system (6–7) and suggested the potential advantage of of patients with an improvement of at least 2 points in an early use of this agent in association with selective the INCAT disability scale (p=0.027), the self evalua- cytokine inhibition (8). However, until randomized tion scale (p=0.016), and 2 subscores of the Short trial data are available, lack of reliable biomarkers and Form-36 questionnaire. Consequently, rituximab has safety concerns limit the use of biological therapies in no evidence-based effi cacy in IgM anti-MAG demy- MG other than refractory disease. S58 Abstracts

1) Meriggioli MN, Sanders DB. Lancet Neurology disease and lead in some cases to stable remission. To 2009;8:475–90 better understand how thymectomy can improve MG 2) Drachman DB et al. Ann NY Acad Sci patients, thymic changes occurring MG thymus will 2008;1132:305–14 be detailed and the rational of new therapeutic issues 3) Suh J et al. Yale J Biol Med 2013;86:255–60 to reverse thymic abnormalities will be considered. 4) Evoli A, Padua L. Autoimmun Rev 2013;12:931– 5 5) Howard JF Jr et al. Muscle Nerve 2013; 48:76– THEMATIC WORKSHOP 6.2 84 Theme: NEW THERAPIES IN MYASTHENIA GRAVIS 6) Sun F et al. Muscle Nerve 2014; 49:487–94 7) Huang H et al. Proc Natl Acad Sci U S A 2010;107:4658–63 TW 6.2.3. Antigen-specifi c apheresis in 8) Mahèvas M et al. Front Immunol 2013;4:494 myasthenia gravis Socrates TZARTOS, Athens (Grèce) Hellenic Pasteur Institute, Athens, Greece THEMATIC WORKSHOP 6.2 Theme: NEW THERAPIES IN MYASTHENIA GRAVIS In Myasthenia Gravis (MG) the pathogenic autoan- tibodies target mainly the muscle acetylcholine recep- TW 6.2.2 New issues in the rational of tor (AChR) and occasionally the MuSK and the LRP4. Our goal is to develop an antigen-specifi c therapy, thymectomy based on the selective extracorporeal autoantibody ROZEN LE PANSE, Paris (France) depletion (immunoadsorption), using sepharose-im- UMRS 974 UPMC - INSERM - FRE 3617 CNRS mobilized extracellular domains (ECDs) of the hu- - AIM man AChR subunits and MuSK. We have previously described the expression of these ECDs in the P. pas- Acquired myasthenia gravis (MG) is an autoim- toris system and their use as effi cient immunoadsor- mune disorder caused by autoantibodies that target bents in vitro. The maximal capacity of the matrix for components of the neuromuscular junction leading to autoantibodies and its ability to be regenerated after muscle weakness and disabling fatigability. Autoanti- each use, by elution of bound autoantibodies has been bodies are mostly against the anti-acetylcholine re- determined. Furthermore, the immobilized AChR and ceptor (AChR), and less frequently the muscle MuSK ECDs were proven effi cient adsorbents in specifi c kinase receptor or the low density lipoprotein fl ow-rate conditions similar to those required during a receptor-related protein 4. therapeutic session. In parallel, the possibility for MG with anti-AChR antibodies is often associated whole-blood adsorption has been investigated. The with thymic abnormalities, such as thymoma or thy- initial screening showed the same adsorption effi cien- mic follicular hyperplasia. Active neoangiogenic pro- cy when whole blood instead of serum was used. We cesses, abnormal chemokine expression and ectopic proceeded to ex vivo whole blood immunoadsorption germinal centres are the main features of thymic hy- using immunized animals to determine the effi ciency perplasia. Recently, we demonstrated that interferon-β and safety of the procedure. Initial results show a re- induced by double-stranded RNA signalisation, plays duction of the antibody titer following a single proce- a central role in thymic events leading to MG. Indeed, dure and no subsequent boost effect. We are currently interferon-β can orchestrates the overexpression of setting up a protocol for repeated immunoadsorption α-AChR probably leading to dendritic cell autosensi- sessions to maximize the observed effect and its dura- tization, the abnormal recruitment of peripheral cells tion. Upon completion of these tests we will proceed and germinal centre formation. to clinical trials of therapeutic immunoadsorption in Thymectomy has become part of the standard ther- MG patients. The successful development and appli- apeutic treatment for MG patients with anti-AChR cation of this antigen-specifi c therapy for MG could antibodies. In patients with thymoma, the thymus is be used as a model for other antibody-mediated auto- removed to avoid spreading of the tumour. In non- immune disorders as well. thymomatous MG, thymectomy is considered to re- duce severity of MG, to slowdown the course of the Abstracts S59

THEMATIC WORKSHOP 6.3 - Tegtmeyer LC et al. Multiple phenotypes in phos- Theme: FOCUS ON NEW INSIGHTS IN METABOLIC MYOPA- phoglucomutase 1 defi ciency. N Engl J Med. 2014 THIES Feb 6;370(6):533–42. - Nilsson J et al. Polyglucosan body myopathy TW 6.3.1 New genes for muscle caused by defective ubiquitin ligase RBCK1. Ann Neurol. 2013;74(6):914–9. glycogenosis Pascal LAFORET, Paris (France) Paris-Est Neuromuscular center THEMATIC WORKSHOP 6.3 U974, Université Pierre et Marie Curie Theme: FOCUS ON NEW INSIGHTS IN METABOLIC MYOPA- Hôpital Pitié-Salpêtrière, 47–83 boulevard de THIES l’Hôpital 75013 Paris

Muscle glycogenosis are a group of metabolic my- TW 6.3.2 Contribution of exercise tests to opathies due to defi ciencies of various enzymes in- study treatment and the phenotypes of volved in the synthesis or degradation of glycogen. metabolic myopathies Patients may present with exercise intolerance and Nicolai PREISLER, Copenhagen (Danemark) rhabdomyolysis episodes, or progressive muscle Neuromuscular Research Unit, 3342 weakness, depending on the defi cient defect. Other Department of Neurology, University Hospital of organs, such as liver or brain, may also be affected. Copenhagen, Rigshospitalet, Denmark Two new glycogenosis have been recently de- scribed, due to mutations in PGM1 and RBCK1 Metabolic myopathies are inborn errors of metabo- genes. Patients with phosphoglucomutase (PGM1) lism affecting muscle. Symptoms and morbidity in defi ciency often present with exercise intolerance and glycogen storage diseases (GSD) and fatty acid oxida- rhabdomyolysis, hepatopathy with hypoglycemia, tion defects (FAOD) are caused by a limitation to use and dysmorphic features. In all cases, abnormal pat- carbohydrates or fatty acids, which are the fuels that tern of protein glycosylation may be detected by iso- supply the energy for skeletal muscle contraction dur- electric focusing, allowing diagnosis, and thus ing aerobic exercise. establishing a connection between glycogenosis and The basis behind the use of exercise to examine and congenital disorders of glycosylation (CDG syn- test patients with GSD and FAOD is that exercise in- dromes). creases the energy requirements in skeletal muscle Patients with RBCK1 mutations exhibit a progres- many-fold, and thus either provokes symptoms or in- sive muscle weakness frequently associated with a duces metabolic derangements that may be detected severe cardiomyopathy which is a major cause of by measuring key substrates, like plasma lactate and death. Pathological hallmark of this disease is the glucose. In addition, by combining exercise with indi- presence of polyglucosan inclusions in skeletal and rect calorimetry and stable isotope methodology im- cardiac muscles. However, the link between muta- portant information on the catabolism and oxidation tions in RBCK1 gene encoding an E3 ubiquitin ligase, of skeletal muscle substrates may be detected in vivo and abnormally branched glycogen accumulation has in humans. not been yet established. Interestingly, mutations in Exercise testing has been used to assess the effects this gene have also been reported in children who died of treatment in patients with GSD and FAOD. The ef- from sepsis due to auto-infl ammatory disease, with fi cacy of carbohydrates as treatments of exercise-in- polyglucosan inclusions identifi ed in skeletal muscle, duced symptoms and as means of improving exercise heart and liver. tolerance has been established with methods applying In addition to extending the list and the clinical exercise testing in McArdle disease (myophosphory- spectrum of muscle glycogenosis, both of these dis- lase defi ciency) and in very long-chain acyl-CoA de- eases pave the way to novel and to date unsuspected hydrogenase (VLCAD) and carnitine physiopathological mechanisms. palmitoyltransferase (CPT) II defi ciencies. More re- cently, cycle-ergometer exercise testing in combina- References: tion with stable isotope methodology and indirect - Stojkovic T et al. Muscle glycogenosis due to calorimetry has been used to assess the effect of bet- phosphoglucomutase 1 defi ciency. N Engl J Med. zafi brate as a treatment to increase fatty acid oxida- 2009;361(4):425–7. tion in CPT II and VLCAD defi ciencies. S60 Abstracts

In addition, exercise testing may be used to explore perature and humidity, and the level of hydration. In the phenotype in a controlled setting in the exercise fact, any healthy individual is probably prone to de- laboratory. In such a setting, we have demonstrated velop rhabdomyolysis in case of suffi cient and severe that debranching enzyme defi ciency (GSD type IIIa) risk factors. is associated with exercise induced symptoms of ex- Recurrent episodes of rhabdomyolysis, a positive cessive fatigue and muscle pain (dynamic symptoms), (family) history for muscle (metabolic) disorders, and which is in contrast to the typical description of this absence of suffi ciently explaining exogenous factor GSD, as mainly being a liver GSD with a static mus- point to a underlying genetic cause. The following cle involvement that develops in the 3rd to 4th de- guidelines can be helpful: rhabdomyolysis 1) evoked cade. by prolonged exercise and/or fasting: defects in fatty Thus, cycle-ergometry exercise in combination acid oxidation; 2) triggered by short-lasting exercise: with stable isotopes and indirect calorimetry contin- myophosphorylase defi ciency (with second wind phe- ues to provide new insight into the metabolic derange- nomenon) or various glycolytic disorders (without ments causing disease in metabolic myopathies. second wind phenomenon); and 3) with triggers of various duration: various muscular dystrophies (ele- vated CK) or RYR1-related disorders (CK usually THEMATIC WORKSHOP 6.3 normal). Theme: FOCUS ON NEW INSIGHTS IN METABOLIC MYOPA- Mutations in the RYR1 gene are one of the most THIES common causes of inherited neuromuscular disease, associated with the malignant hyperthermia suscepti- bility trait, and a variety of congenital myopathies. TW 6.3.3 Exercise-induced RYR1 mutations have been recently identifi ed in 14 rhabdomyolysis: diagnostic guidelines families presenting with rhabdomyolysis episodes and RYR1-related cases mainly evoked by exercise in absence of anesthetic triggers. Rhabdomyolysis was commonly triggered Nicol VOERMANS, Nijmeggen (Pays-Bas) by exercise and heat and, less frequently, viral infec- Radboud university medical centre Nijmegen, The tions, alcohol and drugs. Most patients were normally Netherlands strong and had no personal MH history. Muscle biop- Rhabdomyolysis is a common and potentially lethal sies showed mainly subtle changes. Familial RYR1 clinical syndrome that results from acute muscle fi bre mutations were confi rmed in relatives with similar or necrosis with leakage of muscle constituents into no symptoms. These fi ndings suggest that RYR1 mu- blood (CK increase ≥ 10x, with a rapid decrease and tations may account for a substantial proportion of decline). Underlying myopathy or muscle metabolic patients presenting with unexplained rhabdomyolysis defects are responsible for approximately 10% of the and/or exertional myalgia. adult cases, with frequent recurrence and a low inci- dence of acute renal failure or multiple etiologies. Exercise-induced rhabdomyolysis is defi ned as THEMATIC WORKSHOP 6.4 rhabdomyolysis with exercise preceding the episode Theme: PATHOMECHANISMS UNDERLYING MOTOR NEU- of rhabdomyolysis. Most exercise results in some RON DEATH AND NEW THERAPEUTIC APPROACHES IN ALS/ skeletal muscle damages, and unaccustomed and ec- NMD centric exercise can cause extensive damage. The mechanisms involved in the initiation of the muscle TW 6.4.1 New therapeutic targets for damage is hypothesized to be either mechanical (mus- neuroprotection in ALS/NMD cle damage is the result of direct physical stress on muscle fi bres, especially when exercise involves ec- PAMELA SHAW, SHEFFIELD (Royaume Uni) centric muscle contractions where the muscle is lengthening while trying to contract) or metabolic (the Abstract not received damage is the result of different energetic defi cien- cies, involving primarily metabolic substrates). Vari- ous exogenous factors infl uence this: the intensity of the exercise, the physical condition and fi tness of the individual, including diet and medication, the tem- Abstracts S61

