Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 24
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
2.
Dis Model Mech ; 12(11)2019 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-31582559

RESUMO

Epilepsy is a common primary neurological disorder characterized by the chronic tendency of a patient to experience epileptic seizures, which are abnormal body movements or cognitive states that result from excessive, hypersynchronous brain activity. Epilepsy has been found to have numerous etiologies and, although about two-thirds of epilepsies were classically considered idiopathic, the majority of those are now believed to be of genetic origin. Mutations in genes involved in gamma-aminobutyric acid (GABA)-mediated inhibitory neurotransmission have been associated with a broad range of epilepsy syndromes. Mutations in the GABA-A receptor gamma 2 subunit gene (GABRG2), for example, have been associated with absence epilepsy and febrile seizures in humans. Several rodent models of GABRG2 loss of function depict clinical features of the disease; however, alternative genetic models more amenable for the study of ictogenesis and for high-throughput screening purposes are still needed. In this context, we generated a gabrg2 knockout (KO) zebrafish model (which we called R23X) that displayed light/dark-induced reflex seizures. Through high-resolution in vivo calcium imaging of the brain, we showed that this phenotype is associated with widespread increases in neuronal activity that can be effectively alleviated by the anti-epileptic drug valproic acid. Moreover, these seizures only occur at the larval stages but disappear after 1 week of age. Interestingly, our whole-transcriptome analysis showed that gabrg2 KO does not alter the expression of genes in the larval brain. As a result, the gabrg2-/- zebrafish is a novel in vivo genetic model of early epilepsies that opens new doors to investigate ictogenesis and for further drug-screening assays.


Assuntos
Modelos Animais de Doenças , Receptores de GABA-A/fisiologia , Convulsões/etiologia , Animais , Técnicas de Inativação de Genes , Larva , Luz , Subunidades Proteicas/fisiologia , Receptores de GABA-A/deficiência , Reflexo/fisiologia , Transcriptoma , Ácido Valproico/uso terapêutico , Peixe-Zebra
3.
Neurotherapeutics ; 16(4): 1149-1166, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31342410

RESUMO

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disorder in which the neuromuscular junction progressively degenerates, leading to movement difficulties, paralysis, and eventually death. ALS is currently being treated by only two FDA-approved drugs with modest efficacy in slowing disease progression. Often, the translation of preclinical findings to bedside terminates prematurely as the evaluation of potential therapeutic compounds focuses on a single study or a single animal model. To circumscribe these issues, we screened 3,765 novel small molecule derivatives of pimozide, a recently identified repurposed neuroleptic for ALS, in Caenorhabditis elegans, confirmed the hits in zebrafish and validated the most active compounds in mouse genetic models. Out of the 27 small molecules identified from the high-throughput screen in worms, 4 were found to recover locomotor defects in C. elegans and genetic zebrafish models of ALS. TRVA242 was identified as the most potent compound as it significantly improved efficiency in rescuing locomotor, motorneuron, and neuromuscular junction synaptic deficits in a C. elegans TDP-43 model and in multiple zebrafish genetic (TDP-43, SOD1, and C9ORF72) models of ALS. The actions of TRVA242 were also conserved in a mammalian model as it also stabilized neuromuscular junction deficits in a mouse SOD1 model of ALS. Compounds such as TRVA242 therefore represent new potential therapeutics for the treatment of ALS.


Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/genética , Modelos Animais de Doenças , Junção Neuromuscular/genética , Superóxido Dismutase-1/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Proteínas de Ligação a DNA/administração & dosagem , Proteínas de Ligação a DNA/metabolismo , Humanos , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/metabolismo , Técnicas de Cultura de Órgãos , Pimozida/administração & dosagem , Pimozida/metabolismo , Peixe-Zebra
4.
PLoS One ; 14(5): e0216159, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31048868

