Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 17 de 17
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Sci Rep ; 9(1): 17841, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31780714

RESUMO

Cone snails use separately evolved venoms for prey capture and defence. While most use a harpoon for prey capture, the Gastridium clade that includes the well-studied Conus geographus and Conus tulipa, have developed a net hunting strategy to catch fish. This unique feeding behaviour requires secretion of "nirvana cabal" peptides to dampen the escape response of targeted fish allowing for their capture directly by mouth. However, the active components of the nirvana cabal remain poorly defined. In this study, we evaluated the behavioural effects of likely nirvana cabal peptides on the teleost model, Danio rerio (zebrafish). Surprisingly, the conantokins (NMDA receptor antagonists) and/or conopressins (vasopressin receptor agonists and antagonists) found in C. geographus and C. tulipa venom failed to produce a nirvana cabal-like effect in zebrafish. In contrast, low concentrations of the non-competitive adrenoceptor antagonist ρ-TIA found in C. tulipa venom (EC50 = 190 nM) dramatically reduced the escape response of zebrafish larvae when added directly to aquarium water. ρ-TIA inhibited the zebrafish α1-adrenoceptor, confirming ρ-TIA has the potential to reverse the known stimulating effects of norepinephrine on fish behaviour. ρ-TIA may act alone and not as part of a cabal, since it did not synergise with conopressins and/or conantokins. This study highlights the importance of using ecologically relevant animal behaviour models to decipher the complex neurobiology underlying the prey capture and defensive strategies of cone snails.

2.
JAMA Psychiatry ; 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31268507

RESUMO

Importance: Genome-wide association studies (GWASs) in European populations have identified more than 100 schizophrenia-associated loci. A schizophrenia GWAS in a unique Indian population offers novel findings. Objective: To discover and functionally evaluate genetic loci for schizophrenia in a GWAS of a unique Indian population. Design, Setting, and Participants: This GWAS included a sample of affected individuals, family members, and unrelated cases and controls. Three thousand ninety-two individuals were recruited and diagnostically ascertained via medical records, hospitals, clinics, and clinical networks in Chennai and surrounding regions. Affected participants fulfilled DSM-IV diagnostic criteria for schizophrenia. Unrelated control participants had no personal or family history of psychotic disorder. Recruitment, genotyping, and analysis occurred in consecutive phases beginning January 1, 2001. Recruitment was completed on February 28, 2018, and genotyping and analysis are ongoing. Main Outcomes and Measures: Associations of single-nucleotide polymorphisms and gene expression with schizophrenia. Results: The study population included 1321 participants with schizophrenia, 885 family controls, and 886 unrelated controls. Among participants with schizophrenia, mean (SD) age was 39.1 (11.4) years, and 52.7% were male. This sample demonstrated uniform ethnicity, a degree of inbreeding, and negligible rates of substance abuse. A novel genome-wide significant association was observed between schizophrenia and a chromosome 8q24.3 locus (rs10866912, allele A; odds ratio [OR], 1.27 [95% CI, 1.17-1.38]; P = 4.35 × 10-8) that attracted support in the schizophrenia Psychiatric Genomics Consortium 2 data (rs10866912, allele A; OR, 1.04 [95% CI, 1.02-1.06]; P = 7.56 × 10-4). This locus has undergone natural selection, with the risk allele A declining in frequency from India (approximately 72%) to Europe (approximately 43%). rs10866912 directly modifies the abundance of the nicotinate phosphoribosyltransferase gene (NAPRT1) transcript in brain cortex (normalized effect size, 0.79; 95% CI, 0.6-1.0; P = 5.8 × 10-13). NAPRT1 encodes a key enzyme for niacin metabolism. In Indian lymphoblastoid cell lines, (risk) allele A of rs10866912 was associated with NAPRT1 downregulation (AA: 0.74, n = 21; CC: 1.56, n = 17; P = .004). Preliminary zebrafish data further suggest that partial loss of function of NAPRT1 leads to abnormal brain development. Conclusions and Relevance: Bioinformatic analyses and cellular and zebrafish gene expression studies implicate NAPRT1 as a novel susceptibility gene. Given this gene's role in niacin metabolism and the evidence for niacin deficiency provoking schizophrenialike symptoms in neuropsychiatric diseases such as pellagra and Hartnup disease, these results suggest that the rs10866912 genotype and niacin status may have implications for schizophrenia susceptibility and treatment.

