Loss of Catalytically Inactive Lipid Phosphatase Myotubularin-related Protein 12 Impairs Myotubularin Stability and Promotes Centronuclear Myopathy in Zebrafish

X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by mutations of the myotubularin gene, MTM1. Myotubularin belongs to a large family of conserved lipid phosphatases that include both catalytically active and inactive myotubularin-related proteins (i.e., “MTMRs”). Biochemically, catalytically inactive MTMRs have been shown to form heteroligomers with active members within the myotubularin family through protein-protein interactions. However, the pathophysiological significance of catalytically inactive MTMRs remains unknown in muscle. By in vitro as well as in vivo studies, we have identified that catalytically inactive myotubularin-related protein 12 (MTMR12) binds to myotubularin in skeletal muscle. Knockdown of the mtmr12 gene in zebrafish resulted in skeletal muscle defects and impaired motor function. Analysis of mtmr12 morphant fish showed pathological changes with central nucleation, disorganized Triads, myofiber hypotrophy and whorled membrane structures similar to those seen in X-linked myotubular myopathy. Biochemical studies showed that deficiency of MTMR12 results in reduced levels of myotubularin protein in zebrafish and mammalian C2C12 cells. Loss of myotubularin also resulted in reduction of MTMR12 protein in C2C12 cells, mice and humans. Moreover, XLMTM mutations within the myotubularin interaction domain disrupted binding to MTMR12 in cell culture. Analysis of human XLMTM patient myotubes showed that mutations that disrupt the interaction between myotubularin and MTMR12 proteins result in reduction of both myotubularin and MTMR12. These studies strongly support the concept that interactions between myotubularin and MTMR12 are required for the stability of their functional protein complex in normal skeletal muscles. This work highlights an important physiological function of catalytically inactive phosphatases in the pathophysiology of myotubular myopathy and suggests a novel therapeutic approach through identification of drugs that could stabilize the myotubularin-MTMR12 complex and hence ameliorate this disorder.     

Defective Autophagy and mTORC1 Signaling in Myotubularin Null Mice

Autophagy is a vesicular trafficking pathway that regulates the degradation of aggregated proteins and damaged organelles. Initiation of autophagy requires several multiprotein signaling complexes, such as the ULK1 kinase complex and the Vps34 lipid kinase complex, which generates phosphatidylinositol 3-phosphate [PtdIns(3)P] on the forming autophagosomal membrane. Alterations in autophagy have been reported for various diseases, including myopathies. Here we show that skeletal muscle autophagy is compromised in mice deficient in the X-linked myotubular myopathy (XLMTM)-associated PtdIns(3)P phosphatase myotubularin (MTM1). Mtm1-deficient muscle displays several cellular abnormalities, including a profound increase in ubiquitin aggregates and abnormal mitochondria. Further, we show that Mtm1 deficiency is accompanied by activation of mTORC1 signaling, which persists even following starvation. In vivo pharmacological inhibition of mTOR is sufficient to normalize aberrant autophagy and improve muscle phenotypes in Mtm1 null mice. These results suggest that aberrant mTORC1 signaling and impaired autophagy are consequences of the loss of Mtm1 and may play a primary role in disease pathogenesis.     

Myotubularin regulates the function of the late endosome through the gram domain-phosphatidylinositol 3,5-bisphosphate interaction

Myotubularin and related proteins constitute a large and highly conserved family possessing phosphoinositide 3-phosphatase activity, although not all members possess this activity. This family contains a conserved region called the GRAM domain that is found in a variety of proteins associated with membrane-coupled processes and signal transduction. Mutations of myotubularin are found in X-linked myotubular myopathy, a severe muscle disease. Mutations in the GRAM domain are responsible for this condition, suggesting crucial roles for this region. Here, we show that the GRAM domain of myotubularin binds to phosphoinositide with the highest affinity to phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P(2)). In patients with myotubular myopathy, mutations in the myotubularin GRAM domain eliminate this binding, indicating that the PtdIns(3,5)P(2) binding ability of the GRAM (glucosyltransferases, Rablike GTPase activators and myotubularin) domain is crucial for the functions of myotubularin in vivo. Stimulation of epidermal growth factor recruits myotubularin to the late endosomal compartment in a manner dependent on the phosphoinositide binding. Overexpression of myotubularin inhibits epidermal growth factor receptor trafficking from late endosome to lysosome and induces the large endosomal vacuoles. Thus, our data suggest that myotubularin phosphatase physiologically functions in late endosomal trafficking and vacuolar morphology through interaction with PtdIns(3,5)P(2).     