RESUMO

Glycine receptors (GlyRs) are ligand-gated chloride channels mediating inhibitory neurotransmission in the brain stem and spinal cord. They function as pentamers composed of alpha and beta subunits for which 5 genes have been identified in human (GLRA1, GLRA2, GLRA3, GLRA4, GLRB). Several in vitro studies showed that the pentameric subtype composition as well as its stoichiometry influence the distribution and the molecular function of the receptor. Moreover, mutations in some of these genes are involved in different human conditions ranging from tinnitus to epilepsy and hyperekplexia, suggesting distinct functions of the different subunits. Although the beta subunit is essential for synaptic clustering of the receptor, the specific role of each alpha subtype is still puzzling in vivo. The zebrafish genome encodes for five glycine receptor alpha subunits (glra1, glra2, glra3, glra4a, glra4b) thus offering a model of choice to investigate the respective role of each subtype on general motor behaviour. After establishing a phylogeny of GlyR subunit evolution between human and zebrafish, we checked the temporal expression pattern of these transcripts during embryo development. Interestingly, we found that glra1 is the only maternally transmitted alpha subunit. We also showed that the expression of the different GlyR subunits starts at different time points during development. Lastly, in order to decipher the role of each alpha subunit on the general motor behaviour of the fish, we knocked out individually each alpha subunit by CRISPR/Cas9-targeted mutagenesis. Surprisingly, we found that knocking out any of the alpha2, 3, a4a or a4b subunit did not lead to any obvious developmental or motor phenotype. However, glra1-/- (hitch) embryos depicted a strong motor dysfunction from 3 days, making them incapable to swim and thus leading to their premature death. Our results infer a strong functional redundancy between alpha subunits and confirm the central role played by glra1 for proper inhibitory neurotransmission controlling locomotion. The genetic tools we developed here will be of general interest for further studies aiming at dissecting the role of GlyRs in glycinergic transmission in vivo and the hitch mutant (hic) is of specific relevance as a new model of hyperekplexia.


Assuntos
Receptores de Glicina/genética , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Inativação de Genes/métodos , Atividade Motora/genética , Mutação , Fenótipo , Filogenia , Receptores de Glicina/metabolismo , Transmissão Sináptica/genética , Peixe-Zebra/genética
5.
Front Mol Neurosci ; 12: 44, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30833887

RESUMO

During development of the zebrafish embryo, glycine signaling promotes the differentiation of neural stem cells (NSCs). We found that glycine signaling suppresses the expression of Ligand of Numb X1 (lnx1, Ligand of numb protein-x1), a gene of unknown function during NSC differentiation that is selectively expressed in the embryonic central nervous system (CNS). As a consequence, Numb levels were stabilized and Notch activity (measured as her4.1 expression) was reduced, promoting NSC differentiation. These consequent actions were blocked by knockdown of lnx1. In contrast, lnx1 overexpression increased NSC proliferation and led to defects of neural tube closure at the early stages of development. Thus, our data provide evidence that glycine/lnx1 signaling modulates NSC proliferation by regulation of Notch signaling.

6.
JCI Insight ; 3(21)2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30385710

RESUMO

Glycine encephalopathy (GE), or nonketotic hyperglycinemia (NKH), is a rare recessive genetic disease caused by defective glycine cleavage and characterized by increased accumulation of glycine in all tissues. Here, based on new case reports of GLDC loss-of-function mutations in GE patients, we aimed to generate a zebrafish model of severe GE in order to unravel the molecular mechanism of the disease. Using CRISPR/Cas9, we knocked out the gldc gene and showed that gldc-/- fish recapitulate GE on a molecular level and present a motor phenotype reminiscent of severe GE symptoms. The molecular characterization of gldc-/- mutants showed a broad metabolic disturbance affecting amino acids and neurotransmitters other than glycine, with lactic acidosis at stages preceding death. Although a transient imbalance was found in cell proliferation in the brain of gldc-/- zebrafish, the main brain networks were not affected, thus suggesting that GE pathogenicity is mainly due to metabolic defects. We confirmed that the gldc-/- hypotonic phenotype is due to NMDA and glycine receptor overactivation, and demonstrated that gldc-/- larvae depict exacerbated hyperglycinemia at these synapses. Remarkably, we were able to rescue the motor dysfunction of gldc-/- larvae by counterbalancing pharmacologically or genetically the level of glycine at the synapse.


Assuntos
Glicina Desidrogenase (Descarboxilante)/deficiência , Glicina/sangue , Hiperglicinemia não Cetótica/genética , Transtornos Motores/enzimologia , Transmissão Sináptica/efeitos dos fármacos , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Proteína 9 Associada à CRISPR/metabolismo , Dextrometorfano/administração & dosagem , Dextrometorfano/uso terapêutico , Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Evolução Fatal , Feminino , Conservantes de Alimentos/uso terapêutico , Glicina/líquido cefalorraquidiano , Glicina Desidrogenase (Descarboxilante)/metabolismo , Humanos , Hiperglicinemia não Cetótica/diagnóstico , Hiperglicinemia não Cetótica/enzimologia , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Transtornos Motores/fisiopatologia , Mutação , Fenótipo , Benzoato de Sódio/administração & dosagem , Benzoato de Sódio/uso terapêutico , Resultado do Tratamento , Peixe-Zebra
7.
Epilepsia ; 59(11): 2061-2074, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30324621

RESUMO

OBJECTIVE: In humans, mutations of the γ-aminobutyric acid receptor subunit 1 (GABRA1) cause either mild or severe generalized epilepsy. Although these epilepsy-causing mutations have been shown to disrupt the receptor activity in vitro, their in vivo consequences on brain development and activity are not known. Here, we aim at unraveling the epileptogenesis mechanisms of GABRA1 loss of function. METHODS: We generated a gabra1-/- zebrafish mutant line displaying highly penetrant epileptic seizures. We sought to identify the underlying molecular mechanisms through unbiased whole transcriptomic assay of gabra1-/- larval brains. RESULTS: Interestingly, mutant fish show fully penetrant seizures at juvenile stages that accurately mimic tonic-clonic generalized seizures observed in patients. Moreover, highly penetrant seizures can be induced by light stimulation, thus providing us with the first zebrafish model in which evident epileptic seizures can be induced by nonchemical agents. Our transcriptomic assay identified misregulated genes in several pathways essential for correct brain development. More specifically, we show that the early development of the brain inhibitory network is specifically affected. Although the number of GABAergic neurons is not altered, we observed a drastic reduction in the number of inhibitory synapses and a decreased complexity of the GABAergic network. This is consistent with the disruption in expression of many genes involved in axon guidance and synapse formation. SIGNIFICANCE: Together with the role of GABA in neurodevelopment, our data identify a novel aspect of epileptogenesis, suggesting that the substratum of GABRA1-deficiency epilepsy is a consequence of early brain neurodevelopmental defects, in particular at the level of inhibitory network wiring.


Assuntos
Epilepsia Generalizada/genética , Expressão Gênica/genética , Transtornos do Neurodesenvolvimento/etiologia , Receptores de GABA-A/deficiência , Receptores de GABA-A/genética , Animais , Animais Geneticamente Modificados , Anticonvulsivantes/uso terapêutico , Encéfalo/efeitos dos fármacos , Encéfalo/embriologia , Encéfalo/metabolismo , Encéfalo/patologia , Clonazepam/uso terapêutico , Modelos Animais de Doenças , Embrião não Mamífero , Epilepsia Generalizada/tratamento farmacológico , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Glutamato Descarboxilase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Larva , Luz/efeitos adversos , Mortalidade Prematura , Mutação , Transtornos do Neurodesenvolvimento/genética , Neurônios/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Transcriptoma/fisiologia , Peixe-Zebra
8.
Curr Biol ; 28(12): 1924-1937.e5, 2018 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-29861134