THEMATIC WORKSHOP 6.4 local protein synthesis in axons. These fi ndings point Theme: PATHOMECHANISMS UNDERLYING MOTOR NEU- to common pathomechanisms in SMA and ALS, and RON DEATH AND NEW THERAPEUTIC APPROACHES IN ALS/ to new strategies which can be used to restore altered NMD RNA metabolism in these different forms of motor neuron disease. TW 6.4.2 Dysregulation of RNA processing in motor neuron degeneration THEMATIC WORKSHOP 6.4 Michael SENDTNER, Wuerzburg (Allemagne) Theme: PATHOMECHANISMS UNDERLYING MOTOR NEU- Institute for Clinical Neurobiology, University RON DEATH AND NEW THERAPEUTIC APPROACHES IN ALS/ Hospital Wuerzburg, Germany NMD

Loss of motor endplates, axonal degeneration and cell death of motor neurons are characteristic patho- TW 6.4.3 Cellular autonomy and motor logical features of motor neuron diseases. The identi- neurone degeneration fi cation of gene defects for familial Amyotrophic SIDDHARTHAN CHANDRAN, Edinburgh lateral sclerosis (ALS) and other motor neuron dis- (Royaume Uni) eases has pointed to distinct pathophysiological mechanisms but it is still unclear how they lead to Abstract not received synaptic loss, axonal degeneration and ultimatively to motor neuron cell death. The survival motor neuron (SMN) protein which is defi cient in spinal muscular THEMATIC WORKSHOP 6.5 atrophy (SMA) also interacts with RNA binding pro- teins such as hnRNP R, TDP-43 and FUS. Smn inter- Theme: UBIQUITIN-PROTEASOME COMPLEX IN HEART AND SKELETAL MUSCLES acts with the C-terminus of these proteins where mutations have been identifi ed in familial forms of ALS. A prominent phenotype of SMN defi cient motor TW 6.5.1 Regulation of ubiquitin- neurons is reduced axon elongation in the absence of mediated turnover of sarcomeric proteins altered motor neuron survival pathways. The axonal Mathias GAUTEL, London (Royaume Uni) defects are similar to those observed in motor neurons 1Cardiovascular Division and Randall Division for overexpressing mutant forms of TDP-43, but TDP-43, Cell and Molecular Biophysics, King’s College in particular M337V mutant TDP-43 alters signalling London, London, United Kingdom pathways of endoplasmatic reticulum (ER) stress. The 2Max-Planck-Institute for Heart and Lung Research, subcellular transport of mRNAs for β-actin and other Bad Nauheim, Germany transcripts for axonal growth and presynaptic func- tion is severely affected in Smn defi cient motor neu- The dynamic turnover of muscle sarcomeres is reg- rons, and local translation of these mRNAs in axonal ulated by two major pathways, the ubiquitin-protea- terminals is disturbed. Similar observations have been somal system (UPS) and autophagy-lysosomal (AL) made in motor neurons after depletion of TDP-43. degradation pathways. Muscle-specifi c ubiquitin li- The consequences are disturbed axon elongation, re- gases like MAFBx/atrogin-1 and MURFs target mus- duced growth cone size and functional defi cits in neu- cle proteins for degradation, classically via the UPS, rotransmission that are caused by disturbed integration including sarcomeric proteins like myosin as well as and clustering of voltage-gated calcium channels and signalling proteins, metabolic enzymes and transcrip- other proteins for presynaptic function in axon termi- tion factors. Several UPS and AL-associated proteins nals. The defi cit in clustering of voltage-gated calci- associate directly with the sarcomere, including the um channels in growth cones of SMN-defi cient motor giant sarcomeric muscle protein titin that plays an es- neurons and in motor neurons expressing mutant sential role as a molecular ruler for determining sarco- TDP-43 is accompanied by a signifi cantly reduced mere length. Furthermore, titin has multiple signalling frequency of spontaneous Ca2+ transients. In mild domains, including a C-terminal kinase domain (TK) models of SMA, this defect can be compensated by located in the M-band. This kinase shows unusual se- enlargement of motor units. PTEN signalling restores quence features, including a mechanosensitive regu- defective axon growth and excitability by stimulating S62 Abstracts latory domain, raising the question whether its main sembly and turnover. Dev. Biol., 2011. 351(1): p. 46– function is catalytic, that of a scaffold, or both. Lange 61. et al. [1] reported a signalling complex where TK in- 3. Perera, S., B.S. Mankoo, and M. Gautel, Devel- teracts with the autophagy adaptor protein nbr1, a opmental regulation of MURF E3 ubiquitin ligases in complex implicated in load-dependent muscle protein skeletal muscle. J. Muscle Res. Cell Motil., 2012. turnover regulation. Nbr1 binds a homologous au- 33(2): p. 107–122. tophagy-associated scaffold protein, p62/SQSTM1, 4. Ulbricht, A., et al., Cellular Mechanotransduc- which in turn interacts with at least two ubiquitin E3 tion Relies on Tension-Induced and Chaperone-As- ligases, MURF-1 and -2. This complex is develop- sisted Autophagy. Curr Biol, 2013. 23(5): p. 430–5. mentally tightly regulated, with MURF isogenes also showing fi bre-type specifi c expression patterns [2, 3]. To understand the interplay between sarcomeric ki- THEMATIC WORKSHOP 6.5 nase scaffolding and load-dependent muscle protein Theme: UBIQUITIN-PROTEASOME COMPLEX IN HEART AND turnover via autophagy, we generated genetically al- SKELETAL MUSCLES tered mouse lines carrying truncated versions of Nbr1, lacking either the C-terminal region following its PB1 domain (tr), or lacking the PB1 domain alone (ΔPB1). TW 6.5.2 UPS dysfunction in hereditary These animals can be bred to homozygosity and can cardiomyopathy (MYBPC3 / LMNA) reach over 1 year in age, but show distinct muscle Lucie CARRIER, Hamburg (Allemagne) phenotypes. Unloading and loading experiments in Department of Experimental Pharmacology and skeletal and cardiac muscle show that load-dependent Toxicology, University Medical Center Hamburg- muscle protein turnover is dependent on full-length Eppendorf, Hamburg, Germany Nbr1, whereas complete knockout of p62 is compen- sated. Proteomic analysis reveals altered turnover of Adequate protein turnover is essential for maintain- metabolic enzymes, sarcomeric proteins, and signal- ing cardiac homeostasis. Several protein quality con- ling proteins as well as components of the endosomal trols are involved in protein homeostasis, including pathway. Surprisingly, sarcomeric components whose molecular chaperones and co-chaperones, the autoph- turnover depends on Nbr1 include M-band titin and agy-lysosomal pathway, and the ubiquitin-proteasome myomesin, whereas previously identifi ed cytoskeletal system (UPS). In the last decade, a body of evidence targets for autophagosomal turnover were restricted to has underlined a major role of the UPS in cardiac the peri-myofi brillar Z-disk protein fi lamin-C [4]. In physiology and pathology. Particularly, recent studies conclusion, the sarcomere interacts as a mechano- have shown that dysfunctional proteasomal function sensitive scaffold with autophagy adaptors that are leads to cardiac disorders, including hereditary car- critical and non-redundant for cardiac and skeletal diomyopathies. Hypertrophic and dilated cardiomy- muscle maintenance, as well as for atrophy and repair opathies are the two most prevalent inherited responses. Our studies may have wider implications cardiomyopathies. Both are primarily transmitted as for muscle and cardiac responses to cellular stress, ag- an autosomal-dominant trait and mainly caused by gregate clearance and protein homeostasis. Targeting mutations in genes encoding components of the car- signalling pathways like TK-nbr1-p62 that modulate diac sarcomere, including a relevant striated muscle- muscle protein turnover and remodelling may provide specifi c E3 ubiquitin ligase. A growing body of new approaches to alter the biology of hereditary evidence indicates UPS impairment in inherited car- muscle diseases, muscle wasting, and heart failure. diomyopathies as determined by measurement of the level of ubiquitinated proteins, the activities of the References proteasome and/or the use of fl uorescent UPS reporter 1. Lange, S., et al., The Kinase Domain of Titin substrates. While the paradigm for UPS impairment Controls Muscle Gene Expression and Protein Turn- in HCM is MYBPC3, encoding cardiac myosin-bind- over. Science, 2005. 308: p. 1599–1603, Epub ahead ing protein C (cMyBP-C), this is CRYAB, encoding of print March 31. αB-crystallin for dilated cardiomyopathy (DCM). 2. Perera, S., et al., Developmental regulation of Other genes associated with DCM are also tightly MURF ubiquitin ligases and autophagy proteins nbr1, regulated by the UPS. This is for example the case for p62/SQSTM1 and LC3 during cardiac myofi bril as- LMNA encoding lamins A/C. Mutations in LMNA Abstracts S63 caused Emery-Dreyfuss Muscular Dystrophy, DCM Proteasomal dysfunction is considered to play a with conduction and/or rhythm defects. I will present role in the pathogenesis of protein aggregate myopa- typical examples of UPS impairment in mouse mod- thies (PAM), a wide group of muscular conditions els of inherited cardiomyopathies, suggest potential characterized by accumulation of proteins within mechanisms leading to and consequences of UPS im- muscle fi bers. PAM includes myofi brillar myopathies pairment. Finally, I will underline available therapeu- (MFM), myosin storage myopathy (MSM) and acti- tic options to restore proteasome activity and therefore nopathy, among many others. The vast majority of cardiac homeostasis and function. PAM is caused by mutations in genes coding for cyto- skeletal proteins, including components of the myofi - References brillar apparatus. We have recently identifi ed a new Vignier*, Schlossarek* et al., Circ Res 2009. protein aggregate myopathy, which is associated with Schlossarek et al., Basic Res Cardiol 2012. gene defects in striated muscle-specifi c E3 ubiquitin Schlossarek et al., J Muscle Res Cell Motil 2012. ligases, MuRF1 and MURF3 that mediate the degra- Cattin et al., Hum Mol Genet 2013. dation of myosin heavy chains via the UPS. Schlossarek, Frey & Carrier, J Mol Cell Cardiol This new entity is myopathologically characterized 2014. by subsarcolemmal accumulation of myosin and my- osin-associated proteins and at the ultrastructural lev- el by fragmented sarcomeres with preserved A-bands THEMATIC WORKSHOP 6.5 and M-bands but absent Z-discs. The selective accu- Theme: UBIQUITIN-PROTEASOME COMPLEX IN HEART AND mulation of thick fi laments, that are neither ubiqui- SKELETAL MUSCLES tinated, nor labeled with antibodies against the 20S proteasome subunit, indicates a specifi c impairment of their degradation due to gene defects in the striated TW 6.5.3 Ubiquitin Proteasome system muscle-specifi c E3 ubiquitin ligases. and skeletal myopathies Homa TAJSHARGHI, Gothenburg (Suède) 1Institute of Neuropathology, Department of THEMATIC WORKSHOP 7.1 Pathology