Production of phosphatidylinositol 5-phosphate by the phosphoinositide 3-phosphatase myotubularin in mammalian cells

MTM1, the gene encoding myotubularin (MTM1), is mutated in the X-linked myotubular myopathy (XLMTM), a severe genetic muscular disorder. MTM1 is a phosphoinositide phosphatase hydrolyzing phosphatidylinositol 3-phosphate (PtdIns(3)P) in yeast and in vitro. Because this lipid is implicated in the regulation of vesicular trafficking, we used established cell lines from XLMTM patients to evaluate whether the lack of endogenous MTM1 expression could affect PtdIns(3)P labeling patterns. Our results showed that the vesicular trafficking related to early endosomes was not significantly affected in the XLMTM cell lines compared with control cells. However, in addition to PtdIns(3)P, we found that MTM1 can hydrolyze phosphatidylinositol 3,5-bisphosphate both in vitro and in mammalian cells. Using a mass assay, we demonstrated that the product generated is phosphatidylinositol 5-phosphate (PtdIns(5)P), a recently discovered phosphoinositide, the function of which is still unknown. In L6 myotubes overexpressing MTM1, hyperosmotic shock induced an increase in the mass level of PtdIns(5)P that was reduced by 50% upon overexpression of the MTM1 inactive mutant D278A. These data demonstrate for the first time a role for MTM1 in the production of PtdIns(5)P in mammalian cells, suggesting that the lack of transformation of phosphatidylinositol 3,5-bisphosphate into PtdIns(5)P might be an important component in the etiology of myotubular myopathy.     

A Rho GDP-dissociation inhibitor is involved in cytokinesis of Dictyostelium

Myotubularin-related genes define a novel highly conserved family of eukaryotic proteins of at least 11 human members. The hMTM1 gene that codes for myotubularin is mutated in X-linked myotubular myopathy, a severe congenital disease. Recently, we and others have characterized myotubularin as a potent and specific phosphatidylinositol 3-phosphate 3-phosphatase. In the present study we investigated the lipid phosphatase activity and the subcellular localization of two other members of the family, hMTMR2 protein that is mutated in the demyelinating neuropathy Charcot-Marie-Tooth type 4B and the FYVE-finger containing hMTMR3 protein. Our results show that both proteins are potent phosphatidylinositol 3-phosphate 3-phosphatases either in vitro or in yeast where they interfered with vesicular trafficking. Their localization is mainly cytoplasmic, with however strong labeling of Rac-inducible plasma membrane ruffles. The fact that the ubiquitously expressed hMTM1 and hMTMR2 genes are involved in different pathologies indicates that despite their shared enzymatic activity, they are not functionally redundant, at least in certain cell types. This might be explained by subtle differences in expression and/or in recruitment and regulation at their specific site of action.     

The novel contiguous gene syndrome of myotubular myopathy (MTM1), male hypogenitalism and deletion in Xq28:report of the first familial case.

Hu et al. (1996) and Laporte et al. (1997) recently proposed a novel contiguous gene syndrome of myotubular myopathy, abnormal male genital development and deletion in Xq28. We studied a family where two male infants, both deceased, had myotubular myopathy and intersexual genitalia. Using FISH we detected in the mother a hemizygous deletion including the myotubularin gene MTM1 and F18 (a gene of yet unknown function). DNA studies with STR-markers (short tandem repeats) within and flanking the deleted segment confirmed the deletion in the family and were used for prenatal diagnosis. Our findings confirm the existence of this novel contiguous gene syndrome and support that the deletion of the F18 gene, or a neighboring gene, may cause ambiguous genitalia or severe hypospadias in males. The mother had low muscle power and marked menstrual irregularities which may indicate that she is a manifesting carrier and that the deletion may include a gene (F18 or other) for gonadal function in females.     

Myotubularin phosphatases: policing 3-phosphoinositides

In eukaryotic cells, phosphatidylinositol is subject to differential phosphorylation, resulting in the production of seven distinct phosphatidylinositol phosphates, often referred to as phosphoinositides (PIs). PIs have numerous distinct roles in cellular regulation and membrane trafficking. Recently, myotubularin family PI 3-phosphatases have emerged as key regulators of phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate, two PIs that regulate traffic within the endosomal-lysosomal pathway. Mutations in several myotubularin genes lead to myotubular myopathy and Charcot-Marie-Tooth peripheral neuropathy. Strikingly, nearly half of the members of the human myotubularin family appear to be catalytically inactive. Several inactive myotubularins have essential functions in mammals. Recent work in mammalian cells and model organisms is shedding light on the roles of myotubularins in membrane traffic.     

