WANTED – Dead or alive: Myotubularins, a large disease-associated protein family

Myotubularins define a large family of proteins conserved through evolution. Several members are mutated in different neuromuscular diseases including centronuclear myopathies and Charcot-Marie-Tooth (CMT) neuropathies, or are linked to a predisposition to obesity and cancer. While some members have phosphatase activity against the 3-phosphate of phosphoinositides, regulating the phosphorylation status of PtdIns3P and PtdIns(3,5)P2 implicated in membrane trafficking and autophagy, and producing PtdIns5P, others lack key residues in the catalytic site and are classified as dead-phosphatases. However, these dead phosphatases regulate phosphoinositide-dependent cellular pathways by binding to catalytically active myotubularins. Here we review previous studies on the molecular regulation and physiological roles of myotubularins. We also used the recent myotubularins three-dimensional structures to underline key residues that are mutated in neuromuscular diseases and required for enzymatic activity. In addition, through database mining and analysis, expression profile and specific isoforms of the different myotubularins are described in depth, as well as a revisited protein interaction network. Comparison of the interactome and expression data for each myotubularin highlights specific protein complexes and tissues where myotubularins should have a key regulatory role

Identification of FHL1 as a regulator of skeletal muscle mass: implications for human myopathy

Regulators of skeletal muscle mass are of interest, given the morbidity and mortality of muscle atrophy and myopathy. Four-and-a-half LIM protein 1 (FHL1) is mutated in several human myopathies, including reducing-body myopathy (RBM). The normal function of FHL1 in muscle and how it causes myopathy remains unknown. We find that FHL1 transgenic expression in mouse skeletal muscle promotes hypertrophy and an oxidative fiber-type switch, leading to increased whole-body strength and fatigue resistance. Additionally, FHL1 overexpression enhances myoblast fusion, resulting in hypertrophic myotubes in C2C12 cells, (a phenotype rescued by calcineurin inhibition). In FHL1-RBM C2C12 cells, there are no hypertrophic myotubes. FHL1 binds with the calcineurin-regulated transcription factor NFATc1 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1), enhancing NFATc1 transcriptional activity. Mutant RBM-FHL1 forms aggregate bodies in C2C12 cells, sequestering NFATc1 and resulting in reduced NFAT nuclear translocation and transcriptional activity. NFATc1 also colocalizes with mutant FHL1 to reducing bodies in RBM-afflicted skeletal muscle. Therefore, via NFATc1 signaling regulation, FHL1 appears to modulate muscle mass and strength enhancement

Altered splicing of the BIN1 muscle-specific exon in humans and dogs with highly progressive centronuclear myopathy

Amphiphysin 2, encoded by BIN1, is a key factor for membrane sensing and remodelling in different cell types. Homozygous BIN1 mutations in ubiquitously expressed exons are associated with autosomal recessive centronuclear myopathy (CNM), a mildly progressive muscle disorder typically showing abnormal nuclear centralization on biopsies. In addition, misregulation of BIN1 splicing partially accounts for the muscle defects in myotonic dystrophy (DM). However, the muscle-specific function of amphiphysin 2 and its pathogenicity in both muscle disorders are not well understood. In this study we identified and characterized the first mutation affecting the splicing of the muscle-specific BIN1 exon 11 in a consanguineous family with rapidly progressive and ultimately fatal centronuclear myopathy. In parallel, we discovered a mutation in the same BIN1 exon 11 acceptor splice site as the genetic cause of the canine Inherited Myopathy of Great Danes (IMGD). Analysis of RNA from patient muscle demonstrated complete skipping of exon 11 and BIN1 constructs without exon 11 were unable to promote membrane tubulation in differentiated myotubes. Comparative immunofluorescence and ultrastructural analyses of patient and canine biopsies revealed common structural defects, emphasizing the importance of amphiphysin 2 in membrane remodelling and maintenance of the skeletal muscle triad. Our data demonstrate that the alteration of the muscle-specific function of amphiphysin 2 is a common pathomechanism for centronuclear myopathy, myotonic dystrophy, and IMGD. The IMGD dog is the first faithful model for human BIN1-related CNM and represents a mammalian model available for preclinical trials of potential therapies

Expression of the neuropathy-associated MTMR2 gene rescues MTM1-associated myopathy

Myotubularins (MTMs) are active or dead phosphoinositides phosphatases defining a large protein family conserved through evolution and implicated in different neuromuscular diseases. Loss-of-function mutations in MTM1 cause the severe congenital myopathy called myotubular myopathy (or X-linked centronuclear myopathy) while mutations in the MTM1-related protein MTMR2 cause a recessive Charcot-Marie-Tooth peripheral neuropathy. Here we aimed to determine the functional specificity and redundancy of MTM1 and MTMR2, and to assess their abilities to compensate for a potential therapeutic strategy. Using molecular investigations and heterologous expression of human MTMs in yeast cells and in Mtm1 knockout mice, we characterized several naturally occurring MTMR2 isoforms with different activities. We identified the N-terminal domain as responsible for functional differences between MTM1 and MTMR2. An N-terminal extension observed in MTMR2 is absent in MTM1, and only the short MTMR2 isoform lacking this N-terminal extension behaved similarly to MTM1 in yeast and mice. Moreover, adeno-associated virus-mediated exogenous expression of several MTMR2 isoforms ameliorates the myopathic phenotype owing to MTM1 loss, with increased muscle force, reduced myofiber atrophy, and reduction of the intracellular disorganization hallmarks associated with myotubular myopathy. Noteworthy, the short MTMR2 isoform provided a better rescue when compared with the long MTMR2 isoform. In conclusion, these results point to the molecular basis for MTMs functional specificity. They also provide the proof-of-concept that expression of the neuropathy-associated MTMR2 gene improves the MTM1-associated myopathy, thus identifying MTMR2 as a novel therapeutic target for myotubular myopathy

