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Marc Bitoun - One of the best experts on this subject based on the ideXlab platform.
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A DNM2 Centronuclear Myopathy Mutation Reveals a Link between Recycling Endosome Scission and Autophagy.
Developmental Cell, 2020Co-Authors: Claudia Puri, Marco M. Manni, Mariella Vicinanza, Christine Hilcenko, Ye Zhu, Gautam Runwal, Eleanna Stamatakou, Fiona M. Menzies, Kamel Mamchaoui, Marc BitounAbstract:Summary Autophagy involves engulfment of cytoplasmic contents by double-membraned autophagosomes, which ultimately fuse with lysosomes to enable degradation of their substrates. We recently proposed that the tubular-vesicular recycling endosome membranes were a core platform on which the critical early events of autophagosome formation occurred, including LC3-membrane conjugation to autophagic precursors. Here, we report that the release of autophagosome precursors from recycling endosomes is mediated by DNM2-dependent scission of these tubules. This process is regulated by DNM2 binding to LC3 and is increased by autophagy-inducing stimuli. This scission is defective in cells expressing a centronuclear-myopathy-causing DNM2 mutant. This mutant has an unusual mechanism as it depletes normal-functioning DNM2 from autophagosome formation sites on recycling endosomes by causing increased binding to an alternative plasma membrane partner, ITSN1. This “scission” step is, thus, critical for autophagosome formation, is defective in a human disease, and influences the way we consider how autophagosomes are formed.
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Loss of Dynamin 2 GTPase function results in microcytic anaemia.
British Journal of Haematology, 2017Co-Authors: Fiona C. Brown, Phillip J. Robinson, Marc Bitoun, Michael Collett, Cedric S. Tremblay, Gerhard Rank, Pietro De Camilli, Carmen J. Booth, Benjamin T. Kile, Stephen M. JaneAbstract:Summary In a dominant mouse ethylnitrosurea mutagenesis screen for genes regulating erythropoiesis, we identified a pedigree with a novel microcytic hypochromia caused by a V235G missense mutation in Dynamin 2 (DNM2). Mutations in DNM2, a GTPase, are highly disease-specific and have been implicated in four forms of human diseases: centronuclear myopathy, Charcot-Marie Tooth neuropathy and, more recently, T-cell leukaemia and Hereditary Spastic Paraplegia, but red cell abnormalities have not been reported to date. The V235G mutation lies within a crucial GTP nucleotide-binding pocket of DNM2, and resulted in defective GTPase activity and incompatibility with life in the homozygous state. DNM2 is an essential mediator of clathrin-mediated endocytosis, which is required for the uptake of transferrin (Tf) into red cells for incorporation of haem. Accordingly, we observed significantly reduced Tf uptake by DNM2+/V235G cells, which led to impaired endosome formation. Despite these deficiencies, surprisingly all iron studies were unchanged, suggesting an unexplained alternative mechanism underlies microcytic anaemia in DNM2+/V235G mice. This study provides the first in vivo evidence for the requirements of DNM2 in normal erythropoiesis.
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Calcium homeostasis alterations in a mouse model of the Dynamin 2-related centronuclear myopathy
Biology Open, 2016Co-Authors: Bodvael Fraysse, Pascale Guicheney, Marc BitounAbstract:Autosomal dominant centronuclear myopathy (CNM) is a rare congenital myopathy characterized by centrally located nuclei in muscle fibers. CNM results from mutations in the gene encoding dynamin 2 (DNM2), a large GTPase involved in endocytosis, intracellular membrane trafficking, and cytoskeleton regulation. We developed a knock-in mouse model expressing the most frequent DNM2-CNM mutation; i.e. the KI-DNM2 R465W model. Heterozygous (HTZ) KI-DNM2 mice progressively develop muscle atrophy, impairment of contractile properties, histopathological abnormalities, and elevated cytosolic calcium concentration. Here, we aim at better characterizing the calcium homeostasis impairment in extensor digitorum longus (EDL) and soleus muscles from adult HTZ KI-DNM2 mice. We demonstrate abnormal contractile properties and cytosolic Ca 2+ concentration in EDL but not soleus muscles showing that calcium impairment is correlated with muscle weakness and might be a determinant factor of the spatial muscle involvement. In addition, the elevated cytosolic Ca 2+ concentration in EDL muscles is associated with an increased sarcolemmal permeability to Ca 2+ and releasable Ca 2+ content from the sarcoplasmic reticulum. However, amplitude and kinetics characteristics of the calcium transient appear unchanged. This suggests that calcium defect is probably not a primary cause of decreased force generation by compromised sarcomere shortening but may be involved in long-term deleterious consequences on muscle physiology. Our results highlight the first pathomechanism which may explain the spatial muscle involvement occurring in DNM2-related CNM and open the way toward development of a therapeutic approach to normalize calcium content.
