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Mitochondria are double-membrane subcellular organelles with highly conserved metabolic functions including ATP production. Mitochondria shapes change continually through the combined actions of fission and fusion events rendering mitochondrial network very dynamic. Mitochondria are largely implicated in pathologies and mitochondrial dynamics is often disrupted upon muscle degeneration in various models. Currently, the exact roles of mitochondria in the molecular mechanisms that lead to muscle degeneration remain poorly understood. Here we report a role for DRP-1 in regulating apoptosis induced by dystrophin-dependent muscle degeneration. We found that: (i) dystrophin-dependent muscle degeneration was accompanied by a drastic increase in mitochondrial fragmentation that can be rescued by genetic manipulations of mitochondrial dynamics (ii) the loss of function of the fission gene drp-1 or the overexpression of the fusion genes eat-3 and fzo-1 provoked a reduction of muscle degeneration and an improved mobility of dystrophin mutant worms (iii) the functions of DRP-1 in apoptosis and of others apoptosis executors are important for dystrophin-dependent muscle cell death (iv) DRP-1-mediated apoptosis is also likely to induce age-dependent loss of muscle cell. Collectively, our findings point toward a mechanism involving mitochondrial dynamics to respond to trigger(s) of muscle degeneration via apoptosis in Caenorhabditis elegans.
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Apoptose/genética , Proteínas de Caenorhabditis elegans/metabolismo , Dinaminas/metabolismo , Distrofina/genética , Músculos/metabolismo , Mutação , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Caspases/metabolismo , Locomoção/genética , Mitocôndrias/metabolismo , Dinâmica MitocondrialRESUMO
The Pea Albumin 1 subunit b (PA1b) peptide is an entomotoxin extracted from legume seeds with lethal activity towards several insect pests. Its toxic activity occurs after the perception of PA1b by a plasmalemmic proton pump (V-ATPase) in the insects. Assays revealed that PA1b showed no activity towards mammalian cells displaying high V-ATPase activity. Similarly, PA1b displayed no binding activity and no biological activity towards other non-insect organisms. We demonstrate here that binding to labelled PA1b was found in all the insect families tested, regardless of the sensitivity or insensitivity of the individual species. The coleopteran Bruchidae, which are mainly legume seed pests, were found to be fully resistant. A number of insect species were seen to be insensitive to the toxin although they exhibited binding activity for the labelled PA1b. The fruit fly, Drosophila melanogaster (Diptera), was generally insensitive when maintained on an agar diet, but the fly appeared to be sensitive to PA1b in bioassays using a different diet. In conclusion, the PA1b toxin provides legumes with a major source of resistance to insects, and insects feeding on legume seeds need to overcome this plant resistance by disrupting the PA1b - V-ATPase interaction.
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Fabaceae/química , Insetos/efeitos dos fármacos , Praguicidas/toxicidade , Proteínas de Plantas/toxicidade , ATPases Vacuolares Próton-Translocadoras/química , Sequência de Aminoácidos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Besouros/efeitos dos fármacos , Drosophila melanogaster/efeitos dos fármacos , Humanos , Proteínas de Insetos/química , Resistência a Inseticidas , Células MCF-7 , Camundongos , Dados de Sequência Molecular , Osteoclastos/efeitos dos fármacos , Praguicidas/química , Proteínas de Plantas/química , Sementes/química , Testes de ToxicidadeRESUMO
BACKGROUND: Because of the increasingly concern of consumers and public policy about problems for environment and for public health due to chemical pesticides, the search for molecules more safe is currently of great importance. Particularly, plants are able to fight the pathogens as insects, bacteria or fungi; so that plants could represent a valuable source of new molecules. RESULTS: It was observed that Medicago truncatula seed flour displayed a strong toxic activity towards the adults of the rice weevil Sitophilus oryzae (Coleoptera), a major pest of stored cereals. The molecule responsible for toxicity was purified, by solvent extraction and HPLC, and identified as a saponin, namely 3-GlcA-28-AraRhaxyl-medicagenate. Saponins are detergents, and the CMC of this molecule was found to be 0.65 mg per mL. Neither the worm Caenorhabditis elegans nor the bacteria E. coli were found to be sensitive to this saponin, but growth of the yeast Saccharomyces cerevisiae was inhibited at concentrations higher than 100 µg per mL. The purified molecule is toxic for the adults of the rice weevils at concentrations down to 100 µg per g of food, but this does not apply to the others insects tested, including the coleopteran Tribolium castaneum and the Sf9 insect cultured cells. CONCLUSIONS: This specificity for the weevil led us to investigate this saponin potential for pest control and to propose the hypothesis that this saponin has a specific mode of action, rather than acting via its non-specific detergent properties.
