RESUMEN
In neurons, fast axonal transport (FAT) of vesicles occurs over long distances and requires constant and local energy supply for molecular motors in the form of adenosine triphosphate (ATP). FAT is independent of mitochondrial metabolism. Indeed, the glycolytic machinery is present on vesicles and locally produces ATP, as well as nicotinamide adenine dinucleotide bonded with hydrogen (NADH) and pyruvate, using glucose as a substrate. It remains unclear whether pyruvate is transferred to mitochondria from the vesicles as well as how NADH is recycled into NAD+ on vesicles for continuous glycolysis activity. The optimization of a glycolytic activity test for subcellular compartments allowed the evaluation of the kinetics of vesicular glycolysis in the brain. This revealed that glycolysis is more efficient on vesicles than in the cytosol. We also found that lactate dehydrogenase (LDH) enzymatic activity is required for effective vesicular ATP production. Indeed, inhibition of LDH or the forced degradation of pyruvate inhibited ATP production from axonal vesicles. We found LDHA rather than the B isoform to be enriched on axonal vesicles suggesting a preferential transformation of pyruvate to lactate and a concomitant recycling of NADH into NAD+ on vesicles. Finally, we found that LDHA inhibition dramatically reduces the FAT of both dense-core vesicles and synaptic vesicle precursors in a reconstituted cortico-striatal circuit on-a-chip. Together, this shows that aerobic glycolysis is required to supply energy for vesicular transport in neurons, similar to the Warburg effect.
Asunto(s)
Glucólisis , NAD , NAD/metabolismo , Glucólisis/fisiología , Axones/metabolismo , Adenosina Trifosfato/metabolismo , Piruvatos/metabolismoRESUMEN
Phosphoinositides are a minor class of short-lived membrane phospholipids that serve crucial functions in cell physiology ranging from cell signalling and motility to their role as signposts of compartmental membrane identity. Phosphoinositide 4-phosphates such as phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are concentrated at the plasma membrane, on secretory organelles, and on lysosomes, whereas phosphoinositide 3-phosphates, most notably phosphatidylinositol 3-phosphate (PI(3)P), are a hallmark of the endosomal system. Directional membrane traffic between endosomal and secretory compartments, although inherently complex, therefore requires regulated phosphoinositide conversion. The molecular mechanism underlying this conversion of phosphoinositide identity during cargo exit from endosomes by exocytosis is unknown. Here we report that surface delivery of endosomal cargo requires hydrolysis of PI(3)P by the phosphatidylinositol 3-phosphatase MTM1, an enzyme whose loss of function leads to X-linked centronuclear myopathy (also called myotubular myopathy) in humans. Removal of endosomal PI(3)P by MTM1 is accompanied by phosphatidylinositol 4-kinase-2α (PI4K2α)-dependent generation of PI(4)P and recruitment of the exocyst tethering complex to enable membrane fusion. Our data establish a mechanism for phosphoinositide conversion from PI(3)P to PI(4)P at endosomes en route to the plasma membrane and suggest that defective phosphoinositide conversion at endosomes underlies X-linked centronuclear myopathy caused by mutation of MTM1 in humans.