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Reprogramming the Dynamin 2 mRNA by Spliceosome-mediated RNA Trans-splicing
Molecular Therapy - Nucleic Acids, 2016Co-Authors: Delphine Trochet, Bernard Prudhon, Arnaud Jollet, Stéphanie Lorain, Marc BitounAbstract:Dynamin 2 (DNM2) is a large GTPase, ubiquitously expressed, involved in membrane trafficking and regulation of actin and microtubule cytoskeletons. DNM2 mutations cause autosomal dominant centronuclear myopathy which is a rare congenital myopathy characterized by skeletal muscle weakness and histopathological features including nuclear centralization in absence of regeneration. No curative treatment is currently available for the DNM2-related autosomal dominant centronuclear myopathy. In order to develop therapeutic strategy, we evaluated here the potential of Spliceosome-Mediated RNA Trans-splicing technology to reprogram the DNM2-mRNA in vitro and in vivo in mice. We show that classical 3′-trans-splicing strategy cannot be considered as accurate therapeutic strategy regarding toxicity of the pre-trans-splicing molecules leading to low rate of trans-splicing in vivo. Thus, we tested alternative strategies devoted to prevent this toxicity and enhance frequency of trans-splicing events. We succeeded to overcome the toxicity through a 5′-trans-splicing strategy which also allows detection of trans-splicing events at mRNA and protein levels in vitro and in vivo. These results suggest that the Spliceosome-Mediated RNA Trans-splicing strategy may be used to reprogram mutated DNM2-mRNA but highlight the potential toxicity linked to the molecular tools which have to be carefully investigated during preclinical development.
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G.P.45
Neuromuscular Disorders, 2014Co-Authors: Delphine Trochet, Bernard Prudhon, Arnaud Jollet, Marc BitounAbstract:The autosomal dominant centronuclear myopathy (AD-CNM) is a rare congenital myopathy defined by skeletal muscle weakness and characteristic histopathological changes. Heterozygous mutations in the DNM2 gene are associated with entire clinical spectrum of AD-CNM. DNM2 gene encodes dynamin 2 (DNM2), a large GTPase ubiquitously expressed and involved in membrane trafficking. A Knock-In mouse model (KI-DNM2R465W) expressing the most frequent mutation found in patients has been recently developed in the laboratory. The heterozygous mice progressively develop a muscle phenotype which recapitulates many aspects of the human condition. The purpose of this project is to evaluate the therapeutic potential of DNM2-mRNA repair by Spliceosome-Mediated RNA Trans-splicing (SMarT) technology able to reprogram the 5′, 3′ or internal coding sequence of endogenous mRNA. We have shown that classical 3′ strategy cannot be considered as accurate therapeutic strategy regarding the toxic effect of the Pre-Transplicing Molecules (PTMs) leading to low rate of trans-splicing in vivo. Thus, we tested several alternative strategies in order to prevent this toxicity and enhance frequency of trans-splicing events. We finally succeeded to overcome the toxicity using a 5′ trans-splicing strategy and obtained hopeful results. Indeed, we detected trans-splicing events at mRNA and protein levels in vitro and in vivo.
Arnaud Ferry - One of the best experts on this subject based on the ideXlab platform.
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Allele-specific silencing therapy for Dynamin 2-related dominant centronuclear myopathy.
EMBO Molecular Medicine, 2017Co-Authors: Delphine Trochet, Aymen Rabai, Arnaud Ferry, Bernard Prudhon, Maud Beuvin, Stéphanie Lorain, Kamel Mamchaoui, Cécile Peccate, Laura Julien, Sofia Benkhelifa-ziyyatAbstract:Rapid advances in allele-specific silencing by RNA interference established a strategy of choice to cure dominant inherited diseases by targeting mutant alleles. We used this strategy for autosomal-dominant centronuclear myopathy (CNM), a rare neuromuscular disorder without available treatment due to heterozygous mutations in the DNM2 gene encoding Dynamin 2. Allele-specific siRNA sequences were developed in order to specifically knock down the human and murine DNM2-mRNA harbouring the p.R465W mutation without affecting the wild-type allele. Functional restoration was achieved in muscle from a knock-in mouse model and in patient-derived fibroblasts, both expressing the most frequently encountered mutation in patients. Restoring either muscle force in a CNM mouse model or DNM2 function in patient-derived cells is an essential breakthrough towards future gene-based therapy for dominant centronuclear myopathy.
