Chemical genetics unveils a key role of mitochondrial dynamics, cytochrome c release and IP3R activity in muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by mutations in the dystrophin gene. The subcellular mechanisms of DMD remain poorly understood and there is currently no curative treatment available. Using a Caenorhabditis elegans model for DMD as a pharmacologic and genetic tool, we found that cyclosporine A (CsA) reduces muscle degeneration at low dose and acts, at least in part, through a mitochondrial cyclophilin D, CYN-1. We thus hypothesized that CsA acts on mitochondrial permeability modulation through cyclophilin D inhibition. Mitochondrial patterns and dynamics were analyzed, which revealed dramatic mitochondrial fragmentation not only in dystrophic nematodes, but also in a zebrafish model for DMD. This abnormal mitochondrial fragmentation occurs before any obvious sign of degeneration can be detected. Moreover, we demonstrate that blocking/delaying mitochondrial fragmentation by knocking down the fission-promoting gene drp-1 reduces muscle degeneration and improves locomotion abilities of dystrophic nematodes. Further experiments revealed that cytochrome c is involved in muscle degeneration in C. elegans and seems to act, at least in part, through an interaction with the inositol trisphosphate receptor calcium channel, ITR-1. Altogether, our findings reveal that mitochondria play a key role in the early process of muscle degeneration and may be a target of choice for the design of novel therapeutics for DMD. In addition, our results provide the first indication in the nematode that (i) mitochondrial permeability transition can occur and (ii) cytochrome c can act in cell death.

Caenorhabditis elegans as a chemical screening tool for the study o f neuromuscular disorders. Manual and semi-automated methods.

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.

Evaluation of the therapeutic potential of carbonic anhydrase inhibitors in two animal models of dystrophin deficient muscular dystrophy.

Duchenne Muscular Dystrophy is an inherited muscle degeneration disease for which there is still no efficient treatment. However, compounds active on the disease may already exist among approved drugs but are difficult to identify in the absence of cellular models. We used the Caenorhabditis elegans animal model to screen a collection of 1000 already approved compounds. Two of the most active hits obtained were methazolamide and dichlorphenamide, carbonic anhydrase inhibitors widely used in human therapy. In C. elegans, these drugs were shown to interact with CAH-4, a putative carbonic anhydrase. The therapeutic efficacy of these compounds was further validated in long-term experiments on mdx mice, the mouse model of Duchenne Muscular Dystrophy. Mice were treated for 120 days with food containing methazolamide or dichlorphenamide at two doses each. Musculus tibialis anterior and diaphragm muscles were histologically analyzed and isometric muscle force was measured in M. extensor digitorum longus. Both substances increased the tetanic muscle force in the treated M. extensor digitorum longus muscle group, dichlorphenamide increased the force significantly by 30%, but both drugs failed to increase resistance of muscle fibres to eccentric contractions. Histological analysis revealed a reduction of centrally nucleated fibers in M. tibialis anterior and diaphragm in the treated groups. These studies further demonstrated that a C. elegans-based screen coupled with a mouse model validation strategy can lead to the identification of potential pharmacological agents for rare diseases.

Metabotyping of Caenorhabditis elegans reveals latent phenotypes.

Assigning functions to every gene in a living organism is the next challenge for functional genomics. In fact, 85-90% of the 19,000 genes of the nematode Caenorhabditis elegans genome do not produce any visible phenotype when inactivated, which hampers determining their function, especially when they do not belong to previously characterized gene families. We used (1)H high-resolution magic angle spinning NMR spectroscopy ((1)H HRMAS-NMR) to reveal the latent phenotype associated to superoxide dismutase (sod-1) and catalase (ctl-1) C. elegans mutations, both involved in the elimination of radical oxidative species. These two silent mutations are significantly discriminated from the wild-type strain and from each other. We identify a metabotype significantly associated with these mutations involving a general reduction of fatty acyl resonances from triglycerides, unsaturated lipids being known targets of free radicals. This work opens up perspectives for the use of (1)H HRMAS-NMR as a molecular phenotyping device for model organisms. Because it is amenable to high throughput and is shown to be highly informative, this approach may rapidly lead to a functional and integrated metabonomic mapping of the C. elegans genome at the systems biology level.

The L-type voltage-dependent Ca2+ channel EGL-19 controls body wall muscle function in Caenorhabditis elegans.

Caenorhabditis elegans is a powerful model system widely used to investigate the relationships between genes and complex behaviors like locomotion. However, physiological studies at the cellular level have been restricted by the difficulty to dissect this microscopic animal. Thus, little is known about the properties of body wall muscle cells used for locomotion. Using in situ patch clamp technique, we show that body wall muscle cells generate spontaneous spike potentials and develop graded action potentials in response to injection of positive current of increasing amplitude. In the presence of K+ channel blockers, membrane depolarization elicited Ca2+ currents inhibited by nifedipine and exhibiting Ca2+-dependent inactivation. Our results give evidence that the Ca2+ channel involved belongs to the L-type class and corresponds to EGL-19, a putative Ca2+ channel originally thought to be a member of this class on the basis of genomic data. Using Ca2+ fluorescence imaging on patch-clamped muscle cells, we demonstrate that the Ca2+ transients elicited by membrane depolarization are under the control of Ca2+ entry through L-type Ca2+ channels. In reduction of function egl-19 mutant muscle cells, Ca2+ currents displayed slower activation kinetics and provided a significantly smaller Ca2+ entry, whereas the threshold for Ca2+ transients was shifted toward positive membrane potentials.