Asunto(s)
Endosomas/metabolismo , Exocitosis , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositoles/metabolismo , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Transporte Biológico , Línea Celular , Membrana Celular/metabolismo , Células HeLa , Humanos , Hidrólisis , Fusión de Membrana , Miopatías Estructurales Congénitas/enzimología , Miopatías Estructurales Congénitas/genética , Miopatías Estructurales Congénitas/patología , Monoéster Fosfórico Hidrolasas/deficiencia , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/deficiencia , Proteínas Tirosina Fosfatasas no Receptoras/genética , Proteínas Tirosina Fosfatasas no Receptoras/metabolismoRESUMEN
Dermatomyositis (DM) is an autoimmune disease associated with enhanced type I interferon (IFN) signalling in skeletal muscle, but the mechanisms underlying muscle dysfunction and inflammation perpetuation remain unknown. Transcriptomic analysis of early untreated DM muscles revealed that the main cluster of down-regulated genes was mitochondria-related. Histochemical, electron microscopy, and in situ oxygraphy analysis showed mitochondrial abnormalities, including increased reactive oxygen species (ROS) production and decreased respiration, which was correlated with low exercise capacities and a type I IFN signature. Moreover, IFN-ß induced ROS production in human myotubes was found to contribute to mitochondrial malfunctions. Importantly, the ROS scavenger N-acetyl cysteine (NAC) prevented mitochondrial dysfunctions, type I IFN-stimulated transcript levels, inflammatory cell infiltrate, and muscle weakness in an experimental autoimmune myositis mouse model. Thus, these data highlight a central role of mitochondria and ROS in DM. Mitochondrial dysfunctions, mediated by IFN-ß induced-ROS, contribute to poor exercise capacity. In addition, mitochondrial dysfunctions increase ROS production that drive type I IFN-inducible gene expression and muscle inflammation, and may thus self-sustain the disease. Given that current DM treatments only induce partial recovery and expose to serious adverse events (including muscular toxicity), protecting mitochondria from dysfunctions may open new therapeutic avenues for DM.
Asunto(s)
Dermatomiositis/metabolismo , Inflamación/metabolismo , Interferón beta/metabolismo , Mitocondrias/metabolismo , Músculo Esquelético/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Acetilcisteína/farmacología , Adulto , Anciano , Animales , Línea Celular , Citocinas/sangre , Dermatomiositis/tratamiento farmacológico , Dermatomiositis/patología , Femenino , Depuradores de Radicales Libres/farmacología , Adyuvante de Freund , Humanos , Inflamación/tratamiento farmacológico , Inflamación/patología , Masculino , Ratones Endogámicos BALB C , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Debilidad Muscular/tratamiento farmacológico , Debilidad Muscular/metabolismo , Debilidad Muscular/patología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Enfermedad Autoinmune Experimental del Sistema Nervioso/tratamiento farmacológico , Enfermedad Autoinmune Experimental del Sistema Nervioso/metabolismo , Enfermedad Autoinmune Experimental del Sistema Nervioso/patología , TranscriptomaRESUMEN
Mechanistic target of rapamycin (mTOR) is a central regulator of cell growth, proliferation, survival and metabolism, as part of mTOR complex 1 (mTORC1) and mTORC2. While partial inhibition of mTORC1 using rapamycin was shown to be cardioprotective, genetic studies in mouse models revealed that mTOR is essential for embryonic heart development and cardiac function in adults. However, the physiological role of mTOR during postnatal cardiac maturation is not fully elucidated. We have therefore generated a mouse model in which cardiac mTOR was inactivated at an early postnatal stage. Mutant mTORcmKO mice rapidly developed a dilated cardiomyopathy associated with cardiomyocyte growth defects, apoptosis and fibrosis, and died during their third week. Here, we show that reduced cardiomyocyte growth results from impaired protein translation efficiency through both 4E-BP1-dependent and -independent mechanisms. In addition, infant mTORcmKO hearts displayed markedly increased apoptosis linked to stretch-induced ANKRD1 (Ankyrin repeat-domain containing protein 1) up-regulation, JNK kinase activation and p53 accumulation. Pharmacological inhibition of p53 with pifithrin-α attenuated caspase-3 activation. Cardiomyocyte death did not result from activation of the MST1/Hippo pro-apoptotic pathway as reported in adult rictor/mTORC2 KO hearts. As well, mTORcmKO hearts showed a strong downregulation of myoglobin content, thereby leading to a hypoxic environment. Nevertheless, they lacked a HIF1α-mediated adaptive response, as mTOR is required for hypoxia-induced HIF-1α activation. Altogether, our results demonstrate that mTOR is critically required for cardiomyocyte growth, viability and oxygen supply in early postnatal myocardium and provide insight into the molecular mechanisms involved in apoptosis of mTOR-depleted cardiomyocytes.