and Neuromuscular Unit, Department of Theme: CELL THERAPY IN NEUROMUSCULAR DISEASES Neurology, IDIBELL-Hospital de Bellvitge, Hospitalet de Llobregat, Barcelona, Spain TW 7.1.1 Mouse satellite cells 2CIBERNED, Centro de Investigació n Biomé dica en Red de Enfermedades Neurodegenerativas, Instituto Jennifer MORGAN, London (Royaume Uni) Carlos III, Spain Dubowitz Neuromuscular Centre, UCL Institute of 3Department of Pathology, Sahlgrenska University Child Health, 30 Guilford Street, London Hospital, University of Gothenburg, Gothenburg, WC1N1EH, UK Sweden 4Department of Clinical and Medical Genetics, The classical skeletal muscle stem cell is the satel- University of Gothenburg, Gothenburg, Sweden lite cell. Satellite cells contribute to both muscle growth and regeneration and are capable of self-re- The ubiquitin-proteasomal system (UPS) is pivotal newal (1). for the selective degradation or recycling of cytoplas- We have used an in vivo transplantation model sys- mic and nuclear intracellular proteins and plays a ma- tem to investigate the effect of different environmen- jor role in the regulation of protein homeostasis. One tal factors on the ability of mouse satellite cells to major function of the ubiquitin–proteasome system contribute to muscle regeneration and functionally re- (UPS) is to prevent accumulation of non-functional, constitute the satellite cell pool. Satellite cells from potentially toxic proteins. Degradation via the UPS normal donor mice contribute robustly to muscle re- system requires the attachment of ubiquitin to target generation only when the host satellite cell niche has proteins by a peptide linkage, allowing its transport to been modulated prior to engraftment (2, 3). Although the 26S proteasome complex. Substrate recognition muscle maintenance and regeneration is impaired for ubiquitination is highly specifi c and provided by with increasing age and as a consequence of muscular the E3 ubiquitin ligases. dystrophies, satellite cells from both aged and young, S64 Abstracts male and female normal mice (4) and dystrophic do- René Laennec, 80054 Amiens Cedex 1 nor mice contribute effectively to muscle regeneration 8Service de Neurologie, Consultation de Pathologies when grafted into a permissive host environment. We Neuromusculaires, Institut de myologie, Hôpital are currently investigating the factors that control do- Pitié Salpêtrière, Faculté de Médecine & Université nor satellite cell-mediated muscle regeneration. Paris VI Pierre et Marie Curie, Assistance publique- Hôpitaux de Paris (APHP), Paris, France, References 9CHU de Caen, Centre de Compétences des 1. Collins CA, Olsen I, Zammit PS, Heslop L, Pet- Pathologies Neuro-musculaires, Service de rie A, Partridge TA, et al. Stem cell function, self-re- Médecine Physique et Réadaptation, Avenue de la newal, and behavioral heterogeneity of cells from the Côte de Nacre, CS 30001, 14033 Caen Cedex 9, adult muscle satellite cell niche. Cell. France et INSERM, U 1075 COMETE, France 2005;122(2):289–301. 2. Boldrin L, Zammit PS, Muntoni F, Morgan JE. Cell therapy was fi rst proposed in the 80’s as a po- Mature adult dystrophic mouse muscle environment tential treatment for muscular dystrophies, based upon does not impede effi cient engrafted satellite cell re- early results obtained in mdx mice: dystrophin expres- generation and self-renewal. Stem Cells. sion was restored in this model by intramuscular injec- 2009;27(10):2478–87. tions of normal myoblasts. These results were quickly 3. Boldrin L, Neal A, Zammit PS, Muntoni F, Mor- followed by clinical trials for patients suffering from gan JE. Donor Satellite Cell Engraftment is Signifi - Duchenne Muscular Dystrophy (DMD) in the early cantly Augmented When the Host Niche is Preserved 90’s, based mainly upon intramuscular injections of al- and Endogenous Satellite Cells are Incapacitated. logenic myoblasts. The clinical benefi ts obtained from Stem Cells. 2012; 30(9):1971–84. these trials were minimal, if any, and research pro- 4. Neal A, Boldrin L, Morgan JE. The satellite cell grams concentrated then on the various pitfalls that in male and female, developing and adult mouse mus- hampered these clinical trials. New therapeutic venues cle: distinct stem cells for growth and regeneration. were then explored, such as the use of stem cells with PLoS One. 2012;7(5):e37950. myogenic potential, which have been described in various populations, including bone marrow, circulat- ing blood or muscle itself. These stem cells presented THEMATIC WORKSHOP 7.1 the main advantage to be available and not exhausted by the numerous cycles of degeneration/regeneration, Theme: CELL THERAPY IN NEUROMUSCULAR DISEASES which characterize muscular dystrophies. However, the different stem candidates have also shown their TW 7.1.2 Cell therapy for muscular limits in terms of effi ciency to participate to the regen- dystrophies: update on the autologous eration of the host. A state of the art summary of the myoblast cell therapy trial for various ongoing clinical and preclinical trials and of oculopharyngeal muscular dystrophy the various cell types that are currently available will be presented. In particular, the results of the phase I/IIa Gilian BUTLER-BROWNE, Paris (France) clinical trial (ClinicalTrials.gov NCT00773227) of au- 1Sorbonne Universités, UPMC Univ Paris 06, tologous myoblast transplantation following myotomy Myology Research Center UM76, F-75013, Paris, in pharyngeal muscle of OPMD patients will be de- France scribed. This study included 12 patients with clinical 2INSERM UMRS974, F-75013, Paris, France diagnosis of OPMD and indication for cricopharyn- 3CNRS FRE 3617, F-75013, Paris, France geal myotomy. The feasibility and safety end points of 4Institut de Myologie, F-75013, Paris, France both autologous myoblast transplantation as well as 5Service d’Oto-Rhino-Laryngologie et de Chirurgie the surgical procedure were assessed by videoendos- Cervico-Faciale, Assistance publique-Hôpitaux de copy in addition to physical examinations. Potential Paris (APHP), Hôpital Tenon, Paris, France therapeutic benefi t was also assessed through videoen- 6Unité de Thérapie Cellulaire et CIC-BT501, Hôpital doscopy and videofl uoroscopy of swallowing, quality Saint Louis, Assistance publique-Hôpitaux de Paris of life score, dysphagia grade, and a drink test. Patients (APHP), Université Paris Diderot, Sorbonne Paris were injected with a median of 178 million myoblasts Cité, Paris, France following myotomy. Short and long-term safety and 7Amiens Hospital, Hematology Department, Avenue tolerability were observed in all patients, with no ad- Abstracts S65 verse effects. There was an improvement in the quality formed, not tumorigenic and myogenic. Notably, this of life score for all 12 patients, and no functional deg- strategy allowed the transfer of a novel DYS-HAC into radation in swallowing was observed for the majority reversibly immortalized DMD mesoangioblasts, which of patients. A cell dose dependant improvement in had a normal karyotype after expansion in culture. Pre- swallowing was even observed in this safety study. liminary results also showed that DYS-HAC-corrected This trial supports the hypothesis that a local injection reversibly immortalized DMD mesoangioblasts were of autologous myoblasts in the pharyngeal muscles is integrated into regenerating myofi bers upon transplan- a safe and effi cient procedure for OPMD patients. tation into immunodefi cient mdx mice. However, degeneration-regeneration cycles of dys- trophic muscles can exhaust the pool of adult progen- THEMATIC WORKSHOP 7.1 itors for ex vivo expansion, as we have shown for Theme: CELL THERAPY IN NEUROMUSCULAR DISEASES limb-girdle muscular dystrophy 2D (LGMD2D, char- acterized by α-sarcoglycan defi cit)3. Therefore, LG- MD2D fi broblasts or myoblasts were reprogrammed TW 7.1.3 Human artifi cial chromosomes to induced pluripotent stem (iPS) cells and then dif- and iPS cells for ex vivo gene therapy of ferentiated to mesoangioblast-like cells3. These cells muscular dystrophies and beyond were expanded, genetically corrected and then trans- planted into ad hoc generated α-sarcoglycan-null im- Francesco Saverio TEDESCO, London (Royaume munodefi cient mice. Hybrid human myofi bres Uni) expressing α-sarcoglycan were identifi ed in the xeno- 1 Department of Cell and Developmental Biology, transplants and upon murine intra-specifi c transplan- University College London, London, UK. tation these cells caused functional amelioration of 2 Department of Biomedical Science, Tottori the dystrophic phenotype3,4. Importantly, this ap- University, Yonago, Japan. proach was also extended to DMD using the DYS- 3 Department of Biosciences, University of Milan, HAC for genetic correction. Finally, preliminary Italy. evidence of use of this platform for disease modelling 4 Institute of Infl ammation and Repair, University of and tissue engineering will be discussed. This strategy Manchester, Manchester, UK. offers proof-of-principle of safety and effi cacy of #Current affi liation: UCL Institute of Child Health, HACs and iPS cells for cell therapies of muscular London, UK. dystrophies, providing also encouraging data for their *[email protected] potential use in drug development and tissue engi- neering for muscle disorders. Mutations in the dystrophin gene affect skeletal muscle and cause Duchenne muscular dystrophy 1. Benedetti S et al., FEBS Journal, 2013. (DMD), ultimately resulting in premature death. Gene 2. Tedesco FS et al., Science Translational Medi- and cell therapy for DMD is diffi cult1, since skeletal cine, 2011. muscle is the most abundant human tissue and dystro- 3. Tedesco FS et al., Science Translational Medi- phin is the largest gene in the human genome. A few cine, 2012. years ago we described the amelioration of dystrophic 4. Gerli MFM et al., Journal of Visualized Experi- mdx mice by combining Human Artifi cial Chromo- ments, 2014. some (HAC)-mediated dystrophin gene-replacement with mesoangioblast myogenic stem/progenitor cells, using a HAC containing the entire dystrophin locus (DYS-HAC) and mouse pericyte-derived mesoangio- THEMATIC WORKSHOP 7.2 blast transplantation as a model system2. Neverthe- Theme: NEW PATHOMECHANISMS OF AMYOTROPHIC less, in order to extend this strategy to human LATERAL SCLEROSIS mesoangioblasts their proliferative capacity needs to be extended to ensure survival after HAC transfer. TW 7.2.1 Genetics of Amyotrophic Here we describe the reversible immortalization of Lateral Sclerosis human mesoangioblasts using lentiviral vectors encod- ing human telomerase and Bmi-1 to bypass replicative Leonard H VAN DEN BERG, Utrecht (Pays-Bas) senescence. Cells have been characterised for prolif- UMC Utrecht, Dept of Neurology, Brain Center eration and transgene expression, remaining non-trans- Rudolf Magnus, The Netherlands S66 Abstracts

ALS is usually described as a progressive disorder ic evidence point to considerable overlap with fronto- of upper- and lower motor neurons leading to muscle temporal dementia (FTD). In recognition of this, weakness and death due to respiratory failure on aver- consensus criteria have been proposed to categorize age 3 years after the onset of symptoms. In clinical the various forms of cognitive and behavioural im- practice, however, we experience a large variability in pairment associated with ALS, although recent data the clinical expression of the disease. An important suggest these criteria will require adjustment to in- gap in our knowledge of ALS pathophysiology is clude defi cits in language, theory of mind and social whether motor neurons in ALS die from a complex cognition. interaction between multiple factors or from the man- A prospective population based study of ALS has ifestation of a unique cause. In other words, is ALS shown that 13% of prevalent patients have full blown one disease or just a phenotype of a large number of FTD, characterized by personality change, irritability, diseases with many causes? Filling this gap in knowl- poor insight and pervasive defi cits on frontal execu- edge may have important consequences for molecular tive tests. Up to 25% of incident patients have evi- diagnostic and therapeutic strategies in ALS and pos- dence of executive impairment, characterized by sibly other complex/neurodegenerative diseases. ALS defi cits in verbal fl uency, judgement and impulse con- could be considered either as a collection of single, trol . Executive impairment tends to progress, and is a unique rare diseases or a diagnostic continuum in strong negative prognostic indicator. Up to 40% of which ALS can be sub-classifi ed according to the rel- ALS patients also develop behavioural impairment, ative contribution of genetic, environmental/lifestyle and a proportion of patients also exhibit defi cits in so- and phenotypic factors. Genetics in ALS may help re- cial cognition and theory of mind. Behavioural im- classify ALS into separate disease entities. A family pairment in the absence of cognitive impairment can history of ALS is obtained in about 5 % but the dis- also occur, and both cognitive and behavioural dys- tinction between familial and apparently sporadic function can precede or follow the onset of motor ALS may be artifi cial and genetic factors play a role symptoms. Up to 40% of patients with ALS remain in all types. For several years, only one gene was cognitively intact when tested longitudinally, indicat- known to have a role in ALS pathogenesis, SOD1. In ing that ALS is a clinically heterogeneous condition. the last few years there has been a rapid advance in Structural and functional imaging of groups of pa- our genetic knowledge of the causes of ALS. Rela- tients with ALS also demonstrate extensive extra-mo- tively common mutations in the TDP-43, FUS and tor involvement, including changes in the deep grey C9orf72 genes, and rare mutations in many more matter. These fi ndings are supported by neuropatho- genes have been identifi ed. In this presentation, I will logical studies, although there is also evolving evi- discuss the genetic architecture of ALS, highlight dence to suggest substantial heterogeneity, indicating some of the genes implicated in pathogenesis, and de- that ALS and ALS-FTD represent a syndrome rather scribe their phenotypic range and overlap with other than a single disease entity. diseases. The recently described hexanucleotide repeat ex- pansion on chromosome 9p21, which occurs in up to 11% of those with ALS , is associated with a distinct THEMATIC WORKSHOP 7.2 clinical , imaging and pathological phenotype with Theme: NEW PATHOMECHANISMS OF AMYOTROPHIC higher rates of cognitive and behavioural impairment LATERAL SCLEROSIS and shorter survival. Those carrying the C9orf72 re- peat expansion are also more likely to have family members with neuropsychiatric conditions including TW 7.2.2 Overlap between Amyotrophic psychosis and a history of suicide. Other Mendelian Lateral Sclerosis and Frontotemporal inherited genes are also associated with an ALS-FTD Dementia phenotype, although it remains unclear as to whether distinct phenotypic characteristics can be discrimi- Orla Hardiman, Dublin (Irlande) nated. Trinity College Dublin, Ireland Future studies will aim to further characterize the Amyotrophic Lateral Sclerosis (ALS) has tradition- neuropsychological, imaging and pathological as- ally been considered a neurodegenerative disease ex- pects of the extra motor features of ALS, and will help clusively involving the motor system. However, to provide a robust classifi cation system that is both recent clinical, imaging neuropathological and genet- clinically and biologically relevant. Abstracts S67

THEMATIC WORKSHOP 7.2 vides a roadmap for developing novel treatments Theme: NEW PATHOMECHANISMS OF AMYOTROPHIC based on specifi c targeting of the genetic mutation. LATERAL SCLEROSIS

TW 7.2.3 Molecular mechanisms in THEMATIC WORKSHOP 7.3 Amyotrophic Lateral Sclerosis Theme: NEW THERAPIES IN METABOLIC MYOPATHIES Kevin TALBOT, Oxford (Royaume Uni) Nuffi eld Department of Clinical Neurosciences, TW 7.3.1 Therapeutic approaches in lipid University of Oxford, UK storage myopathies Antonio TOSCANO, Messina (Italie) Amyotrophic lateral sclerosis (ALS), the common- Department of Neurosciences, University of Messina, est form of degenerative motor neuron disease, is a Italy heterogeneous syndrome with clinical, pathological and genetic overlap with frontotemporal dementia Lipid storage myopathy (LSM) is characterized, (FTD). Arresting the disease process in ALS and other from the pathological point of view, by an abnormally neurodegenerative conditions is currently proving to increased lipid storage in muscle fi bers, due to altera- be challenging because i) patients present with end tions of lipid metabolism pathways. stage disease, ii) we have a rudimentary understand- Nowadays, there are three different types of bio- ing of the initiating biological events in motor neuron chemically determined LSMs: 1) Disorders of fatty- death and iii) our disease models are inadequate. No acid oxidation (FAO), 2) Defects of trygliceride longer viewed as one disease with a single unifi ed metabolism, 3) Defects of trygliceride and membrane cause, ALS can be viewed as resulting from a com- phospholipid biosynthesis. plex convergence of genetic susceptibility, age-relat- In the fi rst group there are: Primary carnitine defi - ed loss of cellular homeostasis, and possible ciency (PCD), Multiple acyl-coenzyme A dehydroge- environmental infl uences. The rapid increase in recent nase defi ciency (MADD), Carnitil-Palmitoyl years of the number of genes in which mutations have Transferase 2 (CPT2) (1) and others, whereas Neutral been associated with ALS has led to in vitro and in lipid storage disease with ichthyosis (CGI-58), and vivo models that have generated a wealth of data indi- Neutral lipid storage disease with myopathy cating disruption of specifi c biochemical pathways (PNPLA2) are in the second one; fi nally, in the third and sub-cellular compartments, including protein group, there is the Recurrent acute rhabdomyolysis in misfolding, mRNA splicing, axonal transport, oxida- childhood (LPIN1) (2,3). tive stress, proteosome and mitochondrial dysfunc- The diagnosis of these disorders is not easy but nec- tion. However, clinical observation of characteristic essary because some of them are treatable conditions regional patterns of involvement and progression, to- and, applying specifi c laboratory tests including mor- gether with electrophysiological and brain-imaging phological, biochemical and molecular genetic analy- studies suggest that ALS is a ‘system degeneration’. ses, it is possible to reach the correct diagnosis. This dichotomy between cellular and systems neuro- From the clinical point of view, the infantile forms biology raises the fundamental questions of what ini- are mainly characterized by cardiomyopathy, liver tiates the disease process and how it is propagated. Is failure, hypoglicemia, metabolic acidosis and delayed the essence of ALS a cell-to-cell transmission of pa- motor milestones whereas the most relevant aspects in thology with, for example, a ‘prion-like’ mechanism, adults are myalgia, recurrent episodes of massive or does the cellular pathology follow degeneration of rhabdomyolysis, exercise intolerance and progressive specifi c synaptic networks? Elucidating the interac- myopathy. tion between cellular degeneration and system level A muscle biopsy with a massive lipidosis could degeneration will aid modeling of the disease in the suggest to expand the laboratory investigations to bet- earliest phases, improve the development of sensitive ter defi ne the different types of lipid disorders (4). markers of disease progression and response to thera- In fact, urinary organic acid profi le, measurement py, and expand our understanding of the biological of blood carnitine and acylcarnitines and a search for basis of clinical and pathological heterogeneity. In some morphological aspects in the smear as Jordan’s particular, the identifi cation of the C9orf72 gene pro- anomaly could properly address to the diagnosis. S68 Abstracts

To confi rm the diagnostic suspect, molecular ge- B, Lin J, Zhu W, Luo S, Zhao C, Li D, Lin P, Lu J, Yan netic analysis is required looking for mutations in al- C. - J Inherit Metab Dis. 2013 Dec 20. [Epub ahead of ready known genes as CPT2, ETFDH, ETFA, ETFB, print]. ABDH5, SLC22A5 and PNPLA2 (5). 6) van de Weijer T, Havekes B, Bilet L, Hoeks J, In this complex situation, the therapeutic approach- Sparks L, Bosma M, Paglialunga S, Jorgensen J, Jans- es (3) suggested for lipid storage myopathies are: sen MC, Schaart G, Sauerwein H, Smeets JL, Wild- berger J, Zechner R, Schrauwen-Hinderling VB, a) Individuals with PCD may show striking im- Hesselink MK, Schrauwen P. Effects of bezafi brate provement with high-dose oral L-carnitine supple- treatment in a patient and a carrier with mutations in mentation (100–300 mg/Kg/day for chronic therapy). the PNPLA2 gene, causing neutral lipid storage dis- b) There is also an increasing evidence indicating ease with myopathy. - Circ Res. 2013 Mar that several cases of MADD, due to ETFDH muta- 1;112(5):e51–4. tions, are Ribofl avin-responsive (100–400 mg/day) with a dramatic improvement of patients clinical con- ditions. In case of incomplete rrcovery, patients could THEMATIC WORKSHOP 7.3 benefi t of an additional CoQ10 supplementation. Theme: NEW THERAPIES IN METABOLIC MYOPATHIES c) It is necessary to raise the attention of patients suggesting avoidance of exacerbating factors as fast- ing, prolonged physical exercise and excessive alco- TW 7.3.2 Therapies in glycogen storage hol ingestion and to maintain a normal carbohydrate myopathies diet in case of infections to reduce lipolysis. Benedikt SCHOSER, Munich (Allemagne) d) Specifi c dietary recommendations for patients Friedrich-Baur-Institut with long-chain, very-long chain fatty acid oxidation Ludwig-Maximilians-Universität München defi ciency or with CPT2 defi ciency. Ziemssenstr. 1a, 80336 Munich, Germany e) In addition, there are a number of new therapeu- tic approaches that are ongoing to improve the oppor- Glycogen storage diseases encompass now more tunities to better treat patients with LSMs (6). than sixteen genetically distinct entities. Recent ad- vances in therapy have re-opened the fi eld of muscle In conclusion, Lipid Storage Myopathies are chal- glycogen storage diseases. The pathogenesis of the lenging diseases but a considerable number of them most common muscle glycogen storage diseases, can be satisfactorily treated, in some cases saving life GSD2/Pompe disease, is more complex than origi- of patients. nally thought, as the primary lysosomal dysfunction is also altered by autophagy and lipofuscin build-up pro- Bibliography cesses. These fi ndings are presumably related to the 1) O. Musumeci, M Aguennouz, G P Comi, C R, M partially failure of enzyme replacement therapy in up Autunno, A Bordoni, S Baratta, F Taroni, G Vita, A to 1/3 of patients with exceedingly long-lasting Toscano Identifi cation of the infant-type R631C mu- Pompe disease. However, with esteemed more than tation in patients with the benign muscular form of 2000 worldwide treated Pompe patients and new up- CPT2 defi ciency. Neuromuscular Disorders, 2007, coming modifi ed enzymes, enzyme replacement ther- 17:960–3. apy is still highly encouraging for diminution of the 2) Bruno C, Dimauro S. Lipid Storage Myopathies individual disease burden of Pompe patients at any Curr Opin Neurol. 2008 Oct;21(5):601–6. age. Chaperones are still under investigation for en- 3) Laforêt P, Vianey-Saban C. Disorders of muscle hancement of enzyme replacement therapy, and quite lipid metabolism: diagnostic and therapeutic chal- recently, drugs like albuterol were tested to booster lenges. Neuromuscul Disord. 2010 Nov;20(11):693– the effi cacy of enzyme replacement therapy. Latest 700. results of gene therapy in muscle glycogen storage 4) Liang WC, Nishino I. Lipid storage Myopathy - diseases open the avenue for the translation into hu- Curr Neurol Neurosci Rep. 2011 Feb;11(1):97–103. man protocols. So, Pompe disease is the role model 5) Clinical features and ETFDH mutation spectrum for all types of glycogen storage diseases and beyond. in a cohort of 90 Chinese patients with late-onset mul- E.g., enzyme replacement therapy might become an tiple acyl-CoA dehydrogenase defi ciency. Xi J, Wen option in other glycogen storage diseases. New and Abstracts S69 old drugs are under investigation in a variety of ani- ly corrects the motor defi cit and biochemical abnor- mal models, e.g. in a canine model of glycogen stor- malities of mice characterized by impaired assembly age disease type 3 and primary muscle cells from of cytochrome c oxidase. Other ongoing strategies are human glycogen storage disease type 3 patients, rapa- based on increasing the expression of antiapoptotic mycin, a specifi c inhibitor of mTOR, has proven to be factors, which leads to remarkable clinical improve- an effective therapy. In polyglucosan body disease/ ment in COX-defective animals; or on the ability of glycogen storage disease type 4, rapamycin and star- mitochondrial aminoacyl-tRNA synthetases to correct vation was proven protective against uncontrolled the functional defect in cybrids carrying mt-tRNA elongation of glycogen chains leading to the forma- mutations. In addition, specifi c conditions, e.g. Ethyl- tion of an insoluble form of glycogen, the polygluco- malonic Encephalopathy and MNGIE, characterized san bodies. Thus, the translation for experimental by accumulation of toxic substances derived from mi- fi ndings are on the run. Finally, exercise therapy and tochondrial dysfunction have been effectively treated nutritional aspects are under investigation in more de- in mouse models by AAV-mediated gene targeting to tail for their supplementary treatment impact in a va- the liver. Taken together, our results suggest that a riety of muscle glycogen storage diseases. spectrum of different approaches are effective in ex- Disclosures: BS received advisory board and perimental mitochondrial dysfunction. Some of these speaker honoraria from Genzyme, a Sanofi company can be rapidly transferred to clinical trials in humans. and Biomarin Pharmaceutical Inc.

THEMATIC WORKSHOP 7.4 THEMATIC WORKSHOP 7.3 Theme: PARANEOPLASTIC PERIPHERAL NEUROPATHY Theme: NEW THERAPIES IN METABOLIC MYOPATHIES TW 7.4.1 Paraneoplastic sensory TW 7.3.3 Experimental therapy in neuropathies: clinical and mitochondrial disorders pathophysiological aspects Massimo ZEVIANI, Cambridge (Royaume Uni) Jean-Christophe ANTOINE, Saint-Etienne (France) MRC-Mitochondrial Biology Unit, Cambridge, UK Department of Neurology and Reference Center for Neuromuscular Diseases, University Hospital of The genetic and biochemical intricacy of mitochon- Saint-Etienne, France drial bioenergetics explains in part the extreme het- erogeneity of mitochondrial disorders, a formidable Paraneoplastic sensory neuronopathy belongs to a challenge for both diagnostic workup and treatment. group of infl ammatory disorders affecting the dorsal Although energy failure associated with reduced ATP root ganglia. It is one of the classical paraneoplastic biosynthesis appears to be critical in individual or neurological syndromes and most patients have anti- combined MRC-complex defects, other mechanisms Hu antibodies and small cell lung carcinoma. Cases are also likely to be involved, and are probably pre- with other intracellular onconeural antibodies includ- dominant in the pathogenesis of specifi c syndromes, ing anti-CV2/CRMP5 and anti-amphyphisin antibod- such as alterations of cellular reduction-oxidation sta- ies have been reported and seronegative cases or cases tus, production of reactive oxygen species (ROS), with other cancers may occur. Although immunoglob- compromised Ca2+ homeostasis, and altered mito- ulin deposits containing anti-Hu antibodies can be chondrial dynamics, autophagy or apoptosis. We have found around sensory neurons, the disorder is proba- started a long-term program on the development of bly mediated by cytotoxic T-cells. SNN is the most experimental therapy in recombinant mouse models frequent paraneoplastic neurological syndrome but it with mitochondrial dysfunction and in mutant cy- represents only a small proportion of patients with brids. A fi rst strategy is centered on the activation of sensory neuronopathy. Identifying these patients early the mitochondrial biogenetic program by AMPK or in the course of their disease is important since it may SIRT1 agonists. Both AMPK and SIRT1 induce ex- lead to an early diagnosis of the tumor which is prob- pression of energy genes, partly by targeting the tran- ably the best way of stabilizing the neuropathy and scriptional coactivator PGC-1α. We found that improving the tumor prognosis. Other paraneoplastic pharmacological stimulation of AMPK or SIRT1 part- sensory neuropathies involve antibodies toward pe- S70 Abstracts ripheral nerve membrane antigens and include neu- with, most often mild, autonomic dysfunction, which ropathies with Waldenström’s disease and anti-MAG is not seen in AChR MG. In LEMS associated with monoclonal IgM and small fi ber involvements in pa- SCLC the symptoms of the disease develops much tients with anti-CASPR2 antibodies, Morvan’s syn- faster in a shorter period of time than in the non-tu- drome and thymoma. Contrarily to antibodies directed mour variant. The DELTA-P score can help to predict toward intracellular antigens, antibodies recognizing the presence of an underlying SCLC at the fi rst visit of cell surface antigens are probably pathogenic. the patient. It is an easy score based purely on clinical fi ndings. Treatment of non-tumour LEMS consists of symp- THEMATIC WORKSHOP 7.4 tomatic treatment with 3,4-diaminopyridine or the use Theme: PARANEOPLASTIC PERIPHERAL NEUROPATHY of prednisolone and immunosuppressive agents for more severely affected patients, while patients with LEMS and SCLC are treated by chemotherapy. A de- TW 7.4.2 Update on Lambert-Eaton tailed analysis of the clinical signs and symptoms is myasthenic syndrome very helpful in distinguishing the different forms of myasthenia. Jan VERSCHUUREN, Leiden (Pays-Bas) Dept. Neurology, Leiden University Medical Center, Leiden, The Netherlands THEMATIC WORKSHOP 7.4 Autoimmune disorders of the neuromuscular syn- Theme: PARANEOPLASTIC PERIPHERAL NEUROPATHY apse are associated with different well defi ned anti- bodies, including the acetylcholine receptor (AChR), TW 7.4.3 Paraneoplastic dysautonomia muscle-specifi c kinase (MuSK), low-density lipopro- tein receptor-related protein 4 (Lrp4), or voltage-gat- (from the PNS-Euronetwork Database) ed calcium channels (VGCC). More than 80% of Bruno GIOMETTO, Treviso (Italie) these patients have AChR antibodies (AChR MG) Dept. of Neurology, Ospedale Ca’ Foncello, Azienda with a reported incidence varying between 2 and 20 ULSS9 TREVISO (Italy) per million inhabitants per year, and a prevalence of 1–2 per 10.000 persons. The prevalence of the Lam- Introduction: In paraneoplastic syndromes of the bert-Eaton myasthenic syndrome (LEMS) and the peripheral nervous system (PNS), sensory ganglia are other forms of myasthenia are about 20 times more often targeted by the immunological attack. rare. Subacute sensory neuronopathy (SSN) is a classical Both LEMS and AChR MG have a tumour and non- paraneoplastic syndrome. There is a clear predomi- tumour associated form, where LEMS is associated nance of SSN with well-established clinical features with small cell lung cancer (SCLC) and AChR MG and oncological and immunological associations. with thymoma. The non-tumour related forms of these Dysautonomia can occur as an isolated paraneo- disorders show a bimodal distribution in their relation plastic peripheral neuropathy and is also a classical to sex and age, with the age of 40–50 years as a cut- syndrome. The lack of extensive clinical and epide- off point. Below this age predominantly female pa- miologic studies on these rare disorders prompted es- tients, with a high prevalence of HLA DR3-B8 are tablishment of the PNS Euronetwork by 20 consortium affected, while above this age the majority of the pa- members from 11 European countries. The partner- tients is male with no clear HLA association. Both ship collected information on 979 patients, resulting LEMS and AChR MG are caused by IgG1 or IgG3 in the largest database on PNS to date (Giometto et al, antibodies. LEMS starts almost exclusively with 2010). In this study we report data related to the pa- proximal weakness of the legs, and with disease pro- tients with paraneoplastic dysautonomia. gression weakness extends in a caudal-cranial direc- Results: 33 patients (31 adults and 2 children) with tion. AChR-MG classically starts with ptosis and dysautonomic paraneoplastic neurological syndromes diplopia, followed by generalized, predominantly (dPNS) were recruited (6% of the total study popula- proximal weakness. A characteristic ptosis with oph- tion) from 5 centres. All were defi nite PNS. Most pa- thalmoplegia is rare in LEMS, which makes this dis- tients had an additional PNS, most frequently a order more diffi cult to diagnose. LEMS is associated peripheral, usually sensory, neuropathy. Dysautono- mia was the sole PNS in only 27% of patients. Chron- Abstracts S71 ic pseudo-obstruction was the commonest syndrome Mutations in Amphiphysin-2/BIN1 are associated (73%) . with autosomal recessive All of the 31 adults had serum onconeural antibod- (ARCNM), a muscle disorder characterized by myofi - ies 80% of which were against Hu protein. 76% of the bers with centrally positioned myonuclei, defects in patients had detectable tumours, usually SCLC or T-tubules and abnormal triads. How Amphiphysin-2 NSCLC. Of the 25 Hu antibody-positive adult pa- orchestrates T-tubule formation and ARCNM-associ- tients, 19 had tumours (13 SCLC, 3 NSCLC, 1 liver ated mutations lead to muscle morphological defects carcinoma, and 1 unknown primary) leaving 7 in remains elusive. We developed an in vitro system whom no tumour was found after follow up of up to 8 where myotubes mature into myofi bers with periph- years. CSF was analysed from 12 patients. 10/12 had eral myonuclei, myofi brils, T-tubules and triads. We a cell count of 10 or higher and 9/12 had a total pro- established the main molecular mechanisms involved tein of > 0.5 g/l. 2/4 CSF had oligoclonal bands. in the positioning of myonuclei and triad formation. 5 patients were lost to follow up. Of the other 27 We found that Amphiphysin-2 interacts with N-WASP patients, 77% died: 55% from their dPNS, 15% from in newly formed myofi bers as well as in adult muscle their tumour, 1 from sepsis, and 1 during mediastinos- and that this interaction is disrupted by Amphiphy- copy. Average time from onset to death from PNS was sin-2 ARCNM mutations. We established that N- 8 months (range 1 – 24 months). dPNS improved in WASP and Amphiphysin-2 are required for nuclear 2/14 patients after tumour treatment. Both of these pa- positioning, T-tubule biogenesis and triad formation, tients had NSCLC. dPNS did not improve in any of but not myofi brillogenesis. We also demonstrated that the 13 patients given immunotherapy. N-WASP and Amphiphysin-2 are involved in the Conclusions: We suspect that dysautonomic PNS is maintenance of triads in adult myofi bers. Finally, we underdiagnosed as only 5/19 of our Centres reported showed that N-WASP distribution is disrupted in cases. In addition, the majority of cases (73%) devel- muscle from ARCNM, ADCNM patients and more oped in the setting of at least one other PNS, in par- slightly in XLCNM and DM1 patients. Our results ticular Subacute Sensory Neuronopathy, which we support a role for N-WASP in Amphiphysin-2-depen- speculate may dominate the presentation and mask dent nuclear positioning, T-tubule biogenesis and tri- the autonomic features. Dysautonomic PNS was most ad formation in skeletal muscle formation and in strongly associated with lung tumours, especially CNM pathophysiology. SCLC, and Hu antibodies. It improved only rarely with tumour treatment and none of our 13 patients im- proved with immunotherapy. Prognosis was poor. THEMATIC WORKSHOP 7.5 When dysautonomia developed, 77% died from their Theme: INTERNAL STRUCTURE PNS, all within 2 years of onset.

No disclosure TW 7.5.2 Molecular basis of sarcoplasmic reticulum organization in skeletal muscle Reference fi bers Bruno Giometto, Wolfgang Grisold, Roberta Vi- taliani, et al. Arch Neurol. 2010;67(3):330–335. Vincenzo SORRENTINO, Siena (Italie) Department of Molecular Medicine and Development, University of Siena, 53100 Siena, Italy

THEMATIC WORKSHOP 7.5 The sarcoplasmic reticulum (SR) is a specialized Theme: INTERNAL MUSCLE CELL STRUCTURE form of endoplasmic reticulum that plays a key role in the regulation of muscle contraction by controlling re- TW 7.5.1 Formation and maintenance of lease of Ca2+ following membrane depolarization in a triads during development and process named excitation-contraction (e-c) coupling. In skeletal muscle, e-c coupling occurs at specialized pathological conditions intracellular junctions, named triads, where two termi- Edgar GOMES, Paris (France) nal cisternae of the SR are positioned to fl ank, on op- Myology Center of Research, UMRS 974 UPMC - posite sites, a transverse (T)-tubule. The terminal INSERM, Paris, France cisternae membrane that faces the T-tubule represents S72 Abstracts the junctional SR. This is the SR region where the ry- Spontaneous and depolarization-induccd calcium anodine receptors Ca2+ release channels and addition- releases during excitation- contraction coupling of al proteins, including triadin, junctin and calsequestrin, skeletal muscle fi bres occur at complex sites called assemble into a large multi-protein complex. The do- calcium release units (CRUs). The composition, mains where this machinery assembles are also re- structure and positioning of CRUs in adult muscle are ferred to as Calcium Release Units (CRUs). The stereotyped, although subject to species and fi ber- junctional SR is selectively aligned with the junction type dependent variations. The units are composed of between the A and the I bands of the sarcomere and is a highly complex macromolecular complex involving in direct connection with the longitudinal SR, a large direct molecular interactions between a membrane network of tubules that are interconnected one with component of the transverse tubules (the voltage sens- each other in forming a network that surrounds indi- ing CaV1.1 channels or dihydropyridine receptors, vidually each single myofi bril and that covers both the arranged in tetrads associated with the four subunits A and the I bands of the sarcomere. Regardless of the of RyR1); the calcium release channels of the sarco- distinction in junctional and longitudinal domains, tu- plasmic reticulum, SR (ryanodine receptors, RyRi and bules and cisternae of the SR share a continuous lumen RyR3); other SR membrane proteins (triadin and delimited by a single continuous membrane, thus cor- junctin); the luminal protein calsequestrin; and the responding to a single, though very large, organelle. docking protein junctophilin. CRUs have specifi c po- It would then appear that, to effi ciently position the sitions relative to the cross striations of the myofi brils. Ca2+ store close to the contractile apparatus, the en- A basic if simplifi ed understanding of these arrange- tire SR is organized to surround each individual myo- ments and how they develop is obtained from ultra- fi bril and that proteins of the CRUs are precisely structural observations of differentiating cells in vitro targeted to the junctional SR. In the recent years we and in vivo and from observations of null mutations. have started to identify and characterize some of the A) Positional clues from differentiation: 1) the initial proteins that contribute to stabilize the SR around the position of transverse (T) tubules is longitudinal; 2) myofi brils and to dissect the complex molecular initial CRU complexes are also longitudinal, but spe- mechanisms that target and organize some of the pro- cifi cally located relative to the myofi brils; 3) the shifts teins of the CRU at the junctional SR. Current knowl- from longitudinal to transverse orientations of T tu- edge on these issues will be presented. bules and triads are coordinated events. B) Composi- tion clues from development: 1) CRU differentiate 1. Rossi et al., Biochem J. 2014 458 (part 2), Pages through a hierarchical sequence from peripheral cou- 407–417. plings to longitudinal dyads/triads to transverse triads 2. Randazzo et al., J Cell Biol. 2013 Feb while acquiring their components; 2) early peripheral 18;200(4):523–36. couplings do not have Cav1.1 or RyR. Hence an inde- 3. Golini L et al., J Biol Chem. 2011 Dec pendent docking protein (identifi ed as junctophilin by 23;286(51):43717–25. H. Takeshima) is required; 3) calsequestrin, RyR and 4. Cusimano et al., Proc Natl Acad Sci U S A. 2009 Cav1.1 are acquired in sequence. C) Clues from null Mar 24;106(12):4695–700. mutations: 1) RyR1 are targeted to CRUs and form 5. Rossi et al., Traffi c. 2008 Jul;9(7):1044–9. arrays in the absence of CaV1.1 (in dysgenic mice); 2) 6. Bagnato et al., J Cell Biol. 2003 Jan CaV1.1 are targeted to CRUs but do not form tetrads 20;160(2):245–53. in the absence of RyR1 (in dyspedic mice); 3) triadin is appropriately targeted to CRUs in dyspedic muscle in vitro and in vivo; 4) RyR1 and RyR3 are targeted to THEMATIC WORKSHOP 7.5 CRUs independently of each other when expressed in Theme: INTERNAL MUSCLE CELL STRUCTURE a dyspedic cell line, but the latter do not associate with triadin, they do not restore skeletal type e-c cou- pling and do not induce tetrad assembly by CaV1.1; TW 7.5.3 The molecular basis of the 5) when coexpressed in vivo RyR1 and RyR3 assume stereotyped muscle organization different position (junctional versus parajunctional) within the triads. 6) calsequestrin is targeted to and CLARA FRANZINI-ARMSTRONG, Philadelphia retained in CRUs in the absence of RyRs; 7) a null (Etats-Unis) mutation for triadin ablates the visible CASQ anchors Dept. of Cell and Developmental Biology, University to the jSR membrane. of Pennsylvania, Philadelphia, PA, USA Abstracts S73

Supported by NIH grant HL 48093. uated in Mybpc3-targeted knock-in (KI) mice that Slides: Triads 3 D drawing: the macromolecular carry a homozygous G>A transition in exon 6, result- complex and some of its constituents: RyR. DHPR ing in 3 different aberrant mRNAs and proteins. An tetrads, CASQ, triadin-junctin alternative variant (Var-4) deleted of exons 5–6 was The stereotyoed positioning of triads: at the Z line also identifi ed in wild-type (WT) and KI mice. (zfi sh?) or A-I junction (mammals) 5’-trans-splicing was induced between the endoge- Show feet, tetrads and CASQ network nous mutant Mypbc3 pre-mRNA and an engineered Figurefrom Barine…SORRENTINO TYPE 1 AND pre-trans-splicing molecule (PTM) carrying a WT- TYPE 3 NULL Mybpc3 cDNA sequence. PTMs were packaged into Clues from development: adeno-associated virus (AAV) for specifi c transduc- T tubule are initially longoitudinal tion of cultured cardiac myocytes and the heart in Longituinal Triads are at the A-I junction BEFORE vivo. Full-length repaired Mybpc3 mRNA represent- T tubules are transverse. ed 33% and 0.15% of total Mybpc3 transcripts in car- PRESUMABY SR assocaitin owht myofi brils diac myocytes and in the heart, respectively. Repaired f0drags the T tubules transversely cMyBP-C protein was detected by immunoprecipita- Z fi sh at 48 hrs versus mouse or chicken at severa tion in cells and in vivo. Exon-skipping enhancing weeks. expression of Var-4 mRNA was induced by AON-5 A hierarchical sequence from peripheral couplings and AON-6 that mask exonic splicing enhancer motifs to longitudinal triads to transverse triads. in exons 5 and 6. AONs were inserted into modifi ed U7snRNA and packaged in AAV. Transduction of car- diac myocytes or systemic administration in newborn THEMATIC WORKSHOP 8.1 KI mice markedly increased Var-4 mRNA and pro- Theme: THERAPEUTIC APPROACH TARGETING THE HEART tein, reduced aberrant mRNAs, and rescued the car- IN NEUROMUSCULAR DISEASES diac phenotype in mice. We provide the fi rst evidence of successful 5’-trans-splicing in vivo and of cardiac functional rescue by exon skipping. These fi ndings TW 8.1.1 RNA-based or gene-based open new horizons for causal therapy of severe forms therapies of genetic diseases with cardiac involvement. Lucie CARRIER, Hamburg (Allemagne) Department of Experimental Pharmacology and References Toxicology, University Medical Center Hamburg- Gedicke-Hornung*, Behrens-Gawlik* et al., Eppendorf, Hamburg, Germany EMBO Mol Med 2013. Mearini*, Stimpel* et al., Mol Therapy – Nucl Ac- An overview of gene therapies for heart failure will ids 2013. be fi rst presented. Then, I will focus on RNA-based Behrens-Gawlik et al., Pfl ügers Arch – Eur J Physi- therapeutics, such as exon-skipping using antisense ol 2014. oligoribonucleotides (AONs) or RNA trans-splicing that we recently applied for hypertrophic cardiomy- opathy (HCM). HCM is an autosomal-dominant dis- THEMATIC WORKSHOP 8.1 order and the leading cause of sudden cardiac death in Theme: THERAPEUTIC APPROACH TARGETING THE HEART young athletes. It is mainly characterized by left ven- IN NEUROMUSCULAR DISEASES tricular hypertrophy (LVH), diastolic dysfunction and increased interstitial fi brosis. HCM is often caused by TW 8.1.2 Prevention and treatment of mutations in MYBPC3, encoding cardiac myosin- binding protein C (cMyBP-C). Most MYBPC3 muta- heart involvement in muscle diseases tions result in truncated proteins. Findings in humans Denis Duboc, Henri Marc Becane, Rabba Ben Yaou, and in cat/mouse models indicate that haploinsuffi - Bruno Eymard, Pascal Laforet, Anthony Béhin , ciency is the most prevalent HCM mechanism. More- Tania Stojkovic, Karim Wahbi over, marked reduction of cMyBP-C results in LVH Cardiology, Hospital Cochin and Myology Institute, and dysfunction in mice and in severe neonatal, lethal Hospital de la Salpétrière, Paris, France forms in humans. RNA-based therapeutics were eval- S74 Abstracts