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G.O.22
Neuromuscular Disorders, 2014Co-Authors: Belinda S Cowling, Christine Kretz, Ivana Prokic, Hichem Tasfaout, Arnaud Ferry, Norma B. Romero, T. Chevremont, Catherine Coirault, Vincent Laugel, Jocelyn LaporteAbstract:Centronuclear myopathies (CNM) are associated with muscle weakness and abnormally located nuclei in skeletal muscle. They can be due to mutations in the MTM1 gene encoding myotubularin (X-linked centronuclear myopathy or myotubular myopathy), in the DNM2 gene encoding dynamin 2 (dominant CNM), or in BIN1 (amphiphysin 2), RYR1 or TTN for typical or atypical autosomal forms. Currently, no effective treatments exist for centronuclear myopathies. We and others showed that overexpression of wildtype DNM2 in muscle cause a CNM-like phenotype. We thus hypothesized MTM1 and DNM2 function in a common pathway, where either MTM1 loss-of-function or DNM2 gain-of-function lead to the CNM phenotype. To test this hypothesis, we reduced the expression of DNM2 in Mtm1-/y mice that reproduce a CNM phenotype with a progressive myopathy leading to death by 6–12 weeks. Mtm1-/yDNM2+/− mice survived up to 2 years. CNM histological features including fiber atrophy and nuclei mispositioning were prevented or strongly delayed and reduced, and muscle strength was increased. Downregulation of DNM2 selectively in skeletal muscle during embryogenesis or in young Mtm1-/y mice showed the rescue is cell autonomous and that downregulation of DNM2 can stop and potentially revert the progression of the phenotype. Thus, we identified MTM1 and DNM2 are in a common pathway regulating muscle organization and force. We introduce the original concept of ”cross-therapy” where one form of the disease (X-linked CNM, MTM1) can be rescued by decreasing expression of another gene mutated in CNM (DNM2). Preliminary data will be presented supporting downregulation of DNM2 can also rescue a myopathy not due to MTM1 mutations, enlarging the spectrum of myopathies that could be rescued by this strategy. While DNM2 is a key mechanoenzyme for important cellular processes, its reduction is strongly beneficial for several myopathies and represents a novel potential therapeutic approach.
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Reducing dynamin 2 expression rescues X-linked centronuclear myopathy
Journal of Clinical Investigation, 2014Co-Authors: Belinda Cowling, Christine Kretz, Ivana Prokic, Arnaud Ferry, O.s. Koutsopoulos, T. Chevremont, Catherine Coirault, Vincent Laugel, Norma Romero, Jocelyn LaporteAbstract:Centronuclear myopathies (CNM) are congenital disorders associated with muscle weakness and abnormally located nuclei in skeletal muscle. An autosomal dominant form of CNM results from mutations in the gene encoding dynamin 2 (DNM2), and loss-of-function mutations in the gene encoding myotubularin (MTM1) result in X-linked CNM (XLCNM, also called myotubular myopathy), which promotes severe neonatal hypo-tonia and early death. Currently, no effective treatments exist for XLCNM. Here, we found increased DNM2 levels in XLCNM patients and a mouse model of XLCNM (Mtm1-/y). Generation of Mtm1-/y mice that were heterozygous for DNM2 revealed that reduction of DNM2 in XLCNM mice restored life span, whole-body strength, and diaphragm function and increased muscle strength. Additionally, classic CNM-associated his-tological features, including fiber atrophy and nuclei mispositioning, were absent or reduced. Ultrastructur-al analysis revealed improvement of sarcomere organization and triad structures. Skeletal muscle-specific decrease of DNM2 during embryogenesis or in young mice after disease onset revealed that the rescue associated with downregulation of DNM2 is cell autonomous and is able to stop and potentially revert XLCNM progression. These data indicate that MTM1 and DNM2 regulate muscle organization and force through a common pathway. Furthermore, despite DNM2 being a key mechanoenzyme, its reduction is beneficial for XLCNM and represents a potential therapeutic approach for patients.