3.
Curr Biol ; 27(13): 1968-1981.e7, 2017 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-28648821

RESUMO

The embryonic notochord is a flexible structure present during development that serves as scaffold for formation of the vertebrate spine. This rod-like organ is thought to have evolved in non-vertebrate chordates to facilitate locomotion by providing a rigid but flexible midline structure against which the axial muscles can contract. This hydrostatic "skeleton" is exposed to a variety of mechanical forces during oscillation of the body. There is evidence that caveolae, submicroscopic cup-shaped plasma membrane pits, can buffer tension in cells that undergo high levels of mechanical stress. Indeed, caveolae are particularly abundant in the embryonic notochord. In this study, we used the CRISPR/Cas9 system to generate a mutant zebrafish line lacking Cavin1b, a coat protein required for caveola formation. Our cavin1b-/- zebrafish line exhibits reduced locomotor capacity and prominent notochord lesions characterized by necrotic, damaged, and membrane-permeable cells. Notochord diameter and body length are reduced, but remarkably, the mutants recover and are homozygous viable. By manipulating mechanical stress using a number of different assays, we show that progression of lesion severity in the mutant notochord is directly dependent on locomotion. We also demonstrate changes in caveola morphology in vivo in response to mechanical stress. Finally, induction of a catastrophic collapse of live cavin1b-/- mutant notochord cells provides the first real-time observation of caveolae mediating cellular mechanoprotection.


Assuntos
Cavéolas/metabolismo , Notocorda/embriologia , Peixe-Zebra/embriologia , Animais , Fenômenos Biomecânicos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Estresse Mecânico , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
4.
Hum Mol Genet ; 25(9): 1728-38, 2016 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-26908606

RESUMO

Spinal muscular atrophy (SMA) is an autosomal recessive disease linked to survival motor neuron (SMN) protein deficiency. While SMN protein is expressed ubiquitously, its deficiency triggers tissue-specific hallmarks, including motor neuron death and muscle atrophy, leading to impaired motor functions and premature death. Here, using stable miR-mediated knockdown technology in zebrafish, we developed the first vertebrate system allowing transgenic spatio-temporal control of the smn1 gene. Using this new model it is now possible to investigate normal and pathogenic SMN function(s) in specific cell types, independently or in synergy with other cell populations. We took advantage of this new system to first test the effect of motor neuron or muscle-specific smn1 silencing. Anti-smn1 miRNA expression in motor neurons, but not in muscles, reproduced SMA hallmarks, including abnormal motor neuron development, poor motor function and premature death. Interestingly, smn1 knockdown in motor neurons also induced severe late-onset phenotypes including scoliosis-like body deformities, weight loss, muscle atrophy and, seen for the first time in zebrafish, reduction in the number of motor neurons, indicating motor neuron degeneration. Taken together, we have developed a new transgenic system allowing spatio-temporal control of smn1 expression in zebrafish, and using this model, we have demonstrated that smn1 silencing in motor neurons alone is sufficient to reproduce SMA hallmarks in zebrafish. It is noteworthy that this research is going beyond SMA as this versatile gene-silencing transgenic system can be used to knockdown any genes of interest, filling the gap in the zebrafish genetic toolbox and opening new avenues to study gene functions in this organism.


Assuntos
Embrião não Mamífero/citologia , MicroRNAs/genética , Neurônios Motores/patologia , Músculo Esquelético/patologia , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Neurônios Motores/metabolismo , Músculo Esquelético/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/antagonistas & inibidores , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Peixe-Zebra
5.
Dev Neurobiol ; 76(5): 507-18, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26184457

RESUMO

During early development of the central nervous system (CNS), a subset of yolk-sac derived myeloid cells populate the brain and provide the seed for the microglial cell population, which will self-renew throughout life. As development progresses, individual microglial cells transition from a phagocytic amoeboid state through a transitional morphing phase into the sessile, ramified, and normally nonphagocytic microglia observed in the adult CNS under healthy conditions. The molecular drivers of this tissue-specific maturation profile are not known. However, a survey of tissue resident macrophages identified miR-124 to be expressed in microglia. In this study, we used transgenic zebrafish to overexpress miR-124 in the mpeg1 expressing yolk-sac-derived myeloid cells that seed the microglia. In addition, a systemic sponge designed to neutralize the effects of miR-124 was used to assess microglial development in a miR-124 loss-of-function environment. Following the induction of miR-124 overexpression, microglial motility and phagocytosis of apoptotic cells were significantly reduced. miR-124 overexpression in microglia resulted in the accumulation of residual apoptotic cell bodies in the optic tectum, which could not be achieved by miR-124 overexpression in differentiated neurons. Conversely, expression of the miR-124 sponge caused an increase in the motility of microglia and transiently rescued motility and phagocytosis functions when activated simultaneously with miR-124 overexpression. This study provides in vivo evidence that miR-124 activity has a key role in the development of functionally mature microglia.