Asunto(s)
Apoptosis/genética , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Biosíntesis de Proteínas , Serina-Treonina Quinasas TOR/metabolismo , Proteína p53 Supresora de Tumor/genética , Animales , Biomarcadores , Biopsia , Cardiomiopatía Dilatada/patología , Cardiomiopatía Dilatada/fisiopatología , Puente Cardiopulmonar , Modelos Animales de Enfermedad , Ecocardiografía , Metabolismo Energético/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Pruebas de Función Cardíaca , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Ratones Noqueados , Proteínas Musculares/metabolismo , Mioglobina/metabolismo , Proteínas Nucleares/metabolismo , Proteolisis , Proteínas Represoras/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Centronuclear myopathies are characterized by muscle weakness and abnormal centralization of nuclei in muscle fibers not secondary to regeneration. The severe neonatal X-linked form (myotubular myopathy) is due to mutations in the phosphoinositide phosphatase myotubularin (MTM1), whereas mutations in dynamin 2 (DNM2) have been found in some autosomal dominant cases. By direct sequencing of functional candidate genes, we identified homozygous mutations in amphiphysin 2 (BIN1) in three families with autosomal recessive inheritance. Two missense mutations affecting the BAR (Bin1/amphiphysin/RVS167) domain disrupt its membrane tubulation properties in transfected cells, and a partial truncation of the C-terminal SH3 domain abrogates the interaction with DNM2 and its recruitment to the membrane tubules. Our results suggest that mutations in BIN1 cause centronuclear myopathy by interfering with remodeling of T tubules and/or endocytic membranes, and that the functional interaction between BIN1 and DNM2 is necessary for normal muscle function and positioning of nuclei.
Asunto(s)
Dinamina II/genética , Enfermedades Musculares/genética , Mutación , Proteínas del Tejido Nervioso/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Células COS , Línea Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Dinamina II/metabolismo , Femenino , Genes Recesivos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Haplotipos , Humanos , Masculino , Ratones , Microscopía Confocal , Datos de Secuencia Molecular , Enfermedades Musculares/metabolismo , Enfermedades Musculares/patología , Proteínas del Tejido Nervioso/metabolismo , Polimorfismo de Nucleótido Simple , Unión Proteica , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Multicellular organisms use programmed cell death to eliminate unwanted or potentially harmful cells. Improper cell corpse removal can lead to autoimmune diseases. The development of interventional therapies that increase engulfment activity could represent an attractive approach to treat such diseases. Here, we describe mtm-1, the Caenorhabditis elegans homolog of human myotubularin 1, as a potential negative regulator of apoptotic cell corpse clearance. Loss of mtm-1 function leads to substantially reduced numbers of persistent cell corpses in engulfment mutants, which is a result of a restoration of engulfment function rather than of impaired or delayed programmed cell death. Epistatic analyses place mtm-1 upstream of the ternary GEF complex, which consists of ced-2, ced-5 and ced-12, and parallel to mig-2. Over-activation of engulfment results in the removal of viable cells that have been brought to the verge of death under limiting caspase activity. In addition, mtm-1 also promotes phagosome maturation in the hermaphrodite gonad, potentially through CED-1 receptor recycling. Finally, we show that the CED-12 PH domain can bind to PtdIns(3,5)P(2) (one target of MTM-1 phosphatase activity), suggesting that MTM-1 might regulate CED-12 recruitment to the plasma membrane.
Asunto(s)
Apoptosis/fisiología , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Animales , Animales Modificados Genéticamente , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/genética , Proteínas Portadoras/genética , Proteínas del Citoesqueleto/genética , Regulación del Desarrollo de la Expresión Génica , Genes de Helminto , Humanos , Proteínas de la Membrana/genética , Modelos Biológicos , Mutación , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/deficiencia , Proteínas Tirosina Fosfatasas no Receptoras/genética , Transducción de Señal , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Amphiphysin 2 (BIN1) is a membrane and actin remodeling protein mutated in congenital and adult centronuclear myopathies. Here, we report an unexpected function of this N-BAR domain protein BIN1 in filopodia formation. We demonstrated that BIN1 expression is necessary and sufficient to induce filopodia formation. BIN1 is present at the base of forming filopodia and all along filopodia, where it colocalizes with F-actin. We identify that BIN1-mediated filopodia formation requires IRSp53, which allows its localization at negatively-curved membrane topologies. Our results show that BIN1 bundles actin in vitro. Finally, we identify that BIN1 regulates the membrane-to-cortex architecture and functions as a molecular platform to recruit actin-binding proteins, dynamin and ezrin, to promote filopodia formation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas del Tejido Nervioso , Proteínas Nucleares , Seudópodos , Proteínas Supresoras de Tumor , Humanos , Animales , Células HeLa , Línea Celular , Actinas/metabolismo , Seudópodos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Membrana Celular/metabolismoRESUMEN
The expression of the Huntingtin protein, well known for its involvement in the neurodegenerative Huntington's disease, has been confirmed in skeletal muscle. The impact of HTT deficiency was studied in human skeletal muscle cell lines and in a mouse model with inducible and muscle-specific HTT deletion. Characterization of calcium fluxes in the knock-out cell lines demonstrated a reduction in excitation-contraction (EC) coupling, related to an alteration in the coupling between the dihydropyridine receptor and the ryanodine receptor, and an increase in the amount of calcium stored within the sarcoplasmic reticulum, linked to the hyperactivity of store-operated calcium entry (SOCE). Immunoprecipitation experiments demonstrated an association of HTT with junctophilin 1 (JPH1) and stromal interaction molecule 1 (STIM1), both providing clues on the functional effects of HTT deletion on calcium fluxes. Characterization of muscle strength and muscle anatomy of the muscle-specific HTT-KO mice demonstrated that HTT deletion induced moderate muscle weakness and mild muscle atrophy associated with histological abnormalities, similar to the phenotype observed in tubular aggregate myopathy. Altogether, this study points toward the hypotheses of the involvement of HTT in EC coupling via its interaction with JPH1, and on SOCE via its interaction with JPH1 and/or STIM1.
Asunto(s)
Calcio , Retículo Sarcoplasmático , Ratones , Humanos , Animales , Calcio/metabolismo , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Retículo Sarcoplasmático/metabolismo , Músculo Esquelético/metabolismo , Acoplamiento Excitación-Contracción/fisiologíaRESUMEN
Phosphoinositides (PIs) are lipid second messengers implicated in signal transduction and membrane trafficking. Seven distinct PIs can be synthesized by phosphorylation of the inositol ring of phosphatidylinositol (PtdIns), and their metabolism is accurately regulated by PI kinases and phosphatases. Two of the PIs, PtdIns3P and PtdIns(3,5)P(2), are present on intracellular endosomal compartments, and several studies suggest that they have a role in membrane remodeling and trafficking. We refer to them as 'endosomal PIs'. An increasing number of human genetic diseases including myopathy and neuropathies are associated to mutations in enzymes regulating the turnover of these endosomal PIs. The PtdIns3P and PtdIns(3,5)P(2) 3-phosphatase myotubularin gene is mutated in X-linked centronuclear myopathy, whereas its homologs MTMR2 and MTMR13 and the PtdIns(3,5)P(2) 5-phosphatase SAC3/FIG4 are implicated in Charcot-Marie-Tooth peripheral neuropathies. Mutations in the gene encoding the PtdIns3P 5-kinase PIP5K3/PIKfyve have been found in patients affected with François-Neetens fleck corneal dystrophy. This review presents the roles of the endosomal PIs and their regulators and proposes defects of membrane remodeling as a common pathological mechanism for the corresponding diseases.