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C.P.7 Dynamin 2 in skeletal muscle development and diseases
Neuromuscular Disorders, 2012Co-Authors: Belinda S Cowling, Aurelien Roux, Pascale Koebel, Leonela Amoasii, Arnaud Ferry, O.s. Koutsopoulos, C. Koch, H. Mojzisova, E. Heckel, Christine KretzAbstract:Abstract Dynamins are large GTPases implicated in membrane remodeling and cytoskeletal dynamics. Heterozygous mutations in dynamin 2 (DNM2) have been linked to autosomal dominant centronuclear myopathy (ADCNM) and Charcot–Marie–Tooth peripheral neuropathy (CMT), two discrete progressive neuromuscular disorders, highlighting the importance of dynamin 2 for normal axonal and muscle maintenance. We have now identified the first homozygous mutation in a dynamin protein, DNM2, leading to a congenital syndrome associating fetal hypokinesia and early death. Patient fibroblasts displayed reduced transferrin uptake, and this mutation impacted on DNM2 in vitro membrane tubulation function. Skeletal muscles were strongly affected in these patients, as in patients with dominant ADCNM myopathy, suggesting a key role for DNM2 in this tissue. To gain insight into the function of DNM2 in skeletal muscle, we first exogenously expressed wild-type DNM2 or R465W DNM2, the most common ADCNM mutation, into adult wild-type mouse skeletal muscle by intramuscular Adeno-Associated Virus (AAV) injections. Expression of R465W DNM2 led to the development of a centronuclear myopathy phenotype with abnormal organelle positioning. In addition, expression of both constructs reduced the specific maximal muscle force, suggesting that the ADCNM disease arises from an increased DNM2 function. As adult muscles were injected, it also suggested that DNM2 has an important role in the maintenance of muscle structure. To further decipher the function of DNM2 in skeletal muscle, we created DNM2 knockout mice. Complete knockout of DNM2 in all tissues was lethal before embryonic day 12. Therefore we developed a muscle-specific knockout mouse and have characterized muscle-specific isoforms of DNM2. KO mice were viable to birth and muscle defects led to death within the first weeks of life. Taken together, we conclude DNM2 plays important roles in both development and maintenance of skeletal muscle.
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Increased Expression of Wild-Type or a Centronuclear Myopathy Mutant of Dynamin 2 in Skeletal Muscle of Adult Mice Leads to Structural Defects and Muscle Weakness
The American Journal of Pathology, 2011Co-Authors: Belinda S Cowling, Pascale Koebel, Anne Toussaint, Leonela Amoasii, Arnaud Ferry, Laurianne Davignon, Ichizo Nishino, Jean-louis Mandel, Jocelyn LaporteAbstract:Dynamin 2 (DNM2) is a large GTPase implicated in many cellular functions, including cytoskeleton regulation and endocytosis. Although ubiquitously expressed, DNM2 was found mutated in two genetic disorders affecting different tissues: autosomal dominant centronuclear myopathy (ADCNM; skeletal muscle) and peripheral Charcot-Marie-Tooth neuropathy (peripheral nerve). To gain insight into the function of DNM2 in skeletal muscle and the pathological mechanisms leading to ADCNM, we introduced wild-type DNM2 (WT-DNM2) or R465W DNM2 (RW-DNM2), the most common ADCNM mutation, into adult wild-type mouse skeletal muscle by intramuscular adeno-associated virus injections. We detected altered localization of RW-DNM2 in mouse muscle. Several ADCNM features were present in RW-DNM2 mice: fiber atrophy, nuclear mislocalization, and altered mitochondrial staining, with a corresponding reduction in specific maximal muscle force. The sarcomere and triad structures were also altered. We report similar findings in muscle biopsy specimens from an ADCNM patient with the R465W mutation. In addition, expression of wild-type DNM2 induced some muscle defects, albeit to a lesser extent than RW-DNM2, suggesting that the R465W mutation has enhanced activity in vivo. In conclusion, we show the RW-DNM2 mutation acts in a dominant manner to cause ADCNM in adult muscle, and the disease arises from a primary defect in skeletal muscle rather than secondary to peripheral nerve involvement. Therefore, DNM2 plays important roles in the maintenance of adult muscle fibers.
Jocelyn Laporte - One of the best experts on this subject based on the ideXlab platform.
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Different in vivo impacts of dynamin 2 mutations implicated in Charcot-Marie-Tooth neuropathy or centronuclear myopathy.