Assuntos
MicroRNAs/metabolismo , Microglia/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose/fisiologia , Movimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Técnicas de Silenciamento de Genes , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/genética , Neurônios/metabolismo , Fagocitose/fisiologia , Homologia de Sequência , Colículos Superiores/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
6.
J Proteome Res ; 14(10): 4372-81, 2015 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-26322961

RESUMO

Venomous marine cone snails produce a unique and remarkably diverse range of venom peptides (conotoxins and conopeptides) that have proven to be invaluable as pharmacological probes and leads to new therapies. Conus catus is a hook-and-line fish hunter from clade I, with ∼20 conotoxins identified, including the analgesic ω-conotoxin CVID (AM336). The current study unravels the venom composition of C. catus with tandem mass spectrometry and 454 sequencing data. From the venom gland transcriptome, 104 precursors were recovered from 11 superfamilies, with superfamily A (especially κA-) conotoxins dominating (77%) their venom. Proteomic analysis confirmed that κA-conotoxins dominated the predation-evoked milked venom of each of six C. catus analyzed and revealed remarkable intraspecific variation in both the intensity and type of conotoxins. High-throughput FLIPR assays revealed that the predation-evoked venom contained a range of conotoxins targeting the nAChR, Cav, and Nav ion channels, consistent with α- and ω-conotoxins being used for predation by C. catus. However, the κA-conotoxins did not act at these targets but induced potent and rapid immobilization followed by bursts of activity and finally paralysis when injected intramuscularly in zebrafish. Our venomics approach revealed the complexity of the envenomation strategy used by C. catus, which contains a mix of both excitatory and inhibitory venom peptides.


Assuntos
Bloqueadores dos Canais de Cálcio/isolamento & purificação , Conotoxinas/isolamento & purificação , Caramujo Conus/química , Venenos de Moluscos/isolamento & purificação , Antagonistas Nicotínicos/isolamento & purificação , Bloqueadores dos Canais de Potássio/isolamento & purificação , Sequência de Aminoácidos , Animais , Organismos Aquáticos , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/toxicidade , Canais de Cálcio/metabolismo , Conotoxinas/química , Conotoxinas/toxicidade , Caramujo Conus/fisiologia , Anotação de Sequência Molecular , Dados de Sequência Molecular , Venenos de Moluscos/química , Venenos de Moluscos/toxicidade , Atividade Motora/efeitos dos fármacos , Antagonistas Nicotínicos/química , Antagonistas Nicotínicos/toxicidade , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/toxicidade , Canais de Potássio/metabolismo , Comportamento Predatório/fisiologia , Receptores Nicotínicos/metabolismo , Especificidade da Espécie , Transcriptoma , Peixe-Zebra/fisiologia
7.
Hum Genet ; 134(11-12): 1163-82, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26337422

RESUMO

Protein-coding mutations in the transcription factor-encoding gene ARX cause various forms of intellectual disability (ID) and epilepsy. In contrast, variations in surrounding non-coding sequences are correlated with milder forms of non-syndromic ID and autism and had suggested the importance of ARX gene regulation in the etiology of these disorders. We compile data on several novel and some already identified patients with or without ID that carry duplications of ARX genomic region and consider likely genetic mechanisms underlying the neurodevelopmental defects. We establish the long-range regulatory domain of ARX and identify its brain region-specific autoregulation. We conclude that neurodevelopmental disturbances in the patients may not simply arise from increased dosage due to ARX duplication. This is further exemplified by a small duplication involving a non-functional ARX copy, but with duplicated enhancers. ARX enhancers are located within a 504-kb region and regulate expression specifically in the forebrain in developing and adult zebrafish. Transgenic enhancer-reporter lines were used as in vivo tools to delineate a brain region-specific negative and positive autoregulation of ARX. We find autorepression of ARX in the telencephalon and autoactivation in the ventral thalamus. Fluorescently labeled brain regions in the transgenic lines facilitated the identification of neuronal outgrowth and pathfinding disturbances in the ventral thalamus and telencephalon that occur when arxa dosage is diminished. In summary, we have established a model for how breakpoints in long-range gene regulation alter the expression levels of a target gene brain region-specifically, and how this can cause subtle neuronal phenotypes relating to the etiology of associated neuropsychiatric disease.