Other model organisms for sarcomeric muscle diseases.

Model organisms are vital to our understanding of human muscle biology and disease. The potential of the nematode Caenorhabditis elegans, the fruitfly, Drosophila melanogaster and the zebrafish, Danio rerio, as model genetic organisms for the study of human muscle disease is discussed by examining their muscle biology, muscle genetics and development. The powerful genetic tools available with each organism are outlined. It is concluded that these organisms have already demonstrated potential in facilitating the study of muscle disease and in screening for therapeutic agents.

Dystrophin-dependent muscle degeneration requires a fully functional contractile machinery to occur in C. elegans.

In mammals, the lack of dystrophin leads to a degeneration of skeletal muscles. It has been known for many years that this pathology can be blocked by denervation or immobilization of muscles. It is not yet clear, however, whether this suppressing effect is due to the absence of fiber contraction per se, or to other mechanisms which may be induced by such treatments. We took advantage of the genetic tools available in the animal model Caenorhabditis elegans to address this question. Using RNA interference and existing mutants, we genetically impaired the excitation-contraction cascade at specific points in a dystrophin-deficient C. elegans strain which normally undergoes extensive muscle degeneration. Our data show that reducing sarcomere contraction by slightly impairing the contraction machinery is sufficient to dramatically suppress muscle degeneration. Thus, it is the physical tension exerted on the muscle fibers which is the key deleterious event in the absence of dystrophin.

The SLO-1 BK channel of Caenorhabditis elegans is critical for muscle function and is involved in dystrophin-dependent muscle dystrophy.

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.

Mos as a tool for genome-wide insertional mutagenesis in Caenorhabditis elegans: results of a pilot study.

The sequence of the Caenorhabditis elegans genome contains approximately 19 000 genes. Available mutants currently exist for <20% of these genes. The existence of a Mos-based inducible transposon system in C.elegans could theoretically serve as a tool to saturate the genome with insertions. We report here the results of a pilot study aimed at assaying this strategy. We generated 914 independent random Mos insertions and determined their location by inverse PCR. The distribution of the insertions throughout the genome does not reveal any gross distortion, with the exception of a major hotspot on chromosome I (rDNA locus). Transposons are evenly distributed between the genic and intergenic regions. Within genes, transposons insert preferentially into the introns. We derived the consensus target site for Mos in C.elegans (ATATAT), which is common to Tc1, another mariner element. Finally, we assayed the mutagenic properties of insertions located in exons by comparing the phenotype of homozygous strains to that of known mutations or RNAi of the same gene. This pilot experiment shows that a Mos-based approach is a viable strategy that can contribute to the constitution of genome-wide collections of identified C.elegans mutants.

Characterization of the Caenorhabditis elegans G protein-coupled serotonin receptors.

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.

The stn-1 syntrophin gene of C.elegans is functionally related to dystrophin and dystrobrevin.

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.

Patterns of selection against transposons inferred from the distribution of Tc1, Tc3 and Tc5 insertions in the mut-7 line of the nematode Caenorhabditis elegans.

To identify the factors (selective or mutational) that affect the distribution of transposable elements (TEs) within a genome, it is necessary to compare the pattern of newly arising element insertions to the pattern of element insertions that have been fixed in a population. To do this, we analyzed the distribution of recent mutant insertions of the Tc1, Tc3, and Tc5 elements in a mut-7 background of the nematode Caenorhabditis elegans and compared it to the distribution of element insertions (presumably fixed) within the sequenced genome. Tc1 elements preferentially insert in regions with high recombination rates, whereas Tc3 and Tc5 do not. Although Tc1 and Tc3 both insert in TA dinucleotides, there is no clear relationship between the frequency of insertions and the TA dinucleotide density. There is a strong selection against TE insertions within coding regions: the probability that a TE will be fixed is at least 31 times lower in coding regions than in noncoding regions. Contrary to the prediction of theoretical models, we found that the selective pressure against TE insertions does not increase with the recombination rate. These findings indicate that the distribution of these three transposon families in the genome of C. elegans is determined essentially by just two factors: the pattern of insertions, which is a characteristic of each family, and the selection against insertions within coding regions.

Identification of 1088 new transposon insertions of Caenorhabditis elegans: a pilot study toward large-scale screens.

We explored the feasibility of a strategy based on transposons to generate identified mutants of most Caenorhabditis elegans genes. A total of 1088 random new insertions of C. elegans transposons Tc1, Tc3, and Tc5 were identified by anchored PCR, some of which result in a mutant phenotype.

Genetic evidence for a dystrophin-glycoprotein complex (DGC) in Caenorhabditis elegans.