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Methods that use homologous recombination to engineer the genome of C. elegans commonly use strains carrying specific insertions of the heterologous transposon Mos1. A large collection of known Mos1 insertion alleles would therefore be of general interest to the C. elegans research community. We describe here the optimization of a semi-automated methodology for the construction of a substantial collection of Mos1 insertion mutant strains. At peak production, more than 5,000 strains were generated per month. These strains were then subject to molecular analysis, and more than 13,300 Mos1 insertions characterized. In addition to targeting directly more than 4,700 genes, these alleles represent the potential starting point for the engineered deletion of essentially all C. elegans genes and the modification of more than 40% of them. This collection of mutants, generated under the auspices of the European NEMAGENETAG consortium, is publicly available and represents an important research resource.
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Caenorhabditis elegans/genética , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA , Engenharia Genética/métodos , Genoma/genética , Recombinação Genética , Transposases , Animais , Animais Geneticamente Modificados , Recombinação Homóloga , Mutagênese Insercional , PesquisaRESUMO
We previously reported the use of the cheap and fast-growing nematode Caenorhabditis elegans to search for molecules, which reduce muscle degeneration in a model for Duchenne Muscular Dystrophy (DMD). We showed that Prednisone, a steroid that is generally prescribed as a palliative treatment to DMD patients, also reduced muscle degeneration in the C. elegans DMD model. We further showed that this strategy could lead to the discovery of new and unsuspected small molecules, which have been further validated in a mammalian model of DMD, i.e. the mdx mouse model. These proof-of-principles demonstrate that C. elegans can serve as a screening tool to search for drugs against neuromuscular disorders. Here, we report and discuss two methodologies used to screen chemical libraries for drugs against muscle disorders in C. elegans. We first describe a manual method used to find drugs against DMD. We further present a semi-automated method, which is currently in use for the search of drugs against the Schwartz-Jampel Syndrome (SJS). Both assays are simple to implement and can be readily transposed and/or adapted to screens against other muscle/neuromuscular diseases, which can be modeled in the worm. Finally we discuss, with respect to our experience and knowledge, the different parameters that have to be taken into account before choosing one or the other method.
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Caenorhabditis elegans/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Automação , Modelos Animais de Doenças , Humanos , Distrofia Muscular Animal , OsteocondrodisplasiasRESUMO
Duchenne muscular dystrophy, a genetic disease caused by the absence of functional dystrophin, remains without adequate treatment. Although great hopes are attached to gene and cell therapies, identification of active small molecules remains a valid option for new treatments. We have studied the effect of 20 approved pharmaceutical compounds on the muscles of dystrophin-deficient mdx5Cv mice. These compounds were selected as the result of a prior screen of 800 approved molecules on a dystrophin mutant of the invertebrate animal model Cænorhabditis elegans. Drugs were administered to the mice through maternal feeding since 2weeks of life and mixed in their food after the 3rd week of life. The effects of the drugs on mice were evaluated both at 6weeks and 16weeks. Each drug was tested at two concentrations. Prednisone was added to the molecule list as a positive control. To investigate treatment efficiency, more than 30 histological, biochemical and functional parameters were recorded. This extensive study reveals that tricyclics (Imipramine and Amitriptyline) are beneficial to the fast muscles of mdx mice. It also highlights a great variability of responses according to time, muscles and assays.
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Aprovação de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Distrofia Muscular de Duchenne/tratamento farmacológico , Animais , Creatina Quinase/sangue , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Composição de Medicamentos , Distrofina/genética , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Contração Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/fisiologia , Músculos/efeitos dos fármacos , Músculos/patologia , Distrofia Muscular de Duchenne/sangue , Distrofia Muscular de Duchenne/patologiaRESUMO
Serotonin (5-HT) regulates a wide range of behaviors in Caenorhabditis elegans, including egg laying, male mating, locomotion and pharyngeal pumping. So far, four serotonin receptors have been described in the nematode C. elegans, three of which are G protein-coupled receptors (GPCR), (SER-1, SER-4 and SER-7), and one is an ion channel (MOD-1). By searching the C. elegans genome for additional 5-HT GPCR genes, we identified five further genes which encode putative 5-HT receptors, based on sequence similarities to 5-HT receptors from other species. Using loss-of-function mutants and RNAi, we performed a systematic study of the role of the eight GPCR genes in serotonin-modulated behaviors of C. elegans (F59C12.2, Y22D7AR.13, K02F2.6, C09B7.1, M03F4.3, F16D3.7, T02E9.3, C24A8.1). We also examined their expression patterns. Finally, we tested whether the most likely candidate receptors were able to modulate adenylate cyclase activity in transfected cells in a 5-HT-dependent manner. This paper is the first comprehensive study of G protein-coupled serotonin receptors of C. elegans. It provides a direct comparison of the expression patterns and functional roles for 5-HT receptors in C. elegans.
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Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Expressão Gênica , Receptores Acoplados a Proteínas G/genética , Receptores de Serotonina/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Células COS , Proteínas de Caenorhabditis elegans/metabolismo , Chlorocebus aethiops , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Interferência de RNA , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Serotonina/metabolismo , Comportamento Reprodutivo , Homologia de Sequência de AminoácidosRESUMO
Prevention of muscle fiber degeneration is a key issue in the treatment of muscular dystrophies such as Duchenne Muscular Dystrophy (DMD). It is widely postulated that existing pharmaceutical compounds might potentially be beneficial to DMD patients, but tools to identify them are lacking. Here, by using a Caenorhabditis elegans model of dystrophin-dependent muscular dystrophy, we show that the neurohormone serotonin and some of its agonists are potent suppressors of muscle degeneration. Inhibitors of serotonin reuptake transporters, which prolong the action of endogenous serotonin, have a similar effect. Moreover, reduction of serotonin levels leads to degeneration of non-dystrophic muscles. Our results demonstrate that serotonin is critical to C. elegans striated muscles. These findings reveal a new function of serotonin in striated muscles.
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Músculo Esquelético/efeitos dos fármacos , Distrofia Muscular Animal/tratamento farmacológico , Distrofia Muscular de Duchenne/tratamento farmacológico , Serotoninérgicos/uso terapêutico , Serotonina/uso terapêutico , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Distrofina/deficiência , Distrofina/genética , Distrofina/metabolismo , Cinética , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Distrofia Muscular Animal/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Fatores de TempoRESUMO
The Caenorhabditis elegans SLO-1 channel belongs to the family of calcium-activated large conductance BK potassium channels. SLO-1 has been shown to be involved in neurotransmitter release and ethanol response. Here, we report that SLO-1 also has a critical role in muscles. Inactivation of the slo-1 gene in muscles leads to phenotypes similar to those caused by mutations of the dystrophin homologue dys-1. Notably, slo-1 mutations result in a progressive muscle degeneration when put into a sensitized genetic background. slo-1 localization was observed by gfp reporter gene in both the M-line and the dense bodies (Z line) of the C.elegans body-wall muscles. Using the inside-out configuration of the patch clamp technique on body-wall muscle cells of acutely dissected wild-type worms, we characterized a Ca2+-activated K+ channel that was identified unambiguously as SLO-1. Since neither the abundance nor the conductance of SLO-1 was changed significantly in dys-1 mutants compared to wild-type animals, it is likely that the inactivation of dys-1 causes a misregulation of SLO-1. All in all, these results indicate that SLO-1 function in C.elegans muscles is related to the dystrophin homologue DYS-1.
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Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/metabolismo , Distrofina/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Músculos/metabolismo , Distrofia Muscular Animal/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Caenorhabditis elegans/metabolismo , Músculos/patologia , Distrofia Muscular Animal/patologia , Mutação/genética , Fenótipo , Canais de Potássio Cálcio-Ativados/metabolismoRESUMO
Duchenne muscular dystrophy is a degenerative muscular disease caused by mutations in the dystrophin gene. There is no curative treatment against Duchenne muscular dystrophy. In several countries, the steroid prednisone (or analogs) is prescribed as a palliative treatment. In the model animal Caenorhabditis elegans, mutations of the dys-1 dystrophin-like gene lead to a muscular degenerative phenotype when they are associated with a mild MyoD mutation. This cheap and fast-growing model of dystrophinopathy may be used to screen for molecules able to slow muscle degeneration. In a blind screen of approximately 100 compounds covering a wide spectrum of targets, we found that prednisone is beneficial to the C. elegans dystrophin-deficient muscles. Prednisone reduces by 40% the number of degenerating cells in this animal. This result is a proof-of-principle for the use of C. elegans as a tool in the search for molecules active against the effects of dystrophin-deficiency. Moreover, since C. elegans is not susceptible to inflammation, this suggests that prednisone exerts a direct effect on muscle survival.
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Distrofina/deficiência , Distrofia Muscular de Duchenne/tratamento farmacológico , Prednisona/uso terapêutico , Animais , Anti-Inflamatórios/uso terapêutico , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Distrofina/genética , Distrofina/metabolismo , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Músculos/efeitos dos fármacos , Distrofia Muscular de Duchenne/metabolismo , Mutação , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Fatores de Tempo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
Syntrophins are a family of PDZ domain-containing adaptor proteins required for receptor localization. Syntrophins are also associated with the dystrophin complex in muscles. We report here the molecular and functional characterization of the Caenorhabditis elegans gene stn-1 (F30A10.8), which encodes a syntrophin with homology to vertebrate alpha and beta-syntrophins. stn-1 is expressed in neurons and in muscles of C.elegans. stn-1 mutants resemble dystrophin (dys-1) and dystrobrevin (dyb-1) mutants: they are hyperactive, bend their heads when they move forward, tend to hypercontract, and are hypersensitive to the acetylcholinesterase inhibitor aldicarb. These phenotypes are suppressed when stn-1 is expressed under the control of a muscular promoter, indicating that they are caused by the absence of stn-1 in muscles. These results suggest that the role of syntrophin is linked to dystrophin function in C.elegans.