Assuntos
Variações do Número de Cópias de DNA , Duplicação Gênica , Proteínas de Homeodomínio/genética , Deficiência Intelectual/genética , Fatores de Transcrição/genética , Adulto , Animais , Animais Geneticamente Modificados , Encéfalo/embriologia , Encéfalo/metabolismo , Estudos de Casos e Controles , Embrião não Mamífero , Feminino , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Fatores de Transcrição/metabolismo , Peixe-Zebra
8.
Nat Commun ; 6: 7378, 2015 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-26051838

RESUMO

Although zebrafish is used to model human diseases through mutational and morpholino-based knockdown approaches, there are currently no robust transgenic knockdown tools. Here we investigate the knockdown efficiency of three synthetic miRNA-expressing backbones and show that these constructs can downregulate a sensor transgene with different degrees of potency. Using this approach, we reproduce spinal muscular atrophy (SMA) in zebrafish by targeting the smn1 gene. We also generate different transgenic lines, with severity and age of onset correlated to the level of smn1 inhibition, recapitulating for the first time the different forms of SMA in zebrafish. These lines are proof-of-concept that miRNA-based approaches can be used to generate potent heritable gene knockdown in zebrafish.


Assuntos
Técnicas de Silenciamento de Genes , MicroRNAs/genética , Peixe-Zebra/genética , Regiões 3' não Traduzidas , Animais , Animais Geneticamente Modificados , Proteínas de Peixe-Zebra/genética
9.
Genesis ; 53(5): 321-8, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25864959

RESUMO

miR218-1 and miR218-2 are embedded in introns of SLIT2 and SLIT3, respectively, an arrangement conserved throughout vertebrate genomes. Both miR218 genes are predicted to be transcribed in the same orientation as their host genes and were assumed to be spliced from Slit2/3 primary transcripts. In zebrafish miR218 is active in cranial nerve motor nuclei and spinal cord motor neurons, while slit2 and slit3 are expressed predominantly in the midline. This differential expression pattern suggested independent regulation of miR218 genes by distinct enhancers. We tested conserved noncoding elements for regulatory activity by reporter gene transgenesis in zebrafish. Two human enhancers, 76 kb and 130 kb distant from miR218-2, were identified that drove GFP expression in zebrafish in an almost complete miR218 expression pattern. In the zebrafish slit3 locus, two enhancers with identical activity were discovered. In human SLIT2 one enhancer 52 kb upstream of miR218-1 drove an expression pattern very similar to the enhancers of miR218-2. This establishes that miR218-1/-2 regulatory units are nested within SLIT2/3 and that they are duplicates of an ancestral single locus. Due to the strong activity of the enhancers, unique transgenic lines were created that facilitate morphological and gene functional genetic experiments in motor neurons.


Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Íntrons , MicroRNAs/genética , Neurônios Motores/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
10.
Hum Mol Genet ; 22(22): 4562-78, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-23804750

RESUMO

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.


Assuntos
Ciclofilinas/metabolismo , Ciclosporina/farmacologia , Citocromos c/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Dinâmica Mitocondrial , Distrofia Muscular Animal/tratamento farmacológico , Distrofia Muscular de Duchenne/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Morte Celular , Ciclofilinas/antagonistas & inibidores , Citocromos c/genética , Técnicas de Silenciamento de Genes , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Metazolamida/farmacologia , Dinâmica Mitocondrial/efeitos dos fármacos , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Filogenia , Homologia de Sequência , Peixe-Zebra/embriologia , Peixe-Zebra/genética
11.
BMC Dev Biol ; 12: 37, 2012 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-23244389

RESUMO

BACKGROUND: Genetic studies in mouse have demonstrated the crucial function of PAX4 in pancreatic cell differentiation. This transcription factor specifies ß- and δ-cell fate at the expense of α-cell identity by repressing Arx gene expression and ectopic expression of PAX4 in α-cells is sufficient to convert them into ß-cells. Surprisingly, no Pax4 orthologous gene can be found in chicken and Xenopus tropicalis raising the question of the function of pax4 gene in lower vertebrates such as in fish. In the present study, we have analyzed the expression and the function of the orthologous pax4 gene in zebrafish. RESULTS: pax4 gene is transiently expressed in the pancreas of zebrafish embryos and is mostly restricted to endocrine precursors as well as to some differentiating δ- and ε-cells but was not detected in differentiating ß-cells. pax4 knock-down in zebrafish embryos caused a significant increase in α-cells number while having no apparent effect on ß- and δ-cell differentiation. This rise of α-cells is due to an up-regulation of the Arx transcription factor. Conversely, knock-down of arx caused to a complete loss of α-cells and a concomitant increase of pax4 expression but had no effect on the number of ß- and δ-cells. In addition to the mutual repression between Arx and Pax4, these two transcription factors negatively regulate the transcription of their own gene. Interestingly, disruption of pax4 RNA splicing or of arx RNA splicing by morpholinos targeting exon-intron junction sites caused a blockage of the altered transcripts in cell nuclei allowing an easy characterization of the arx- and pax4-deficient cells. Such analyses demonstrated that arx knock-down in zebrafish does not lead to a switch of cell fate, as reported in mouse, but rather blocks the cells in their differentiation process towards α-cells. CONCLUSIONS: In zebrafish, pax4 is not required for the generation of the first ß- and δ-cells deriving from the dorsal pancreatic bud, unlike its crucial role in the differentiation of these cell types in mouse. On the other hand, the mutual repression between Arx and Pax4 is observed in both mouse and zebrafish. These data suggests that the main original function of Pax4 during vertebrate evolution was to modulate the number of pancreatic α-cells and its role in ß-cells differentiation appeared later in vertebrate evolution.