The myopathy-causing mutation DNM2-S619L leads to defective tubulation in vitro and in developing zebrafish

DNM2 is a ubiquitously expressed GTPase that regulates multiple subcellular processes. Mutations in DNM2 are a common cause of centronuclear myopathy, a severe disorder characterized by altered skeletal muscle structure and function. The precise mechanisms underlying disease-associated DNM2 mutations are unresolved. We examined the common DNM2-S619L mutation using both in vitro and in vivo approaches. Expression of DNM2-S619L in zebrafish led to the accumulation of aberrant vesicular structures and to defective excitation-contraction coupling. Expression of DNM2-S619L in COS7 cells resulted in defective BIN1-dependent tubule formation. These data suggest that DNM2-S619L causes disease, in part, by interfering with membrane tubulation.KEY WORDS: Dynamin-2, Excitation-contraction coupling, Myopathy     

The structure and regulation of myotubularin phosphatases

The human neuromuscular diseases X-linked myotubular myopathy and Charcot-Marie-Tooth disease type 4B are caused by mutations in myotubularin family proteins. The myotubularins are a unique subfamily of protein tyrosine phosphatases that utilize inositol phospholipids, rather than phosphoproteins, as substrates. Recent structural studies, including the first crystal structure of a myotubularin family protein, have defined the structural features that are characteristic of the family and revealed the molecular basis of their unique substrate specificity. Interestingly, the myotubularin family contains a subgroup of proteins that are catalytically inactive. Recent biochemical studies have established that the inactive myotubularins function as adaptors for the active members and play an important regulatory role within the family.     

Expression of myotubularin by an adenoviral vector demonstrates its function as a phosphatidylinositol 3-phosphate [PtdIns(3)P] phosphatase in muscle cell lines: involvement of PtdIns(3)P in insulin-stimulated glucose transport

X-linked myotubular myopathy is a muscle disorder caused by mutations on the myotubular myopathy-1 (MTM-1) gene, coding for myotubularin a 65-kDa polypeptide similar to protein phosphatases. Biochemical and in vivo studies define myotubularin as a phosphatidylinositol 3-phosphate [PtdIns(3)P] phosphatase. To efficiently express myotubularin in muscle cell lines and adipocytes, we used an adenoviral genome recombinogenic to pcDNA3, and to other widely used expression vectors, to produce adenoviruses expressing wild-type (wt), catalytically inactive C375S, and substrate trap D278A myotubularin.[32P]Orthophosphate labeling followed by phosphoinositide analysis of differentiated L6 and C2C12 cells expressing myotubularin demonstrated increased PtdIns(3)P levels upon expression of the C375S and D278A mutants. In keeping with its biochemical function, overexpression of wt myotubularin as an enhanced green fluorescent protein fusion disrupted the endosomal punctuated staining of the FYVE (Fab1p/YOTB Vac1p/EEA1)-domain-containing PtdIns(3)P binding protein early endosomal antigen 1 as well as of a gluathione-S-transferase-FYVE probe directed to PtdIns(3)P. Expression of wt myotubularin, although not affecting activation of proximal insulin signal transduction targets such as protein kinase B and MAPK, induced a decrease in insulin-induced glucose uptake, whereas basal glucose uptake was augmented by expression of D278A (DA) and C375S (CS) mutants. Moreover, overexpression of myotubularin in 3T3-L1 adipocytes impaired insulin-induced translocation at the plasma membrane of green fluorescent protein-tagged glucose transporter 4. These data indicate that PtdIns(3)P is required to direct glucose transporter 4 to insulin-responsive compartments and/or to allow the translocation of the latter at the plasma membrane.We conclude that myotubularin, by modulating the intracellular levels of PtdIns(3)P, plays a role in the control of vesicular traffic related to glucose transport, by counteracting the activities of the PtdIns(3)P-producing phosphatidylinositol 3-kinases.     

SPEG Interacts with Myotubularin,and Its Deficiency Causes Centronuclear Myopathy with Dilated Cardiomyopathy

Centronuclear myopathies (CNMs) are characterized by muscle weakness and increased numbers of central nuclei within myofibers. X-linked myotubular myopathy, the most common severe form of CNM, is caused by mutations in MTM1, encoding myotubularin (MTM1), a lipid phosphatase. To increase our understanding of MTM1 function, we conducted a yeast two-hybrid screen to identify MTM1-interacting proteins. Striated muscle preferentially expressed protein kinase (SPEG), the product of SPEG complex locus (SPEG), was identified as an MTM1-interacting protein, confirmed by immunoprecipitation and immunofluorescence studies. SPEG knockout has been previously associated with severe dilated cardiomyopathy in a mouse model. Using whole-exome sequencing, we identified three unrelated CNM-affected probands, including two with documented dilated cardiomyopathy, carrying homozygous or compound-heterozygous SPEG mutations. SPEG was markedly reduced or absent in two individuals whose muscle was available for immunofluorescence and immunoblot studies. Examination of muscle samples from Speg-knockout mice revealed an increased frequency of central nuclei, as seen in human subjects. SPEG localizes in a double line, flanking desmin over the Z lines, and is apparently in alignment with the terminal cisternae of the sarcoplasmic reticulum. Examination of human and murine MTM1-deficient muscles revealed similar abnormalities in staining patterns for both desmin and SPEG. Our results suggest that mutations in SPEG, encoding SPEG, cause a CNM phenotype as a result of its interaction with MTM1. SPEG is present in cardiac muscle, where it plays a critical role; therefore, individuals with SPEG mutations additionally present with dilated cardiomyopathy.     

RANKL induces NFATc1 acetylation and stability via histone acetyltransferases during osteoclast differentiation

The MTM (myotubularin)/MTMR (myotubularin-related) protein family is comprised of 15 lipid phosphatases, of which nine members are catalytically active. MTMs are known to play a fundamental role in human physiology as gene mutations can give rise to X-linked myotubular myopathy or Charcot-Marie-Tooth disease, which manifest in skeletal muscle or in peripheral neurons respectively. Interestingly, studies have shown MTMR2 and MTMR5, two MTM family members, to be highly expressed in the testis, particularly in Sertoli and germ cells, and knockout of either gene resulted in spermatogenic defects. Other studies have shown that MTMR2 functions in endocytosis and membrane trafficking. In the testis, MTMR2 interacts and co-localizes with c-Src/phospho-Src-(Tyr?1?), a non-receptor protein tyrosine kinase that regulates the phosphorylation state of proteins at the apical ES (ectoplasmic specialization), a unique type of cell junction found between Sertoli cells and elongating/elongated spermatids. In the present review, we highlight recent findings that have made a significant impact on our understanding of this protein family in normal cell function and in disease, with the emphasis on the role of MTMs and MTMRs in spermatogenesis. We also describe a working model to explain how MTMR2 interacts with other proteins such as c-Src, dynamin 2, EPS8 (growth factor receptor pathway substrate 8) and ARP2/3 (actin-related protein 2/3) at the apical ES and the apical TBC (tubulobulbar complex; tubular-like invaginations that function in the disassembly of the apical ES and in the recycling of its components) to regulate spermiation at late stage VIII of the seminiferous epithelial cycle.     

Crystal structure of a phosphoinositide phosphatase, MTMR2: insights into myotubular myopathy and Charcot-Marie-Tooth syndrome

Myotubularin-related proteins are a large subfamily of protein tyrosine phosphatases (PTPs) that dephosphorylate D3-phosphorylated inositol lipids. Mutations in members of the myotubularin family cause the human neuromuscular disorders myotubular myopathy and type 4B Charcot-Marie-Tooth syndrome. The crystal structure of a representative member of this family, MTMR2, reveals a phosphatase domain that is structurally unique among PTPs. A series of mutants are described that exhibit altered enzymatic activity and provide insight into the specificity of myotubularin phosphatases toward phosphoinositide substrates. The structure also reveals that the GRAM domain, found in myotubularin family phosphatases and predicted to occur in approximately 180 proteins, is part of a larger motif with a pleckstrin homology (PH) domain fold. Finally, the MTMR2 structure will serve as a model for other members of the myotubularin family and provide a framework for understanding the mechanism whereby mutations in these proteins lead to disease.     