Many muscle diseases are concerned by cardiovas- consequences of pathological cardiac stress, confer- cular complications. ring clinical benefi t to the millions of patients with The most dramatic are cardiac sudden death and heart failure. heart failure. But atrial fi brillation with the risk of sys- temic emboli and the venous thrombo embolism pa- thology take an important place in the morbidity of THEMATIC WORKSHOP 8.2 these diseases. Theme: TREAT NMD ALLIANCE: OMICS TECHNOLOGIES In this presentation we will show the specifi cities of FOR TRANSLATIONAL RESEARCH IN NEUROMUSCULAR these complications and propose some guidelines to DISORDERS prevent them, and particularly to prevent the risk of sudden death in DM1 and in laminopathies and to TW 8.2.1 Genomics and gene discovery in limit the progression of left ventricular dysfunction in dystrophinopathies. hereditary neuropathies Jan SENDEREK, Munich (Allemagne) Friedrich-Baur-Institut, Department of Neurology, THEMATIC WORKSHOP 8.1 Ludwig Maximilians University, 80336 Munich, Theme: THERAPEUTIC APPROACH TARGETING THE HEART Germany IN NEUROMUSCULAR DISEASES Peripheral neuropathy is one of the most common referrals to neurologic clinics. Most cases are ac- TW 8.1.3 Targeting HDACs (histone quired (due to diabetes mellitus, alcoholism or auto- deacetylase) in cardiovascular disease immune or infectious diseases), however, several Joseph A HILL, Dallas (Etats-Unis) genetic forms do exist as well. Mutations in more than University of Texas Southwestern Medical Center 50 genes affecting diverse glial or neuronal functions have been associated with different forms of heredi- Heart failure, a syndrome culminating the patho- tary neuropathies. This has led to a substantial im- genesis of many forms of heart disease, is highly provement in diagnostics of the disease and in the prevalent and projected to be increasingly so for years understanding of implicated pathophysiological to come. Major efforts are directed at identifying mechanisms. As for other Mendelian conditions, gene means of preventing, slowing, or possibly reversing discovery in hereditary neuropathies has been largely the unremitting progression of pathological stress fostered by the decoding of the complete human ge- leading to myocardial injury and ultimately heart fail- nome at the turn of the Millennium. More recently, ure. Indeed, despite widespread use of evidence-based highly-parallel genetic technologies (e.g., whole therapies, heart failure morbidity and mortality re- exome sequencing and whole genome sequencing) main high. Recent work has uncovered a fundamental have been adapted for the study of inherited neuropa- role of reversible protein acetylation in the regulation thies. Application of these technologies has enabled of many biological processes, including pathological researchers and clinicians to identify numerous new remodeling of the heart. This reversible acetylation is neuropathy genes and to recognize previously unpre- governed by enzymes that attach (histone acetyltrans- dicted genotype-phenotype relationships. It has also ferases, HAT) or remove (histone deacetylases, been proposed that hereditary neuropathies may oc- HDACs) acetyl groups. In the case of the latter, small casionally be inherited in a more complex fashion, as molecule inhibitors of HDACs are currently being an oligogenic disorder. A number of patients has been tested for a variety of oncological indications. Now, described who carried mutations at more than one lo- evidence has revealed that HDAC inhibitors blunt cus, but at present the extent to which possible oligo- pathological cardiac remodeling in the settings of genic inheritance accounts for the phenotype is pressure overload and ischemia/reperfusion, dimin- unknown. In addition to providing an overview of ge- ishing the emergence of heart failure. Mechanistically, netic and functional data concerning various neuropa- HDAC inhibitors reduce stress-induced cardiomyo- thy forms, this review focuses on the current and cyte death, hypertrophy, and ventricular fi brosis. future impact of advances in DNA sequencing on Looking to the future, HDAC inhibitor therapy may gene discovery and diagnostics of hereditary neuropa- emerge as a novel means of arresting the untoward thies. Abstracts S75

THEMATIC WORKSHOP 8.2 Our proteomic data provide important new insights Theme: TREAT NMD ALLIANCE: OMICS TECHNOLOGIES into the composition of pathological protein aggre- FOR TRANSLATIONAL RESEARCH IN NEUROMUSCULAR gates in MFM and expand our knowledge about pro- DISORDERS teins and pathways that seem to be involved in pathogenesis. The identifi cation of specifi c proteomic TW 8.2.2 Proteomics and profi les in different MFM subtypes and the detection pathophysiological signatures in of mutations directly on the protein level can be use- ful in differential diagnosis of protein aggregation myofi brillar myopathies myopathies. RUDOLF A KLEY, Bochum (Allemagne) Department of Neurology, Neuromuscular Centre Ruhrgebiet, University Hospital Bergmannsheil, THEMATIC WORKSHOP 8.2 Ruhr-University Bochum, Germany Theme: TREAT NMD ALLIANCE: OMICS TECHNOLOGIES FOR TRANSLATIONAL RESEARCH IN NEUROMUSCULAR Myofi brillar myopathies (MFM) are a group of usu- DISORDERS ally autosomal dominant inherited muscle disorders characterized by focal disintegration of myofi brils TW 8.2.3 Biomarker discovery in and by the formation of intramyoplasmic protein ag- gregates. Known diseases genes encode proteins that Duchenne Muscular Dystrophy are located at or associated with the Z-disc. We ap- Annemieke AARTSMA-RUS, Leiden (Pays-Bas) plied a highly sensitive proteomic approach to deci- 1Department of Human Genetics, Leiden University pher the composition of pathological protein Medical Center, Leiden, The Netherlands aggregates in different MFM subtypes. The aim was 2Center for Proteomics & Metabolomics, Leiden to identify novel disease-relevant proteins and sub- University Medical Center, Leiden, The Netherlands type-specifi c proteomic profi les. 3Institute of Genetic Medicine, Newcastle University, Skeletal muscle samples from 67 MFM patients Newcastle upon Tyne, UK were included in this study. Aggregate samples and intraindividual control samples (from normally look- The lack of non-invasive biomarkers to be used as ing muscle fi bers) were collected from 10μm muscle surrogate endpoints in Duchenne muscular dystrophy sections by laser microdissection and analyzed by a (DMD) patients is one of the unmet needs in clinical label-free mass spectrometric approach for identifi ca- practice. The availability of objective measures able tion and relative quantifi cation of proteins. We detect- to correlate with or being predictive of clinical benefi t ed 4470 different proteins in the samples and 287 of could be used as secondary clinical endpoints, thus these showed a statistically signifi cant accumulation reducing the time and money needed to map response in aggregate samples with a ratio >1.5 compared to to treatment while speeding up drug approval. controls (mean ratio 4.5). Z-disc and Z-disc-associat- We have set up a multi-step discovery pipeline to ed proteins, especially desmin, fi lamin C and their identify proteins and metabolites present in body fl u- binding partners, constituted the most abundant group ids (serum, plasma and urine) associated with DMD of over-represented aggregate proteins followed by pathology. Candidates are identifi ed with high- proteins involved in protein quality control and pro- throughput proteomic and metabolomics technologies tein degradation, extracellular and sarcolemmal pro- in cross-sectional studies (cases vs controls); the most teins, components of signaling pathways and promising candidates are fi rst validated with an inde- regulators of myofi brillar organization. Subgroup pendent assay and then tested in 2 independent patient analysis revealed a characteristic basic pattern of ag- cohorts. Multivariate analysis is then used to identify gregate composition but also signifi cant differences biomarkers correlating with clinical scores. As an ex- regarding the accumulation ratio, order and propor- tra validation layer, candidate biomarkers are studied tion of individual proteins that enabled the defi nition in 2 large longitudinal cohorts. of subtype-specifi c proteomic profi les. In addition, Matrix Metalloproteinase-9 (MMP-9) is our most some disease-causing mutations could be detected di- advanced candidate. Levels of MMP-9 were mea- rectly on the protein level. sured in serum of 116 patients followed up in New- castle (NCL), Leiden (LUMC) and London (UCL). MMP-9 levels were higher in DMD patients com- S76 Abstracts pared to age-matched healthy controls. A strong sig- Limb girdle muscular dystrophies, inherited by a nifi cant correlation was present between serum and recessive trait, encompass a group of muscle diseases plasma MMP-9 levels in DMD patients. Elevated that originally were lumped together according to cri- MMP-9 levels were confi rmed by gelatin zymogra- teria of 1) proximal weakness and atrophy, 2) dystro- phy, showing a strong correlation between the results phic appearance of muscle tissue, and 3) elevated obtained with the ELISA and with gelatin zymogra- creatine kinase levels. Many patients in this category phy. Longitudinal data from 66 DMD patients fol- can have prominent involvement of distal muscle lowed up in Leiden and Newcastle for over 5 years groups as well (for instance dysferlinopathies and (168 samples) showed that MMP-9 serum levels sig- anoctamine 5 defi ciency), yet still are designated a nifi cantly increase over time. Hence, MMP-9 is a limb girdle muscular dystrophy. Conversely, patients good candidate biomarker to monitor disease progres- who are not coined as a limb girdle muscular dystro- sion in DMD patients. These data are supportive to phy can have severe proximal atrophy and weakness. evaluate the potential of MMP-9 as a biomarker to A number of these have a clear underlying pathology, determine the early response to treatments. which is very much unlike a muscular dystrophy (for Disclosures: AAR discloses being employed by instance, polymyositis and some muscle glycogenosis LUMC which has patent applications on exon skip- and endocrine myopathies). However, several disease ping. As co-inventor on some of these patients, AAR entities within the muscular dystrophy domain also is entitled to a share of potential royalties. can present with pronounced proximal affection of muscles, yet are not classifi ed as limb girdle muscular dystrophy. Examples of that are several types of late- THEMATIC WORKSHOP 8.3 onset forms of congenital muscular dystrophies, myo- Theme: DIAGNOSTIC BOUNDARIES OF LIMB GIRDLE fi brillar myopathies and facioscapulohumeral MUSCULAR DYSTROPHIES muscular dystrophy. The symposium and current presentation will discuss these overlapping phenotypes, and make suggestions TW 8.3.1 Proximal weakness and for some adjustments as to how we classify muscular atrophy: is this a limb girdle muscular dystrophies based on their clinical appearance. dystrophy? John VISSING, Copenhagen (Danemark) Neuromuscular Research Unit, Department of THEMATIC WORKSHOP 8.3 Neurology, Rigshospitalet, University of Theme: DIAGNOSTIC BOUNDARIES OF LIMB GIRDLE Copenhagen, Denmark MUSCULAR DYSTROPHIES