Assuntos
Embrião não Mamífero/citologia , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/metabolismo , Proteínas de Homeodomínio/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Animais , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Morfolinos/farmacologia , Fatores de Transcrição Box Pareados/biossíntese , Fatores de Transcrição Box Pareados/genética , Pâncreas/embriologia , Processamento de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/biossíntese , Proteínas de Peixe-Zebra/biossíntese
12.
Methods ; 56(1): 103-13, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22041718

RESUMO

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.


Assuntos
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 , Osteocondrodisplasias
13.
Br J Pharmacol ; 160(2): 204-16, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20423335

RESUMO

Current high-throughput screening methods for drug discovery rely on the existence of targets. Moreover, most of the hits generated during screenings turn out to be invalid after further testing in animal models. To by-pass these limitations, efforts are now being made to screen chemical libraries on whole animals. One of the most commonly used animal model in biology is the murine model Mus musculus. However, its cost limit its use in large-scale therapeutic screening. In contrast, the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the fish Danio rerio are gaining momentum as screening tools. These organisms combine genetic amenability, low cost and culture conditions that are compatible with large-scale screens. Their main advantage is to allow high-throughput screening in a whole-animal context. Moreover, their use is not dependent on the prior identification of a target and permits the selection of compounds with an improved safety profile. This review surveys the versatility of these animal models for drug discovery and discuss the options available at this day.


Assuntos
Sistemas de Liberação de Medicamentos , Desenho de Drogas , Ensaios de Triagem em Larga Escala/métodos , Animais , Descoberta de Drogas , Humanos , Modelos Animais , Bibliotecas de Moléculas Pequenas
14.
Hum Mol Genet ; 18(21): 4089-101, 2009 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-19648295

RESUMO

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.


Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Modelos Animais de Doenças , Distrofina/deficiência , Distrofia Muscular Animal/prevenção & controle , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Inibidores da Anidrase Carbônica/metabolismo , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Diclorofenamida/farmacologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Distrofina/genética , Humanos , Metazolamida/farmacologia , Camundongos , Camundongos Endogâmicos mdx , Atividade Motora , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/fisiopatologia , Interferência de RNA , Fatores de Tempo
15.
J Proteome Res ; 8(5): 2542-50, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19267476

RESUMO

In this study, we present a methodology for metabotyping of C. elegans using 1H high resolution magic angle spinning (HRMAS) whole-organism nuclear magnetic resonance (NMR). We demonstrate and characterize the robustness of our metabolic phenotyping method, discriminating wild-type N2 from mutant sod-1(tm776) animals, with the latter being an otherwise silent mutation, and we identify and quantify several confounding effects to establish guidelines to ensure optimal quality of the raw data across time and space. We monitor the sample stability under experimental conditions and examine variations arising from effects that can potentially confuse the biological interpretation or prevent the automation of the protocol, including sample culture (breeding of the worms by two biologists), sample preparation (freezing), NMR acquisition (acquisition by different spectroscopists, acquisition in different facilities), and the effect of the age of the animals. When working with intact model organisms, some of these exogenous effects are shown to be significant and therefore require control through experimental design and sample randomization.


Assuntos
Caenorhabditis elegans/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Metaboloma , Metabolômica/métodos , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Modelos Biológicos , Mutação
16.
Proc Natl Acad Sci U S A ; 104(50): 19808-12, 2007 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-18077412

RESUMO

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.


Assuntos
Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Fenótipo , Animais , Caenorhabditis elegans/classificação , Genoma Helmíntico , Genômica , Espectroscopia de Ressonância Magnética , Mutação , Estresse Oxidativo/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1
17.
J Muscle Res Cell Motil ; 27(3-4): 253-8, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16791712

RESUMO

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.


Assuntos
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 de Captação de Serotonina/farmacologia , Fatores de Tempo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA