Prion-like protein aggregation of desmin myofibrillar myopathies

Abstracts 2017 / Neuromuscular Disorders 27 (2017) S96–S249 X-Linked centronuclear myopathy (XLCNM) is a rare and severe congenital myopathy characterised by generalised muscle weakness and abnormal nuclei positioning. Most affected boys die in their first year of life and survivors fail to achieve independent ambulation. It is caused by mutations in the Mtm1 gene encoding myotubularin, a ubiquitously expressed phosphoinositide phosphatase. No cure exists and very few pharmacological avenues are being explored. Here, we treated Mtm1-null mice with tamoxifen (TAM), a drug that modulates estrogen actions and that we have shown earlier to be efficacious in dystrophic (mdx5Cv) mice, a model of Duchenne muscular dystrophy (DMD). We report that TAM is also effective in Mtm1-null mice, a model of XLCNM. Wild type and Mtm1-null mice were given normal chow or a TAM-supplemented chow starting at weaning. Non-treated Mtm1-null mice died at around 40 days. By contrast, about half of the Mtm1-null mice treated with clinically relevant doses of TAM survived beyond 365 days of age. Clinical scoring showed that the motor function of the affected mice was markedly improved. In vivo force recordings performed at D40 and D80 revealed that the force of treated Mtm1-null was significantly improved after only 3 weeks of treatment. Histological and electron microscopy analyses show partial rescue of muscle structure and triads, consistent with improved calcium homeostasis in FDB fibres. Quantitative PCR and western blots demonstrate reduction of BIN1 and DNM2, which act downstream of MTM1. In conclusion, we found that tamoxifen extends the lifespan of Mtm1-null mice up to 10-fold and rescues their motor skills. Collectively, these findings suggest that estrogen signalling is a key pathway that modifies disease severity in unrelated myopathies as diverse as DMD and XLCNM. Tamoxifen is safe and readily available. We believe that it deserves clinical evaluation for XLCNM. http://dx.doi.org/10.1016/j.nmd.2017.06.545

TH.O.20 Exhaustive characterization of the newly developed Duchenne muscular dystrophy rat model: a unique animal model for DMD which mimics the human disease at both the muscular and the cardiac levels C. Huchet 1, G. Toumaniantz 2, T. Larcher 3, B. Fraysse 4, A. Lafoux 5, S. Remy 6, D. Caudal 5, M. Allais 4, E. Amosse 2, I. Anegon 6, C. Le Guiner 4 1 Université de Nantes, Nantes, France; 2 UMR INSERM U1087, Nantes, France; 3 UMR INRA 703, Nantes, France; 4 UMR INSERM U1089, Nantes, France; 5 Therassay Capacites, Nantes, France; 6 UMR INSERM U1064, Nantes, France Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder caused by mutations in the gene encoding dystrophin. The evaluation of potential therapeutic products requires relevant animal models exhibiting a phenotype very close to those observed in human patients. If both large and small animal species deficient for dystrophin (especially mice and dogs) have been extensively used for preclinical studies of DMD, they present some limitations, including the absence or very delayed development of cardiomyopathy. We recently generated a line of dmd mutated-rats (Dmdmdx) using TALENs and initially characterized it at two stages of development. To complete this characterization, we performed a large natural history study, during which different groups of Dmdmdx rats and littermate wild-type controls were followed over several generations and exhaustively evaluated at different time points (1.5, 3, 4.5, 7, 10 and 12 months). One supplemental group of Dmdmdx rats was also used to document the lifespan, as well as the causes of death. We showed that life span of Dmdmdx rats is significantly reduced. Weight, blood biomarkers concentrations, muscle strength and fatigue measured by grip force test, muscle calcium homeostasis and histology (including quantification of fibrosis) in skeletal muscles, diaphragm and heart, are all significantly impaired as soon as the age of 1.5 months and show a clear stepwise evolution along with age. Moreover, echo and electrocardiography approaches highlighted a significant and rapid concentric remodeling associated to an alteration of diastolic function, which progressed unfavorably with age towards systolic heart failure with rhythm disorders. In conclusion, with systematic and stepwise aggressive phenotypes at both the muscular and the cardiac levels, similar to what occurs in DMD patients, this unique and newly developed

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Dmd rat model is now one of the best animal model for the preclinical evaluations of new treatments for DMD. http://dx.doi.org/10.1016/j.nmd.2017.06.546