Human Molecular Genetics, 2019Co-Authors: Xènia Massana Muñoz, Jocelyn Laporte, Pascale Koebel, Suzie Buono, Belinda S CowlingAbstract:Dynamin 2 (DNM2) is a ubiquitously expressed GTPase implicated in many cellular functions such as membrane trafficking and cytoskeleton regulation. Dominant mutations in DNM2 result in tissue-specific diseases affecting peripheral nerves (Charcot-Marie-Tooth neuropathy, CMT) or skeletal muscles (centronuclear myopathy, CNM). However, the reason for this tissue specificity is unknown, and it remains unclear if these diseases share a common pathomechanism. To compare the disease pathophysiological mechanisms in skeletal muscle, we exogenously expressed wild-type DNM2 (WT-DNM2), the DNM2-CMT mutation K562E or DNM2-CNM mutations R465W and S619L causing adult and neonatal forms, respectively, by intramuscular adeno-associated virus (AAV) injections. All muscles expressing exogenous WT-DNM2 and CNM or CMT mutations exhibited reduced muscle force. However, only expression of CNM mutations and WT-DNM2 correlated with CNM-like histopathological hallmarks of nuclei centralization and reduced fiber size. The extent of alterations correlated with clinical severity in patients. Ultrastructural and immunofluorescence analyses highlighted defects of the triads, mitochondria and costameres as major causes of the CNM phenotype. Despite the reduction in force upon expression of the DNM2-CMT mutation, muscle histology and ultrastructure were almost normal. However, the neuromuscular junction was affected in all DNM2-injected muscles, with the DNM2-CMT mutation inducing the most severe alterations, potentially explaining the reduction in force observed with this mutant. In conclusion, expression of WT and CNM mutants recreate a CNM-like phenotype, suggesting CNM mutations are gain-of-function. Histological, ultrastructural and molecular analyses pointed to key pathways uncovering the different pathomechanisms involved in centronuclear myopathy or Charcot-Marie-Tooth neuropathy linked to DNM2 mutations.
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single intramuscular injection of aav shrna reduces DNM2 and prevents myotubular myopathy in mice
Molecular Therapy, 2018Co-Authors: Hichem Tasfaout, Christine Kretz, Valentina M Lionello, Pascale Koebel, Nadia Messaddeq, Deborah Bitz, Jocelyn Laporte, Belinda S CowlingAbstract:Myotubular myopathy, or X-linked centronuclear myopathy, is a severe muscle disorder representing a significant burden for patients and their families. It is clinically characterized by neonatal and severe muscle weakness and atrophy. Mutations in the myotubularin (MTM1) gene cause myotubular myopathy, and no specific curative treatment is available. We previously found that dynamin 2 (DNM2) is upregulated in both Mtm1 knockout and patient muscle samples, whereas its reduction through antisense oligonucleotides rescues the clinical and histopathological features of this myopathy in mice. Here, we propose a novel approach targeting DNM2 mRNA. We screened and validated in vitro and in vivo several short hairpin RNA (shRNA) sequences that efficiently target DNM2 mRNA. A single intramuscular injection of AAV-shDNM2 resulted in long-term reduction of DNM2 protein level and restored muscle force, mass, histology, and myofiber ultrastructure and prevented molecular defects linked to the disease. Our results demonstrate a robust DNM2 knockdown and provide an alternative strategy based on reduction of DNM2 to treat myotubular myopathy.
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Antisense oligonucleotide-mediated DNM2 knockdown prevents and reverts myotubular myopathy in mice.
Nature Communications, 2017Co-Authors: Hichem Tasfaout, Christine Kretz, Belinda S Cowling, Nadia Messaddeq, Suzie Buono, Shuling Guo, Brett P. Monia, Sheri L. Booten, Sarah Greenlee, Jocelyn LaporteAbstract:Centronuclear myopathies (CNM) are non-dystrophic muscle diseases for which no effective therapy is currently available. The most severe form, X-linked CNM, is caused by myotubularin 1 (MTM1) loss-of-function mutations, while the main autosomal dominant form is due to dynamin2 (DNM2) mutations. We previously showed that genetic reduction of DNM2 expression in Mtm1 knockout (Mtm1KO) mice prevents development of muscle pathology. Here we show that systemic delivery of DNM2 antisense oligonucleotides (ASOs) into Mtm1KO mice efficiently reduces DNM2 protein level in muscle and prevents the myopathy from developing. Moreover, systemic ASO injection into severely affected mice leads to reversal of muscle pathology within 2 weeks. Thus, ASO-mediated DNM2 knockdown can efficiently correct muscle defects due to loss of MTM1, providing an attractive therapeutic strategy for this disease.
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DNM2 mutations in a cohort of sporadic patients with centronuclear myopathy.
Genetics and Molecular Biology, 2015Co-Authors: Osorio Abath Neto, Jocelyn Laporte, C.a. Martins, Mary S. Carvalho, Gerson Chadi, Katia Werneck Seitz, Acary Souza Bulle Oliveira, Umbertina Conti Reed, Edmar ZanoteliAbstract:Centronuclear myopathy (CNM) is a rare congenital muscle disease characterized by fibers with prominent centralized nuclei in muscle biopsies. The disease is clinically heterogeneous, ranging from severe neonatal hypotonic phenotypes to adult-onset mild muscle weakness, and can have multiple modes of inheritance in association with various genes, including MTM1, DNM2, BIN1 and RYR1. Here we analyzed 18 sporadic patients with clinical and histological diagnosis of CNM and sequenced the DNM2 gene, which codes for the dynamin 2 protein. We found DNM2 missense mutations in two patients, both in exon 8, one known (p.E368K) and one novel (p.F372C), which is found in a position of presumed pathogenicity and appeared de novo. The patients had similar phenotypes characterized by neonatal signs followed by improvement and late childhood reemergence of slowly progressive generalized muscle weakness, elongated face with ptosis and ophthalmoparesis, and histology showing fibers with radiating sarcoplasmic strands (RSS). These patients were the only ones in the series to present this histological marker, which together with previous reports in the literature suggest that, when RSS are present, direct sequencing of DNM2 mutation hot spot regions should be the first step in the molecular diagnosis of CNM, even in sporadic cases.
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G.O.22
Neuromuscular Disorders, 2014Co-Authors: Belinda S Cowling, Christine Kretz, Ivana Prokic, Hichem Tasfaout, Arnaud Ferry, Norma B. Romero, T. Chevremont, Catherine Coirault, Vincent Laugel, Jocelyn LaporteAbstract:Centronuclear myopathies (CNM) are associated with muscle weakness and abnormally located nuclei in skeletal muscle. They can be due to mutations in the MTM1 gene encoding myotubularin (X-linked centronuclear myopathy or myotubular myopathy), in the DNM2 gene encoding dynamin 2 (dominant CNM), or in BIN1 (amphiphysin 2), RYR1 or TTN for typical or atypical autosomal forms. Currently, no effective treatments exist for centronuclear myopathies. We and others showed that overexpression of wildtype DNM2 in muscle cause a CNM-like phenotype. We thus hypothesized MTM1 and DNM2 function in a common pathway, where either MTM1 loss-of-function or DNM2 gain-of-function lead to the CNM phenotype. To test this hypothesis, we reduced the expression of DNM2 in Mtm1-/y mice that reproduce a CNM phenotype with a progressive myopathy leading to death by 6–12 weeks. Mtm1-/yDNM2+/− mice survived up to 2 years. CNM histological features including fiber atrophy and nuclei mispositioning were prevented or strongly delayed and reduced, and muscle strength was increased. Downregulation of DNM2 selectively in skeletal muscle during embryogenesis or in young Mtm1-/y mice showed the rescue is cell autonomous and that downregulation of DNM2 can stop and potentially revert the progression of the phenotype. Thus, we identified MTM1 and DNM2 are in a common pathway regulating muscle organization and force. We introduce the original concept of ”cross-therapy” where one form of the disease (X-linked CNM, MTM1) can be rescued by decreasing expression of another gene mutated in CNM (DNM2). Preliminary data will be presented supporting downregulation of DNM2 can also rescue a myopathy not due to MTM1 mutations, enlarging the spectrum of myopathies that could be rescued by this strategy. While DNM2 is a key mechanoenzyme for important cellular processes, its reduction is strongly beneficial for several myopathies and represents a novel potential therapeutic approach.
Kamel Mamchaoui - One of the best experts on this subject based on the ideXlab platform.
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A DNM2 Centronuclear Myopathy Mutation Reveals a Link between Recycling Endosome Scission and Autophagy.
Developmental Cell, 2020Co-Authors: Claudia Puri, Marco M. Manni, Mariella Vicinanza, Christine Hilcenko, Ye Zhu, Gautam Runwal, Eleanna Stamatakou, Fiona M. Menzies, Kamel Mamchaoui, Marc BitounAbstract:Summary Autophagy involves engulfment of cytoplasmic contents by double-membraned autophagosomes, which ultimately fuse with lysosomes to enable degradation of their substrates. We recently proposed that the tubular-vesicular recycling endosome membranes were a core platform on which the critical early events of autophagosome formation occurred, including LC3-membrane conjugation to autophagic precursors. Here, we report that the release of autophagosome precursors from recycling endosomes is mediated by DNM2-dependent scission of these tubules. This process is regulated by DNM2 binding to LC3 and is increased by autophagy-inducing stimuli. This scission is defective in cells expressing a centronuclear-myopathy-causing DNM2 mutant. This mutant has an unusual mechanism as it depletes normal-functioning DNM2 from autophagosome formation sites on recycling endosomes by causing increased binding to an alternative plasma membrane partner, ITSN1. This “scission” step is, thus, critical for autophagosome formation, is defective in a human disease, and influences the way we consider how autophagosomes are formed.
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Allele‐specific silencing therapy for Dynamin 2‐related dominant centronuclear myopathy
EMBO Molecular Medicine, 2018Co-Authors: Aymen Rabai, Bernard Prudhon, Maud Beuvin, Delphine Trochet, Stéphanie Lorain, Kamel Mamchaoui, Cécile Peccate, Laura Julien, Sofia Benkhelifa-ziyyat, Sofia Benkhelifa‐ziyyatAbstract:Rapid advances in allele-specific silencing by RNA interference established a strategy of choice to cure dominant inherited diseases by targeting mutant alleles. We used this strategy for autosomal-dominant centronuclear myopathy (CNM), a rare neuromuscular disorder without available treatment due to heterozygous mutations in the DNM2 gene encoding Dynamin 2. Allele-specific siRNA sequences were developed in order to specifically knock down the human and murine DNM2-mRNA harbouring the p.R465W mutation without affecting the wild-type allele. Functional restoration was achieved in muscle from a knock-in mouse model and in patient-derived fibroblasts, both expressing the most frequently encountered mutation in patients. Restoring either muscle force in a CNM mouse model or DNM2 function in patient-derived cells is an essential breakthrough towards future gene-based therapy for dominant centronuclear myopathy.
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Allele-specific silencing therapy for Dynamin 2-related dominant centronuclear myopathy.
EMBO Molecular Medicine, 2017Co-Authors: Delphine Trochet, Aymen Rabai, Arnaud Ferry, Bernard Prudhon, Maud Beuvin, Stéphanie Lorain, Kamel Mamchaoui, Cécile Peccate, Laura Julien, Sofia Benkhelifa-ziyyatAbstract:Rapid advances in allele-specific silencing by RNA interference established a strategy of choice to cure dominant inherited diseases by targeting mutant alleles. We used this strategy for autosomal-dominant centronuclear myopathy (CNM), a rare neuromuscular disorder without available treatment due to heterozygous mutations in the DNM2 gene encoding Dynamin 2. Allele-specific siRNA sequences were developed in order to specifically knock down the human and murine DNM2-mRNA harbouring the p.R465W mutation without affecting the wild-type allele. Functional restoration was achieved in muscle from a knock-in mouse model and in patient-derived fibroblasts, both expressing the most frequently encountered mutation in patients. Restoring either muscle force in a CNM mouse model or DNM2 function in patient-derived cells is an essential breakthrough towards future gene-based therapy for dominant centronuclear myopathy.
Bernard Prudhon - One of the best experts on this subject based on the ideXlab platform.
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Allele‐specific silencing therapy for Dynamin 2‐related dominant centronuclear myopathy
EMBO Molecular Medicine, 2018Co-Authors: Aymen Rabai, Bernard Prudhon, Maud Beuvin, Delphine Trochet, Stéphanie Lorain, Kamel Mamchaoui, Cécile Peccate, Laura Julien, Sofia Benkhelifa-ziyyat, Sofia Benkhelifa‐ziyyatAbstract:Rapid advances in allele-specific silencing by RNA interference established a strategy of choice to cure dominant inherited diseases by targeting mutant alleles. We used this strategy for autosomal-dominant centronuclear myopathy (CNM), a rare neuromuscular disorder without available treatment due to heterozygous mutations in the DNM2 gene encoding Dynamin 2. Allele-specific siRNA sequences were developed in order to specifically knock down the human and murine DNM2-mRNA harbouring the p.R465W mutation without affecting the wild-type allele. Functional restoration was achieved in muscle from a knock-in mouse model and in patient-derived fibroblasts, both expressing the most frequently encountered mutation in patients. Restoring either muscle force in a CNM mouse model or DNM2 function in patient-derived cells is an essential breakthrough towards future gene-based therapy for dominant centronuclear myopathy.
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Allele-specific silencing therapy for Dynamin 2-related dominant centronuclear myopathy.
EMBO Molecular Medicine, 2017Co-Authors: Delphine Trochet, Aymen Rabai, Arnaud Ferry, Bernard Prudhon, Maud Beuvin, Stéphanie Lorain, Kamel Mamchaoui, Cécile Peccate, Laura Julien, Sofia Benkhelifa-ziyyatAbstract:Rapid advances in allele-specific silencing by RNA interference established a strategy of choice to cure dominant inherited diseases by targeting mutant alleles. We used this strategy for autosomal-dominant centronuclear myopathy (CNM), a rare neuromuscular disorder without available treatment due to heterozygous mutations in the DNM2 gene encoding Dynamin 2. Allele-specific siRNA sequences were developed in order to specifically knock down the human and murine DNM2-mRNA harbouring the p.R465W mutation without affecting the wild-type allele. Functional restoration was achieved in muscle from a knock-in mouse model and in patient-derived fibroblasts, both expressing the most frequently encountered mutation in patients. Restoring either muscle force in a CNM mouse model or DNM2 function in patient-derived cells is an essential breakthrough towards future gene-based therapy for dominant centronuclear myopathy.
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Reprogramming the Dynamin 2 mRNA by Spliceosome-mediated RNA Trans-splicing
Molecular Therapy - Nucleic Acids, 2016Co-Authors: Delphine Trochet, Bernard Prudhon, Arnaud Jollet, Stéphanie Lorain, Marc BitounAbstract:Dynamin 2 (DNM2) is a large GTPase, ubiquitously expressed, involved in membrane trafficking and regulation of actin and microtubule cytoskeletons. DNM2 mutations cause autosomal dominant centronuclear myopathy which is a rare congenital myopathy characterized by skeletal muscle weakness and histopathological features including nuclear centralization in absence of regeneration. No curative treatment is currently available for the DNM2-related autosomal dominant centronuclear myopathy. In order to develop therapeutic strategy, we evaluated here the potential of Spliceosome-Mediated RNA Trans-splicing technology to reprogram the DNM2-mRNA in vitro and in vivo in mice. We show that classical 3′-trans-splicing strategy cannot be considered as accurate therapeutic strategy regarding toxicity of the pre-trans-splicing molecules leading to low rate of trans-splicing in vivo. Thus, we tested alternative strategies devoted to prevent this toxicity and enhance frequency of trans-splicing events. We succeeded to overcome the toxicity through a 5′-trans-splicing strategy which also allows detection of trans-splicing events at mRNA and protein levels in vitro and in vivo. These results suggest that the Spliceosome-Mediated RNA Trans-splicing strategy may be used to reprogram mutated DNM2-mRNA but highlight the potential toxicity linked to the molecular tools which have to be carefully investigated during preclinical development.
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G.P.45
Neuromuscular Disorders, 2014Co-Authors: Delphine Trochet, Bernard Prudhon, Arnaud Jollet, Marc BitounAbstract:The autosomal dominant centronuclear myopathy (AD-CNM) is a rare congenital myopathy defined by skeletal muscle weakness and characteristic histopathological changes. Heterozygous mutations in the DNM2 gene are associated with entire clinical spectrum of AD-CNM. DNM2 gene encodes dynamin 2 (DNM2), a large GTPase ubiquitously expressed and involved in membrane trafficking. A Knock-In mouse model (KI-DNM2R465W) expressing the most frequent mutation found in patients has been recently developed in the laboratory. The heterozygous mice progressively develop a muscle phenotype which recapitulates many aspects of the human condition. The purpose of this project is to evaluate the therapeutic potential of DNM2-mRNA repair by Spliceosome-Mediated RNA Trans-splicing (SMarT) technology able to reprogram the 5′, 3′ or internal coding sequence of endogenous mRNA. We have shown that classical 3′ strategy cannot be considered as accurate therapeutic strategy regarding the toxic effect of the Pre-Transplicing Molecules (PTMs) leading to low rate of trans-splicing in vivo. Thus, we tested several alternative strategies in order to prevent this toxicity and enhance frequency of trans-splicing events. We finally succeeded to overcome the toxicity using a 5′ trans-splicing strategy and obtained hopeful results. Indeed, we detected trans-splicing events at mRNA and protein levels in vitro and in vivo.
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A Centronuclear Myopathy - Dynamin 2 Mutation Impairs Autophagy in Mice
Traffic, 2012Co-Authors: Anne-cécile Durieux, Bernard Prudhon, Stéphane Vassilopoulos, Bodvael Fraysse, Jeanne Lainé, Laura Briñas, Gisèle Bonne, Josiane Castells, Damien Freyssenet, Pascale GuicheneyAbstract:Dynamin 2 (DNM2) is involved in endocytosis and intracellular membrane trafficking through its function in vesicle formation from distinct membrane compartments. Heterozygous mutations in the DNM2 gene cause dominant centronuclear myopathy or Charcot-Marie-Tooth neuropathy. We generated a knock-in KI-DNM2 R465W mouse model expressing the most frequent human mutation and recently reported that heterozygous mice progressively developed a myopathy. We investigated here the cause of neonatal lethality occurring in homozygous mice. We show that homozygous mice present at birth with a reduced body weight, hypoglycemia, increased liver glycogen content and hepatomegaly, in agreement with a defect in neonatal autophagy. In vitro studies performed in homozygous embryonic fibroblasts point out to a decrease in the autophagy flux prior to degradation at the autolysosome. We show that starved homozygous cells have a higher number of autophagy-related structures which remain in immature stages probably due to a defect of acidification. Our results highlight the role of DNM2 in the crosstalk between endosomal and autophagic pathways and evidence a new role of DNM2-dependent membrane trafficking in autophagy which may be relevant in DNM2-related human diseases.