Phosphatase-dead myotubularin ameliorates X-linked centronuclear myopathy phenotypes in mice

Myotubularin MTM1 is a phosphoinositide (PPIn) 3-phosphatase mutated in X-linked centronuclear myopathy (XLCNM; myotubular myopathy). We investigated the involvement of MTM1 enzymatic activity on XLCNM phenotypes. Exogenous expression of human MTM1 in yeast resulted in vacuolar enlargement, as a consequence of its phosphatase activity. Expression of mutants from patients with different clinical progression and determination of PtdIns3P and PtdIns5P cellular levels confirmed the link between vacuolar morphology and MTM1 phosphatase activity, and showed that some disease mutants retain phosphatase activity. Viral gene transfer of phosphatase-dead myotubularin mutants (MTM1(C375S) and MTM1(S376N)) significantly improved most histological signs of XLCNM displayed by a Mtm1-null mouse, at similar levels as wild-type MTM1. Moreover, the MTM1(C375S) mutant improved muscle performance and restored the localization of nuclei, triad alignment, and the desmin intermediate filament network, while it did not normalize PtdIns3P levels, supporting phosphatase-independent roles of MTM1 in maintaining normal muscle performance and organelle positioning in skeletal muscle. Among the different XLCNM signs investigated, we identified only triad shape and fiber size distribution as being partially dependent on MTM1 phosphatase activity. In conclusion, this work uncovers MTM1 roles in the structural organization of muscle fibers that are independent of its enzymatic activity. This underlines that removal of enzymes should be used with care to conclude on the physiological importance of their activity

Defective Membrane Remodeling in Neuromuscular Diseases: Insights from Animal Models

Proteins involved in membrane remodeling play an essential role in a plethora of cell functions including endocytosis and intracellular transport. Defects in several of them lead to human diseases. Myotubularins, amphiphysins, and dynamins are all proteins implicated in membrane trafficking and/or remodeling. Mutations in myotubularin, amphiphysin 2 (BIN1), and dynamin 2 lead to different forms of centronuclear myopathy, while mutations in myotubularin-related proteins cause Charcot-Marie-Tooth neuropathies. In addition to centronuclear myopathy, dynamin 2 is also mutated in a dominant form of Charcot-Marie-Tooth neuropathy. While several proteins from these different families are implicated in similar diseases, mutations in close homologues or in the same protein in the case of dynamin 2 lead to diseases affecting different tissues. This suggests (1) a common molecular pathway underlying these different neuromuscular diseases, and (2) tissue-specific regulation of these proteins. This review discusses the pathophysiology of the related neuromuscular diseases on the basis of animal models developed for proteins of the myotubularin, amphiphysin, and dynamin families. A better understanding of the common mechanisms between these neuromuscular disorders will lead to more specific health care and therapeutic approaches

Cellular functions of myotubularins, amphiphysins, and dynamins implicated in human diseases and their related pathological mechanisms.

<p>(A) Human diseases, (B) their related pathological mechanisms. Membrane fission is necessary for vesicle formation and subsequent trafficking, while inhibition of membrane fission or membrane addition at the T-tubules in muscle may be necessary for their formation and maintenance.</p

Protein domains and disease-causing mutations in the myotubularin, amphiphysin, and dynamin families.

<p>Myotubularin contains a PH-GRAM domain that may bind lipids and a coil-coiled-PDZ binding site to form homo- and hetero-dimers with other members of the myotubularin family. Only the disease-causing missense mutations in MTM1 are represented, based on the international UMD-MTM1 database, existing currently in a local version in Strasbourg (France). MTM1 mutations identified in more than two patients are R69C(9 families), P205L(5), V227M(3), R241C(13), G378R(4), E404K(4), and Y397C(5). AMPH1 and BIN1 possess an N-BAR domain able to sense and eventually curve membrane and a C-terminal SH3 domain binding to proteins with proline-rich domains, such as dynamins <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002595#pgen.1002595-Peter1" target="_blank">[48]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002595#pgen.1002595-Owen1" target="_blank">[88]</a>. In addition some isoforms have clathrin-binding and Myc-binding domains (CBD, MBD); a phosphoinositide-binding motif is present between the BAR and MBD domains specifically in skeletal muscle. DNM2 contains a GTPase domain, a central middle (MID) domain, a Pleckstrin Homology (PH) domain, a GTPase Effector Domain (GED), and a C-terminal Proline Rich Domain (PRD). Dominant mutations in DNM2 lead to either centronuclear myopathy (above), or peripheral CMT neuropathy (below). Only coding mutations are listed for all genes.</p

Myotubularin/amphiphysin/dynamin protein functions in specific tissues, based on animal and cell models.

<p>Myotubularin/amphiphysin/dynamin protein functions in specific tissues, based on animal and cell models.</p

Phylogenetic relationships.

<p>Phylogenetic relationships within the amphiphysin (A), dynamin (B), and myotubularin (C) protein families. Sequences were collected using the eggNOG database, which groups genes into families at different taxonomic levels. A high quality multiple sequence alignment was computed for each protein family on all proteins members including, respectively, 91 myotubularin protein sequences, 23 dynamin protein sequences, and 13 amphiphysin protein sequences. For a more detailed description, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002595#pgen.1002595.s001" target="_blank">Protocol S1</a>. Scale represents the percentage of divergence.</p