TH.O.21 Connexin-based hemichannels are key factors in the pathological mechanism underlying dysferlinopathy G. Fernández 1, J. Bevilacqua 1, A. Cardenas 2, J. Sáez 3, P. Caviedes 1, L. Cea 1 1 University of Chile, Santiago, Chile; 2 University of Valparaiso, Valparaiso, Chile; 3 Pontificia Universidad Católica De Chile, Santiago, Chile Dysferlinopathy onsets during the second and third decades of life, usually as progressive lower-limb weakness that later involves trunk and upper-limbs. Dysferlin is localized mainly in the sarcolemma and participates in membrane repair. However, in dysferlin-deficient (DD) mice the recovery of the membrane resealing function by expression of a mini-dysferlin does not arrest progressive muscular damage. The latter suggests the presence of dysferlin-dependent pathogenic mechanisms still unknown. In this regard, we have demonstrated a persistent de novo expression of functional connexin-based hemichannels (Cx HCs) in pathological conditions that affect skeletal muscles. Such membrane channels are permeable to Ca2+ and contribute critically to muscular damage. Connexins 40.1, 43 and 45 were localized in the sarcolemma of myofibers of human muscle biopsies from 5 unrelated dysferlinopathy patients,. DD mice myofibers also exhibited positive immunostaining for Cxs 39, 43 and 45. In addition, an elevated Cx HCs activity, concomitant with elevated resting intracellular free Ca2+ levels, was observed in myofibers obtained from DD mice, compared to control myofibers. Further, we detected a lower performance of DD mice in rota-rod motor testing compared to control mice. Moreover, all these changes were prevented in triple knockout mice deficient in Cx 43 Cx 45, and dysferlin, suggesting that Cxs are relevant in the pathogenic mechanism of dysferlinopathy. Therefore, Cx HCs could be a most suitable candidate for pharmacological therapy. http://dx.doi.org/10.1016/j.nmd.2017.06.547

TH.O.22 Prion-like protein aggregation of desmin myofibrillar myopathies C. Weihl, J. Bieschke Washington University in St. Louis, St. Louis, USA Protein aggregate myopathies (PAMs) are a large class of myodegenerative diseases. Their pathologies are due to the misfolding and aggregation of intracellular proteins in muscle cells. In some cases, these inclusions stain with Congo Red suggesting they are true amyloid. Desminopathies are a prototypical PAM that is caused by dominantly inherited mutations of the DES gene, which codes for the protein desmin. Healthy desmin forms type III intermediate filaments in muscle fibers; whereas desmin disease mutations affect intermediate filament structure by interfering with intermediate filament assembly. This leads to desmin aggregation in desminopathies, although desmin is also a principal component of aggregates in other PAMs. We hypothesize that desmin can intrinsically form amyloidogenic aggregates that template the conversion of nonaggregated desmin in vitro and in vivo. This pathomechanism would be similar to neurodegenerative disorders and suggest a prion-like aggregate phenomenom in myopathies. Consistent with this hypothesis, we demonstrate, for the first time, 1) that recombinant desmin and/or desmin fragments form amyloid in vitro; 2) that disease-associated point mutations dramatically increase desmin amyloid formation; 3) that aggregated desmin amyloids can “seed” the aggregation of naïve unaggregated desmin fragments in vitro; 4) that desmin aggregate “seeds” can be taken up by cells and incorporate into an existing desmin network in vivo; and 5) that aggregated desmin expressed in cell culture can “seed” the aggregation of naïve unaggregated desmin fragments in vitro. These data support a novel pathogenic mechanism of disease in PAMs in which desmin amyloids can

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template the aggregation of normal desmin. This pathomechanism may be amenable to therapies aimed at decreasing protein aggregate transmission and propagation from myofiber to myofiber. http://dx.doi.org/10.1016/j.nmd.2017.06.548

NEW INSIGHTS INTO MUSCLE FUNCTION, IMAGING, THERAPY AND PREVENTION NI.O.23 Sh3kbp1 involvement during skeletal muscle fibers formation: a new candidate for centronuclear myopathies A. Guiraud 1, N. Couturier 1, V. Buchman 2, A. Durieux 3, D. Arnould 3, E. Christin 1, S. Janczarski 4, M. Bitoun 5, V. Gache 1 1 Inserm, Lyon, France; 2 Cardiff University, Cardiff, UK; 3 Université de Lyon, Saint-Etienne, France; 4 CNRS, Lyon, France; 5 Université Pierre et Marie Curie-Paris 6, Paris, France Centronuclear myopathies (CNM) are a group of congenital myopathies characterized by skeletal muscle weakness, fatigability and atrophy. In affected muscles, myonuclei are abnormally located at the center of mature muscle fibers since they never migrate to the cell periphery as in healthy muscles. Initially considered as a consequence of the pathology, myonuclear mispositioning has recently emerged as one of the possible causes of muscle functionality defects observed in CNM. So far, the entire molecular machinery responsible for myonuclear positioning during myofibers formation is not known but recent clues involve cytoskeleton proteins. To discover new actors of myonuclear positioning, we performed a siRNA screen on potential cytoskeleton regulators and identified Sh3kbp1 as a new key regulator protein. Sh3kbp1 is ubiquitously expressed, but is up regulated upon muscle differentiation in vitro and specifically reactivated during in vivo muscle regeneration in mice. In in vitro conditions (C2C12 and primary muscle cells) and in mouse muscle in vivo (Tibialis anterior), Sh3kbp1 accumulates progressively both around myonuclei and at triads. Sh3kbp1 knockdown using siRNA in primary myoblasts or stable shRNA C2C12 cells reveals that the protein is not essential for the activation of myogenesis, but is required for normal myoblasts fusion and muscle fibers maturation. Indeed Sh3kbp1 downregulation increases fusion capacity and induces defects in T-tubules establishment and myonuclear positioning, which are typical features of CNM. Additionally, we demonstrated that Sh3kbp1 is interacting with Dynamin2 and is upregulated in a Dynamin2 mouse model of CNM. Further experiments are currently on-going to elucidate the involvement of the two partners in the CNM phenotype. Altogether our results show that Sh3kbp1 is a new key regulator of both T-tubules maturation and myonuclear positioning, and identify Sh3kbp1 as a central player to better understand CNM physiopathology. http://dx.doi.org/10.1016/j.nmd.2017.06.549