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Endosomas/metabolismo , Enfermedades Genéticas Congénitas/genética , Miopatías Estructurales Congénitas/genética , Fosfatidilinositoles/metabolismo , Animales , Caenorhabditis elegans , Membrana Celular/metabolismo , Enfermedad de Charcot-Marie-Tooth/diagnóstico , Drosophila , Enfermedades Genéticas Congénitas/diagnóstico , Humanos , Modelos Biológicos , Miopatías Estructurales Congénitas/diagnóstico , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilación , Transporte de Proteínas , Saccharomyces cerevisiae/metabolismoRESUMEN
Membrane dynamics is necessary for cell homeostasis and signal transduction and is in part regulated by phosphoinositides. Pikfyve/Fab1p is a phosphoinositide kinase that phosphorylates phosphatidylinositol 3-monophosphate into phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] and is implicated in membrane homeostasis in yeast and in mammalian cells. These two phosphoinositides are substrates of myotubularin phosphatases found mutated in neuromuscular diseases. We studied the roles of phosphatidylinositol phosphate kinase 3 (PPK-3), the orthologue of PIKfyve/Fab1p, in a multicellular organism, Caenorhabditis elegans. Complete loss of ppk-3 function induces developmental defects characterized by embryonic lethality, whereas partial loss of function leads to growth retardation. At the cellular level, ppk-3 mutants display a striking enlargement of vacuoles positive for lysosome-associated membrane protein 1 in different tissues. In the intestine, RAB-7-positive late endosomes are also enlarged. Membranes of the enlarged lysosomes originate at least in part from smaller lysosomes, and functional and genetic analyses show that the terminal maturation of lysosomes is defective. Protein degradation is not affected in the hypomorphic ppk-3 mutant and is thus uncoupled from membrane retrieval. We measured the level of PtdIns(3,5)P2 and showed that its production is impaired in this mutant. This work strongly suggests that the main function of PPK-3 is to mediate membrane retrieval from matured lysosomes through regulation of PtdIns(3,5)P2.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/enzimología , Membranas Intracelulares/ultraestructura , Lisosomas/ultraestructura , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/ultraestructura , Proteínas de Caenorhabditis elegans/análisis , Proteínas de Caenorhabditis elegans/genética , Membranas Intracelulares/enzimología , Lisosomas/enzimología , Datos de Secuencia Molecular , Mutación , Fosfatos de Fosfatidilinositol/análisis , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/análisis , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Vacuolas/enzimología , Vacuolas/ultraestructuraRESUMEN
Genome editing with the CRISPR/Cas9 technology has emerged recently as a potential strategy for therapy in genetic diseases. For dominant mutations linked to gain-of-function effects, allele-specific correction may be the most suitable approach. In this study, we tested allele-specific inactivation or correction of a heterozygous mutation in the Dynamin 2 (DNM2) gene that causes the autosomal dominant form of centronuclear myopathies (CNMs), a rare muscle disorder belonging to the large group of congenital myopathies. Truncated single-guide RNAs targeting specifically the mutated allele were tested on cells derived from a mouse model and patients. The mutated allele was successfully targeted in patient fibroblasts and Dnm2R465W/+ mouse myoblasts, and clones were obtained with precise genome correction or inactivation. Dnm2R465W/+ myoblasts showed an alteration in transferrin uptake and autophagy. Specific inactivation or correction of the mutated allele rescued these phenotypes. These findings illustrate the potential of CRISPR/Cas9 to target and correct in an allele-specific manner heterozygous point mutations leading to a gain-of-function effect, and to rescue autosomal dominant CNM-related phenotypes. This strategy may be suitable for a large number of diseases caused by germline or somatic mutations resulting in a gain-of-function mechanism.
RESUMEN
Centronuclear myopathies (CNMs) are severe diseases characterized by muscle weakness and myofiber atrophy. Currently, there are no approved treatments for these disorders. Mutations in the phosphoinositide 3-phosphatase myotubularin (MTM1) are responsible for X-linked CNM (XLCNM), also called myotubular myopathy, whereas mutations in the membrane remodeling Bin/amphiphysin/Rvs protein amphiphysin 2 [bridging integrator 1 (BIN1)] are responsible for an autosomal form of the disease. Here, we investigated the functional relationship between MTM1 and BIN1 in healthy skeletal muscle and in the physiopathology of CNM. Genetic overexpression of human BIN1 efficiently rescued the muscle weakness and life span in a mouse model of XLCNM. Exogenous human BIN1 expression with adeno-associated virus after birth also prevented the progression of the disease, suggesting that human BIN1 overexpression can compensate for the lack of MTM1 expression in this mouse model. Our results showed that MTM1 controls cell adhesion and integrin localization in mammalian muscle. Alterations in this pathway in Mtm1 -/y mice were associated with defects in myofiber shape and size. BIN1 expression rescued integrin and laminin alterations and restored myofiber integrity, supporting the idea that MTM1 and BIN1 are functionally linked and necessary for focal adhesions in skeletal muscle. The results suggest that BIN1 modulation might be an effective strategy for treating XLCNM.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adhesiones Focales/patología , Miopatías Estructurales Congénitas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Animales Recién Nacidos , Adhesiones Focales/metabolismo , Humanos , Integrina beta1/metabolismo , Longevidad , Masculino , Ratones Transgénicos , Fuerza Muscular , Músculos/patología , Músculos/fisiopatología , Músculos/ultraestructura , Miopatías Estructurales Congénitas/patología , Miopatías Estructurales Congénitas/fisiopatología , Proteínas Nucleares/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/metabolismoRESUMEN
Mutations in dynamin 2 (DNM2), an ubiquitously-expressed large GTPase, cause autosomal dominant centronuclear myopathy (DNM2-CNM) and AD Charcot-Marie-Tooth disease type 2B (DNM2-CMT2B). We report a series of 5 patients from the same family who all presented with dominant centronuclear myopathy, mild cognitive impairment, mild axonal peripheral nerve involvement, and the novel E368Q mutation in the DNM2 gene. This study suggests that the phenotypes of dynamin 2 related centronuclear myopathy and Charcot-Marie-Tooth disease overlap and that DNM2 mutations may alter cerebral function. This report extends the clinical knowledge of DNM2-centronuclear myopathy and shows that the role of DNM2 mutations in the central nervous system should be further studied.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Dinamina II/genética , Predisposición Genética a la Enfermedad/genética , Mutación/genética , Miopatías Estructurales Congénitas/genética , Adolescente , Adulto , Axones/metabolismo , Axones/patología , Corteza Cerebral/metabolismo , Corteza Cerebral/fisiopatología , Enfermedad de Charcot-Marie-Tooth/metabolismo , Enfermedad de Charcot-Marie-Tooth/fisiopatología , Trastornos del Conocimiento/genética , Trastornos del Conocimiento/metabolismo , Trastornos del Conocimiento/fisiopatología , Comorbilidad , Análisis Mutacional de ADN , Femenino , Marcadores Genéticos/genética , Pruebas Genéticas , Genotipo , Humanos , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Miopatías Estructurales Congénitas/metabolismo , Miopatías Estructurales Congénitas/fisiopatología , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades del Sistema Nervioso Periférico/metabolismo , Enfermedades del Sistema Nervioso Periférico/fisiopatologíaRESUMEN
Regulation of skeletal muscle development and organization is a complex process that is not fully understood. Here, we focused on amphiphysin 2 (BIN1, also known as bridging integrator-1) and dynamin 2 (DNM2), two ubiquitous proteins implicated in membrane remodeling and mutated in centronuclear myopathies (CNMs). We generated Bin1-/- Dnm2+/- mice to decipher the physiological interplay between BIN1 and DNM2. While Bin1-/- mice die perinatally from a skeletal muscle defect, Bin1-/- Dnm2+/- mice survived at least 18 months, and had normal muscle force and intracellular organization of muscle fibers, supporting BIN1 as a negative regulator of DNM2. We next characterized muscle-specific isoforms of BIN1 and DNM2. While BIN1 colocalized with and partially inhibited DNM2 activity during muscle maturation, BIN1 had no effect on the isoform of DNM2 found in adult muscle. Together, these results indicate that BIN1 and DNM2 regulate muscle development and organization, function through a common pathway, and define BIN1 as a negative regulator of DNM2 in vitro and in vivo during muscle maturation. Our data suggest that DNM2 modulation has potential as a therapeutic approach for patients with CNM and BIN1 defects. As BIN1 is implicated in cancers, arrhythmia, and late-onset Alzheimer disease, these findings may trigger research directions and therapeutic development for these common diseases.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Dinamina II/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Dinamina II/genética , Ratones , Ratones Noqueados , Fibras Musculares Esqueléticas/patología , Miopatías Estructurales Congénitas/genética , Miopatías Estructurales Congénitas/metabolismo , Miopatías Estructurales Congénitas/patología , Miopatías Estructurales Congénitas/terapia , Proteínas del Tejido Nervioso/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
That osteocalcin (OCN) is necessary for hippocampal-dependent memory and to prevent anxiety-like behaviors raises novel questions. One question is to determine whether OCN is also sufficient to improve these behaviors in wild-type mice, when circulating levels of OCN decline as they do with age. Here we show that the presence of OCN is necessary for the beneficial influence of plasma from young mice when injected into older mice on memory and that peripheral delivery of OCN is sufficient to improve memory and decrease anxiety-like behaviors in 16-mo-old mice. A second question is to identify a receptor transducing OCN signal in neurons. Genetic, electrophysiological, molecular, and behavioral assays identify Gpr158, an orphan G protein-coupled receptor expressed in neurons of the CA3 region of the hippocampus, as transducing OCN's regulation of hippocampal-dependent memory in part through inositol 1,4,5-trisphosphate and brain-derived neurotrophic factor. These results indicate that exogenous OCN can improve hippocampal-dependent memory in mice and identify molecular tools to harness this pathway for therapeutic purposes.
Asunto(s)
Cognición/fisiología , Osteocalcina/fisiología , Receptores Acoplados a Proteínas G/fisiología , Envejecimiento/fisiología , Animales , Región CA3 Hipocampal/efectos de los fármacos , Región CA3 Hipocampal/fisiología , Cognición/efectos de los fármacos , Electrofisiología , Femenino , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Memoria/efectos de los fármacos , Memoria/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteocalcina/farmacologíaRESUMEN
Nucleus positioning is key for intracellular organization, cell differentiation, and organ development and is affected in many diseases, including myopathies due to alteration in amphiphysin-2 (BIN1). The actin and microtubule cytoskeletons are essential for nucleus positioning, but their crosstalk in this process is sparsely characterized. Here, we report that impairment of amphiphysin/BIN1 in Caenorhabditis elegans, mammalian cells, or muscles from patients with centronuclear myopathy alters nuclear position and shape. We show that AMPH-1/BIN1 binds to nesprin and actin, as well as to the microtubule-binding protein CLIP170 in both species. Expression of the microtubule-anchoring CAP-GLY domain of CLIP170 fused to the nuclear-envelope-anchoring KASH domain of nesprin rescues nuclear positioning defects of amph-1 mutants. Amphiphysins thus play a central role in linking the nuclear envelope with the actin and microtubule cytoskeletons. We propose that BIN1 has a direct and evolutionarily conserved role in nuclear positioning, altered in myopathies.
Asunto(s)
Núcleo Celular/genética , Proteínas de Microfilamentos/genética , Proteínas Asociadas a Microtúbulos/genética , Miopatías Estructurales Congénitas/genética , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Membrana Nuclear/genética , Proteínas Nucleares/genética , Actinas/genética , Animales , Células COS , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Forma de la Célula/genética , Chlorocebus aethiops , Citoplasma/metabolismo , Citoesqueleto/genética , Citoesqueleto/metabolismo , Citoesqueleto/patología , Células HEK293 , Humanos , Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Complejos Multiproteicos , Músculo Esquelético/metabolismo , Miopatías Estructurales Congénitas/metabolismo , Miopatías Estructurales Congénitas/patología , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismoRESUMEN
The autophagy receptor NBR1 (neighbor of BRCA1 gene 1) binds UB/ubiquitin and the autophagosome-conjugated MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) proteins, thereby ensuring ubiquitinated protein degradation. Numerous neurodegenerative and neuromuscular diseases are associated with inappropriate aggregation of ubiquitinated proteins and GSK3 (glycogen synthase kinase 3) activity is involved in several of these proteinopathies. Here we show that NBR1 is a substrate of GSK3. NBR1 phosphorylation by GSK3 at Thr586 prevents the aggregation of ubiquitinated proteins and their selective autophagic degradation. Indeed, NBR1 phosphorylation decreases protein aggregation induced by puromycin or by the DES/desmin N342D mutant found in desminopathy patients and stabilizes ubiquitinated proteins. Importantly, decrease of protein aggregates is due to an inhibition of their formation and not to their autophagic degradation as confirmed by data on Atg7 knockout mice. The relevance of NBR1 phosphorylation in human pathology was investigated. Analysis of muscle biopsies of sporadic inclusion body myositis (sIBM) patients revealed a strong decrease of NBR1 phosphorylation in muscles of sIBM patients that directly correlated with the severity of protein aggregation. We propose that phosphorylation of NBR1 by GSK3 modulates the formation of protein aggregates and that this regulation mechanism is defective in a human muscle proteinopathy.
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Glucógeno Sintasa Quinasa 3/metabolismo , Agregado de Proteínas/fisiología , Proteínas/metabolismo , Animales , Autofagia/fisiología , Proteína 7 Relacionada con la Autofagia , Línea Celular , Células Cultivadas , Femenino , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Biológicos , Miositis por Cuerpos de Inclusión/metabolismo , Miositis por Cuerpos de Inclusión/patología , Fosforilación , Proteínas/química , Proteínas/genética , Deficiencias en la Proteostasis/genética , Deficiencias en la Proteostasis/metabolismo , Deficiencias en la Proteostasis/patología , UbiquitinaciónAsunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Mutación , Miopatías Estructurales Congénitas/genética , Proteínas Nucleares/genética , Proteínas Supresoras de Tumor/genética , Genes Recesivos , Humanos , Músculo Esquelético/patología , Miopatías Estructurales Congénitas/patologíaRESUMEN
X-linked centronuclear myopathy (XLMTM), also called myotubular myopathy, is a severe congenital myopathy characterized by generalized hypotonia and weakness at birth and the typical histological finding of centralization of myo-nuclei. It is caused by mutations in the MTM1 gene encoding the 3-phosphoinositides phosphatase myotubularin. Mutations in dynamin 2 and amphiphysin 2 genes lead to autosomal forms of centronuclear myopathy (CNM). While XLMTM is the most frequent and severe form of CNM, no mutations are found in about 30% of patients by sequencing all MTM1 exons. Moreover, the impact of MTM1 sequence variants is sometimes difficult to assess. It is thus important to devise a complete molecular diagnostic strategy that includes analysis of the myotubularin transcript and protein expression. We therefore developed novel antibodies against human myotubularin and showed that they are able to detect the endogenous protein by direct Western blot from muscle samples and from cultured cells. In conjunction with RT-PCR analysis we validated the consequences of missense and splice mutations on transcript integrity and protein level. We also detected and characterized a novel deep intronic mutation consisting of a single nucleotide change that induces exonisation of a conserved intronic sequence. Patients with centronuclear myopathy and no molecular diagnosis should be investigated for MTM1 defects at the cDNA and protein level.
Asunto(s)
Genes Ligados a X/genética , Intrones/genética , Miopatías Estructurales Congénitas/diagnóstico , Miopatías Estructurales Congénitas/genética , Secuencia de Bases , Western Blotting , Células Cultivadas , ADN/genética , Exones/genética , Humanos , Datos de Secuencia Molecular , Mutación/genética , Mutación/fisiología , Proteínas Tirosina Fosfatasas no Receptoras/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The recently completed Caenorhabditis elegans genome sequence allows application of high-throughput (HT) approaches for phenotypic analyses using RNA interference (RNAi). As large phenotypic data sets become available, "phenoclustering" strategies can be used to begin understanding the complex molecular networks involved in development and other biological processes. The current HT-RNAi resources represent a great asset for phenotypic profiling but are limited by lack of flexibility. For instance, existing resources do not take advantage of the latest improvements in RNAi technology, such as inducible hairpin RNAi. Here we show that a C. elegans ORFeome resource, generated with the Gateway cloning system, can be used as a starting point to generate alternative HT-RNAi resources with enhanced flexibility. The versatility inherent to the Gateway system suggests that additional HT-RNAi libraries can now be readily generated to perform gene knockdowns under various conditions, increasing the possibilities for phenome mapping in C. elegans.