Genetic Linkage Heterogeneity in Myotubular Myopathy

Myotubular myopathy is a severe congenital disease inherited as an X-linked trait (MTM1; McKusick 31040). It has been mapped to the long arm of chromosome X, to the Xq27-28 region. Significant linkage has subsequently been established for the linkage group comprised of DXS304, DXS15, DXS52, and F8C in several studies. To date, published linkage studies have provided no evidence of genetic heterogeneity in severe neonatal myotubular myopathy (XLMTM). We have investigated a family with typical XLMTM in which no linkage to these markers was found. Our findings strongly suggest genetic heterogeneity in myotubular myopathy and indicate that great care should be taken when using Xq28 markers in linkage studies for prenatal diagnosis and genetic counseling.     

Two Dynamin-2 Genes Are Required for Normal Zebrafish Development

Dynamin-2 (DNM2) is a large GTPase involved in clathrin-mediated endocytosis and related trafficking pathways. Mutations in human DNM2 cause two distinct neuromuscular disorders: centronuclear myopathy and Charcot-Marie-Tooth disease. Zebrafish have been shown to be an excellent animal model for many neurologic disorders, and this system has the potential to inform our understanding of DNM2-related disease. Currently, little is known about the endogenous zebrafish orthologs to human DNM2. In this study, we characterize two zebrafish dynamin-2 genes, dnm2 and dnm2-like. Both orthologs are structurally similar to human DNM2 at the gene and protein levels. They are expressed throughout early development and in all adult tissues examined. Knockdown of dnm2 and dnm2-like gene products resulted in extensive morphological abnormalities during development, and expression of human DNM2 RNA rescued these phenotypes. Our findings suggest that dnm2 and dnm2-like are orthologs to human DNM2, and that they are required for normal zebrafish development.     

The myotubularin family: from genetic disease to phosphoinositide metabolism

The myotubularin-related genes define a large family of eukaryotic proteins, most of them initially characterized by the presence of a ten-amino acid consensus sequence related to the active sites of tyrosine phosphatases, dual-specificity protein phosphatases and the lipid phosphatase PTEN. Myotubularin (hMTM1), the founder member, is mutated in myotubular myopathy, and a close homolog (hMTMR2) was recently found mutated in a recessive form of Charcot-Marie-Tooth neuropathy. Although myotubularin was thought to be a dual-specificity protein phosphatase, recent results indicate that it is primarily a lipid phosphatase, acting on phosphatidylinositol 3-monophosphate, and might be involved in the regulation of phosphatidylinositol 3-kinase (PI 3-kinase) pathway and membrane trafficking.     

Phosphoinositide regulation of integrin trafficking required for muscle attachment and maintenance

Muscles must maintain cell compartmentalization when remodeled during development and use. How spatially restricted adhesions are regulated with muscle remodeling is largely unexplored. We show that the myotubularin (mtm) phosphoinositide phosphatase is required for integrin-mediated myofiber attachments in Drosophila melanogaster, and that mtm-depleted myofibers exhibit hallmarks of human XLMTM myopathy. Depletion of mtm leads to increased integrin turnover at the sarcolemma and an accumulation of integrin with PI(3)P on endosomal-related membrane inclusions, indicating a role for Mtm phosphatase activity in endocytic trafficking. The depletion of Class II, but not Class III, PI3-kinase rescued mtm-dependent defects, identifying an important pathway that regulates integrin recycling. Importantly, similar integrin localization defects found in human XLMTM myofibers signify conserved MTM1 function in muscle membrane trafficking. Our results indicate that regulation of distinct phosphoinositide pools plays a central role in maintaining cell compartmentalization and attachments during muscle remodeling, and they suggest involvement of Class II PI3-kinase in MTM-related disease.     

Knockdown of cathepsin D in zebrafish fertilized eggs determines congenital myopathy

CD (cathepsin D) is a ubiquitous lysosomal hydrolase involved in a variety of pathophysiological functions, including protein turnover, activation of pro-hormones, cell death and embryo development. CD-mediated proteolysis plays a pivotal role in tissue and organ homoeostasis. Altered expression and compartmentalization of CD have been observed in diseased muscle fibres. Whether CD is actively involved in muscle development, homoeostasis and dystrophy remains to be demonstrated. Zebrafish (Danio rerio) is emerging as a valuable ‘in vivo’ vertebrate model for muscular degeneration and congenital myopathies. In this work, we report on the perturbance of the somitic musculature development in zebrafish larvae caused by MPO (morpholino)-mediated silencing of CD in oocytes at the time of fertilization. Restoring CD expression, using an MPO-non-matching mutated mRNA, partially rescued the normal phenotype, confirming the indispensable role of CD in the correct development and integrity of the somitic musculature. This is the first report showing a congenital myopathy caused by CD deficiency in a vertebrate experimental animal model.