With the advent of progress in unravelling the mo- TW 8.3.2 Overlap of phenotypes in lecular background of a great variety of muscle dis- dominantly inherited limb girdle eases, it has become increasingly clear that what was believed to be a single disease with a clear phenotype muscular dystrophies vs. other can have multiple genetic backgrounds. The opposite dystrophies is often also the case, i.e. that distinct aberrations in Kate BUSHBY, Newcastle (Royaume Uni) single genes may show a wide range of phenotypes. MRC Centre for Neuromuscular Diseases at Thus, classical lumping of disease entities according Newcastle, Institute of Genetic Medicine, to phenotypic presentation has been challenged by International Centre for Life, Newcastle University molecular genetics that often split such entities. One NE1 3BZ could then argue that the current classifi cation system of inherited muscle diseases could be replaced by a The original classifi cation of the dominantly inher- classifi cation that relies exclusively on the molecular ited limb girdle muscular dystrophies has been the genetic defect. However, lumping diseases according basis for the identifi cation of a number of different to phenotype has many advantages, because clinical disease genes. Within this group of disorders however appearance is what the clinicians look at the fi rst time it is abundantly clear that phenotypic and genetic het- he/she sees the patient, and therefore is a pivotal entry erogeneity is the norm, challenging the basis on which port for onward diagnostic strategy. we make these diagnoses and extending the pheno- types seen in association with mutations in specifi c Abstracts S77 genes. This is especially true for the disorders over- which are common causes for adult onset LGMD; and lapping genetically with the myofi brillar myopathies, also for LMNA mutations, a nuclear envelope protein where several disease genes now have been reported more frequently affected in an adult onset muscular in association with an “LGMD” presentation as well dystrophy but also now described in CMD. as the more typical mixed proximal/ distal presenta- In my presentation I will describe our experience so tion. This is likely to become even more the case as far with the diagnosis of these conditions and provide patients are no longer diagnosed by a targeted gene some insight on when to suspect them. approach but by next generation sequencing. Disclosure. The Author receives funding from the The way that this impacts on our classifi cation of Muscular Dystrophy Campaign UK; the EU Neu- these diseases but even more importantly on the infor- romics research program; the NIH and the UK Na- mation that we can give to patients to help with man- tional National Health System for his work on agement and prognosis will be discussed. congenital muscular dystrophies.

THEMATIC WORKSHOP 8.3 THEMATIC WORKSHOP 8.4 Theme: DIAGNOSTIC BOUNDARIES OF LIMB GIRDLE Theme: ARTHROGRYPOSIS MUSCULAR DYSTROPHIES TW 8.4.1 Arthrogryposis a general TW 8.3.3 Phenotypic transitions from overview and syndromes congenital to limb girdle muscular dystrophies Mar TULINIUS, Gothenburg (Suède) Department of Pediatrics, University of Gothenburg, Francesco MUNTONI, London (Royaume Uni) The Queen Silvia Children’s Hospital, Gothenburg, Dubowitz Neuromuscular Centre, Sweden UCL Institute of Child Health & Great Ormond Street Hospital for Children, London, UK Arthrogryposis, or arthrogryposis multiplex con- genita, is a descriptive term for the presence of mul- Congenital muscular dystrophies (CMD) are de- tiple congenital contractures at birth. The etiology is fi ned as conditions with onset at birth or within the heterogeneous and more than 200 conditions are fi rst 6 months of life. The last 20 years have seen a known in which multiple congenital contractures are a dramatic improvement of the understanding of the predominant sign. The birth prevalence in western molecular basis of these conditions with now more Sweden was found to be 1 in 5100 live births (Darin than 20 molecularly defi ned variant, mostly related to et al 2002) and has been estimated to be between 1 in mutations in genes encoding for extracellular matrix 3000 to 1 in 12000 live births in different population- proteins or proteins with defi nitive or suspected gly- based studies. cosyltransferase function; or structural components of All forms of arthrogryposis are associated with de- the nuclear envelope. Several of these proteins have a creased movements in utero and can be classifi ed from clear role in allowing adequate muscle formation / a clinical point of view into three categories: involve- satellite cell function, providing an explanation for ment of the limbs only, limb involvement with other the prenatal onset of muscle pathology, abnormalities, or limb involvement with major central The availability of robust genetic testing has al- nervous system dysfunction. Arthrogryposis can also lowed to explore the boundaries of the phenotypic be classifi ed from an etiologic point of view into dis- spectrum of these conditions, allowing to identify in orders of the developing motor system (brain, spinal several instance the occurrence of milder, late onset cord, neuromuscular system or connective tissue) or phenotypes which are for all purposes limb girdle to abnormalities in the environment of the fetus. muscular dystrophy (LGMD) variants. The most common cause of arthrogryposis is amyo- This is clearly the case both for protein of the extra- plasia representing 1/3 to 1/4 of all affected individu- cellular matrix such as laminin alpha2, responsible als (Kroksmark et al 2006, Hall et 2014), followed by both for a severe CMD but also a –so far not frequent- dysfunction of the central nervous system and distal ly recognized- LGMD form; mutations in the glycos- arthrogryposes syndromes. Amyoplasia and the distal yltransferases, with a few of the mutant genes (such as arthrogryposes syndromes will be dealt with by other FKRP or ISPD, but more recently also GMPPB) speakers in this workshop. The patients with central S78 Abstracts nervous system dysfunction and arthrogryposis were tures are at their maximum at birth, and are usually more severe compared to other subgroups of patients not progressive. In ¾ of all cases there are joint con- with arthrogryposis in terms of joint contractures at tractures symmetrically in all four limbs. Involvement birth, feeding diffi culties during infancy, onset of in- of only upper limbs or only lower limbs occurs, as dependent walking and mortality. Mental retardation well as asymmetrical involvement. There may also be is common in this subgroup. The aim of this presenta- involvement of the jaws and spine. Typical joint posi- tion is to give a general overview on arthrogryposis, tions at birth are inward rotation and adduction of the to discuss the group with major central nervous sys- shoulders, extended elbows, fl exed wrists and fi ngers, tem involvement and other syndromatic causes, such varying contractures in lower limbs with external ro- as fetal akinesia deformation sequence and pre- and tation and abduction of the hips with fl exion in the perinatal disorders of neuromuscular transmission as- knees or fl exion of the hips with extended or fl exed sociated with arthrogryposis. knees and bilateral club feet. The face often has a No disclosure. rounded appearance. There is often a midline facial hemangioma and also dimples over affected joints. In References: about 10% of all cases abdominal wall defects are Darin et al. Multiple congenital contractures: Birth also present at birth. Children with amyoplasia usual- prevalence, etiology and outcome. J Pediatr ly have no intellectual impairment, and are very adapt 2002;140:61–7. at fi nding solutions and ways to compensate for their Hall et al. Amyoplasia revisited. Am J Med Genet motor disabilities. They often need extensive treat- Part A 2014;164A:700–30. ment with physiotherapy, orthoses, orthopedic sur- Kroksmark AK, et al. Muscle involvement and mo- gery etc, and the treatment involves a team with tor function in amyoplasia. Am J Med Genet Part A experience of diagnosis and treatment in arthrogrypo- 2006;140A:1757–67. sis. Stretching and muscle activation are important mainstays in treatment, and especially important in early life. THEMATIC WORKSHOP 8.4 No disclosures. Theme: ARTHROGRYPOSIS Hall et al. Amyoplasia revisited. Am J Med Genet Part A 2014;164A:700–30. Kroksmark AK, et al. Muscle involvement and mo- TW 8.4.2 Amyoplasia: current concepts tor function in amyoplasia. Am J Med Genet Part A Eva KIMBER, Uppsala (Suède) 2006;140A:1757–67. Department of Women´s and Children´s Health, Uppsala University Children´s Hospital, Uppsala, Sweden. THEMATIC WORKSHOP 8.4 Theme: ARTHROGRYPOSIS Amyoplasia is the most common cause of arthro- gryposis, about 1/4 - 1/3 of all cases. Amyoplasia is a highly specifi c clinical diagnosis. The term amyopla- TW 8.4.3 Distal arthrogryposis: a sia implies no muscle growth/ development, and, typ- developmental myopathy ically, some muscles are absent or very thin and Anders OLDFORS, Gothenburg (Suède) replaced by fatty or connective tissue, while other Department of Pathology, Institute of Biomedicine, muscles are normal. Amyoplasia is a sporadically oc- University of Gothenburg, Sweden curing condition, and there is no known genetic cause. There is no recurrence risk. As in all arthrogryposis, Distal arthrogryposis (DA) belongs to a group of the cause of joint contractures in amyoplasia is de- syndromes, arthrogryposis multiplex congenita, with creased fetal mobility. Case studies have demonstrat- congenital contractures affecting several joints in dif- ed that vascular compromise/decreased blood fl ow to ferent body areas. Distal arthrogryposis involves the fetus in early pregnancy, gestational week 9–12, is mainly hands and feet but additional features such as involved in the pathogenesis. It is thought that anteri- facial involvement, scoliosis, ophthalmoplegia and or horn cells in the spinal cord are damaged, resulting hearing defi cits have formed the basis for a clinical in muscle hypoplasia and joint contractures. Contrac- classifi cation including at least 10 different forms. Abstracts S79

The most common forms are classifi ed as DA1 and form of myosin heavy chain that is frequently mutated DA2B. DA1 is restricted to hands and feet whereas in cases of DA1 and DA2 is not expressed postnatally. DA2B also shows facial involvement and corresponds However, abnormal expression of perinatal myosin as to the Sheldon-Hall syndrome. A more severe form well as abnormal fi ber size variability among type 1 known as the Freeman-Sheldon syndrome is classi- fi bers have been observed in patients with DA associ- fi ed as DA2A. ated with MYH3 mutations. In cases where the mu- Recent progress in genetics have disclosed that sev- tated protein is expressed postnatally, such as troponin eral of the DA syndromes are associated with domi- I (TNNI2), more clear evidence of myopathic changes nant mutations in genes encoding skeletal muscle have been obtained. sarcomeric proteins that are expressed during early In DA1 and DA2 there is no clear correlation be- development such as embryonic and perinatal myosin tween the clinical phenotype and the type of mutated (MYH3 and MYH8), beta-tropomyosin (TPM2), tro- protein, since mutations in different genes can be ponin I (TNNI2), troponin T (TNNT3) and myosin found in the same clinical syndrome and different binding protein C (MYBPC1). These observations syndromes can be associated with mutations in the have lead to the hypothesis that the pathogenesis of same gene. DA syndromes in many cases is caused by a develop- No disclosure mental myopathy. Muscle biopsy performed in patients with DA has supported this concept although the embryonic iso-