Dystrophin is the product of the gene mutated in Duchenne muscular dystrophy (DMD). Neither the function of dystrophin nor the physiopathology of the disease have been clearly established so far. In mammals, the dystrophin-glycoprotein complex (DGC) includes dystrophin, as well as transmembrane and cytoplasmic proteins. Since Caenorhabditis elegans possesses a dystrophin-like gene (dys-1), we investigated whether homologues of the DGC members could also be found in the C. elegans genome. Conserved homologues were found for dystroglycan, delta/gamma-sarcoglycan and syntrophin. Divergent but related proteins were found for alpha- and beta-sarcoglycans. No sarcospan counterpart was found. The expression of the conserved homologues was inactivated using the RNA interference technique. Phenotypes similar to that of dys-1 were obtained, both in the wild-type background and in combination with other mutations. These results strongly suggest that a protein complex comprising functional analogies with the DGC exists in C. elegans.

Dystrophin and functionally related proteins in the nematode Caenorhabditis elegans.

We investigated the function of dystrophin in the nematode Caenorhabditis elegans. Although nematodes and mammals diverged early in evolution, their muscles share many structural and molecular features, thus rendering C. elegans relevant as a model to study muscle function. Dystrophin, dystrobrevin, dystroglycans and several sarcoglycans have conserved homologues in the genome of C. elegans. The major strength of the model comes from its genetic tractability, which allows the quick and easy manipulation of gene expression, either to inactivate genes, or to create transgenic animals. Over the last 2 years, work on C. elegans dystrophin has led to the identification of a putative new member of the dystrophin-glycoprotein complex, and has brought additional data suggesting that dystrophin mutations affect ion channel function.

Overexpression of dystrobrevin delays locomotion defects and muscle degeneration in a dystrophin-deficient Caenorhabditis elegans.

Duchenne muscular dystrophy is one of the most common neuromuscular diseases. It is caused by mutations in the dystrophin gene. Dystrobrevins are dystrophin-associated proteins potentially involved in signal transduction. The nematode Caenorhabditis elegans possesses one dystrophin-like (dys-1) and one dystrobrevin-like (dyb-1) gene. Mutations of dyb-1 and dys-1 lead to similar phenotypes, comprising hyperactivity and a tendency to hypercontract, which suggest that these proteins may participate in a common function. We show here that overexpression of the Dyb-1 protein delays the onset of the myopathy observed in the C. elegans double mutant (dys-1; hlh-1 mutations). This finding indicates that, in C. elegans, (1) the absence of dystrophin can be partly compensated for by extra doses of dystrobrevin, and (2) dystrobrevin is partly functional in absence of dystrophin.

Prednisone reduces muscle degeneration in dystrophin-deficient Caenorhabditis elegans.

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.

[C. elegans as a model for human inherited degenerative diseases]. / C. elegans comme modèle pour les maladies dégénératives héréditaires humaines.

The nematode C. elegans is an established model for developmental biology. Since the early 90's, this simple model organism has been increasingly used for studying human disease pathogenesis. C. elegans models based either on the mutagenesis of human disease genes conserved in this nematode or transgenesis with disease genes not conserved in C. elegans show several features that are observed in mammalian models. These observations suggest that the genetic dissection and pharmacological manipulation of disease-like phenotypes in C. elegans will shed light on the cellular mechanisms that are altered in human diseases, and the compounds that may be used as drugs. This review illustrates these aspects by commenting on two inherited degenerative diseases, Duchenne's muscular dystrophy and Huntington's neurodegenerative disease.

ZYX-1, the unique zyxin protein of Caenorhabditis elegans, is involved in dystrophin-dependent muscle degeneration.

In vertebrates, zyxin is a LIM-domain protein belonging to a family composed of seven members. We show that the nematode Caenorhabditis elegans has a unique zyxin-like protein, ZYX-1, which is the orthologue of the vertebrate zyxin subfamily composed of zyxin, migfilin, TRIP6, and LPP. The ZYX-1 protein is expressed in the striated body-wall muscles and localizes at dense bodies/Z-discs and M-lines, as well as in the nucleus. In yeast two-hybrid assays ZYX-1 interacts with several known dense body and M-line proteins, including DEB-1 (vinculin) and ATN-1 (α-actinin). ZYX-1 is mainly localized in the middle region of the dense body/Z-disk, overlapping the apical and basal regions containing, respectively, ATN-1 and DEB-1. The localization and dynamics of ZYX-1 at dense bodies depend on the presence of ATN-1. Fluorescence recovery after photobleaching experiments revealed a high mobility of the ZYX-1 protein within muscle cells, in particular at dense bodies and M-lines, indicating a peripheral and dynamic association of ZYX-1 at these muscle adhesion structures. A portion of the ZYX-1 protein shuttles from the cytoplasm into the nucleus, suggesting a role for ZYX-1 in signal transduction. We provide evidence that the zyx-1 gene encodes two different isoforms, ZYX-1a and ZYX-1b, which exhibit different roles in dystrophin-dependent muscle degeneration occurring in a C. elegans model of Duchenne muscular dystrophy.