NI.O.24 Centronuclear myopathy-causing mutations in dynamin-2 impair actin-dependent trafficking in muscle cells A. González-Jamett 1, X. Baez-Matus 1, M. Bui 2, P. Guicheney 3, N. Romero 4, P. Caviedes 5, M. Bitoun 6, J. Bevilacqua 7, A. Cárdenas 1 1 Facultad de Ciencias, Universidad de Valparaíso, Valparaíso, Chile; 2 Institut de Myologie, GH Pitié-Salpêtrière, Paris, France; 3 INSERM, UMR_S1166, Sorbonne Universités, UPMC Univ Paris 06, Paris, France; 4 Institut de Myologie, UPMC Paris 6, UM74, Inserm UMRS 974, Paris, France; 5 ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile; 6 UPMC Univ Paris 06 and INSERM UMRS 974, Institute of Myology, Paris, France; 7 Hospital Clínico Universidad de Chile, Santiago, Chile Dynamin-2 is a large GTP-ase that mediates membrane remodeling and actin dynamics in different cell types. It is composed by five highly conserved

domains: a GTP-ase domain, a middle structural domain, a PH domain that binds phosphoinisitides a GTP-ase effector domain and a proline-enricheddomain that binds SH3-containing partners. Mutations mainly localized in the middle and PH domains of dynamin-2 cause dominant centronuclear myopathy (CNM), a congenital disorder characterized by progressive weakness and atrophy of skeletal muscles. However, how these mutations affect the role of dynamin-2 in muscle cells is still unclear. In the present work, we demonstrate that dynamin catalytic activity is required for de novo actin polymerization and to promote actin-mediated trafficking of the GLUT4 glucose transporter in muscle cells. These dynamin functions are impaired in myoblasts expressing dynamin-2 constructs carrying CNM-linked middle domain mutations. Similar effects were observed in mature muscle fibers isolated from a mouse-animal model of CNM. Furthermore, GLUT4 displays aberrant perinuclear accumulation in biopsies from CNM patients carrying middle-domain mutations in dynamin-2 suggesting intracellular trafficking defects. Together, these data present dynamin-2 as a key regulator of actin remodeling and GLUT4 trafficking in muscle cells. In addition, these findings support a model in which an impaired actin-dependent trafficking could contribute to the pathological mechanism in dynamin-2-associated CNM. http://dx.doi.org/10.1016/j.nmd.2017.06.550

NI.O.25 Dynamic assessment of muscle perfusion, deoxymyoglobin and phosphorylated metabolites concentrations through fast interleaved NMR acquisitions with a clinical 3T scanner A. Lopez Kolkovsky, B. Marty, B. Coppa, E. Giacomini, P. Carlier Institute of Myology, Paris, France NMR allows to quantify in vivo multiple aspects of physiological parameters such as regional perfusion, blood and tissue oxygenation, intracellular pH or high-energy phosphate metabolism. Classical NMR acquisition schemes rarely explore more than a few biological parameters during a dynamic paradigm, such as exercise or leg ischemia, and thus multiple separate experimental sessions are required at the expense of adding experimental variability on biological processes which are already multifactorial, increased length of scan time and patient discomfort. We present an interleaved NMR pulse sequence simultaneously measuring a perfusion image, 1H deoxy-myoglobin (dMb) and 31P MR spectra on a standard 3T Prisma scanner which was successfully evaluated in healthy subjects either in the ischemic calf muscle (8-mins cuff on the thigh) or in the exercising quadriceps muscle (8-mins of quadriceps contraction by lifting a load placed on the foot every 3 s). Using a dual-tuned 1H/31P surface coil, one 31P and 80 1H dMb non-localized MR spectra were acquired during the evolution time (820 ms) of a pulsed-ASL (SATIR) sequence. Images were acquired using FLASH (radial read-out, 256 points, 128 spokes). Data sets were generated every 2 seconds (ischemia) or 3 seconds (exercise) during the 20-min experiment. The biochemical responses in both paradigms were in agreement with the literature although peak perfusion values during hyperaemia were lower (25.2 mL/min/ 100g at peak versus 0.22 ± 2.80 mL/min/100 g during ischemia), likely due to the reduced ASL tagging efficacy of the surface coil. This work shows the feasibility of dynamic interleaved measurements with a high temporal resolution in a clinical setting and with different experimental paradigms. This setup opens new possibilities to investigate non-invasively complex or subtle alterations of the coupling between microcirculatory regulation and muscle energetics in NMDs, and in particular in DMD, glycogenoses or even centronuclear congenital myopathies. http://dx.doi.org/10.1016/j.nmd.2017.06.551