RESUMO

Mutations in DEPDC5 are causal factors for a broad spectrum of focal epilepsies, but the underlying pathogenic mechanisms are still largely unknown. To address this question, a zebrafish depdc5 knockout model showing spontaneous epileptiform events in the brain, increased drug-induced seizure susceptibility, general hypoactivity, premature death at 2-3 weeks post-fertilization, as well as the expected hyperactivation of mTOR signaling was developed. Using this model, the role of DEPDC5 in brain development was investigated using an unbiased whole-transcriptomic approach. Surprisingly, in addition to mTOR-associated genes, many genes involved in synaptic function, neurogenesis, axonogenesis, and GABA network activity were found to be dysregulated in larval brains. Although no gross defects in brain morphology or neuron loss were observed, immunostaining of depdc5-/- brains for several GABAergic markers revealed specific defects in the fine branching of the GABAergic network. Consistently, some defects in depdc5-/- could be compensated for by treatment with GABA, corroborating that GABA signaling is indeed involved in DEPDC5 pathogenicity. Further, the mTOR-independent nature of these neurodevelopmental defects was demonstrated by the inability of rapamycin to rescue the GABAergic network defects observed in depdc5-/- brains and, conversely, the inability of GABA to rescue the hypoactivity in another genetic model showing mTOR hyperactivation. This study hence provides the first in vivo evidence that DEPDC5 plays previously unknown roles apart from its canonical function as an mTOR inhibitor. Moreover, these results propose that defective neurodevelopment of GABAergic networks could be a key factor in epileptogenesis when DEPDC5 is mutated.


Assuntos
Epilepsias Parciais/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteínas de Peixe-Zebra/antagonistas & inibidores , Peixe-Zebra/genética , Animais , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação com Perda de Função , Sirolimo/farmacologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
9.
Hum Mol Genet ; 27(10): 1754-1762, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29528390

RESUMO

Large expansions of hexanucleotide GGGGCC (G4C2) repeats (hundreds to thousands) in the first intron of the chromosome 9 open reading frame 72 (C9orf72) locus are the strongest known genetic factor associated with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Different hypotheses exist about the underlying disease mechanism including loss of function by haploinsufficiency, toxicity arising as a result of RNA or dipeptide repeats (DPRs). Five different DPRs are produced by repeat-associated non-ATG-initiated translation of the G4C2 repeats. Though earlier studies have indicated toxicity of the DPRs in worms, flies, primary cultured cells and cell lines, the effect of expressing DPRs of amyotrophic lateral sclerosis-relevant length has not been tested on motor behaviour in vertebrate models. In this study, by expressing constructs with alternate codons encoding different lengths of each DPR (40, 200 and 1000) in the vertebrate zebrafish model, the GR DPR was found to lead to the greatest developmental lethality and morphological defects, and GA, the least. However, expressing 1000 repeats of any DPR, including the 'non-toxic' GA DPR led to locomotor defects. Based on these observations, a transgenic line stably expressing 100 GR repeats was generated to allow specific regional and temporal expression of GR repeats in vivo. Expression of GR DPRs ubiquitously resulted in severe morphological defects and reduced swimming. However, when expressed specifically in motor neurons, the developmental defects were significantly reduced, but the swimming phenotype persisted, suggesting that GR DPRs have a toxic effect on motor neuron function. This was validated by the reduction in motor neuron length even in already formed motor neurons when GR was expressed in these. Hence, the expression of C9orf72-associated DPRs can cause significant motor deficits in vertebrates.


Assuntos
Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/genética , Expansão das Repetições de DNA/genética , Degeneração Lobar Frontotemporal/genética , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Animais Geneticamente Modificados/genética , Dipeptídeos/genética , Modelos Animais de Doenças , Degeneração Lobar Frontotemporal/fisiopatologia , Regulação da Expressão Gênica , Humanos , Locomoção/genética , Locomoção/fisiologia , Neurônios Motores/patologia , Neurônios Motores/fisiologia , Peixe-Zebra/genética
10.
Front Mol Neurosci ; 11: 463, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30618614

RESUMO

Amyotrophic lateral sclerosis (ALS) is a late-onset progressive neurodegenerative disorder that affects both upper and lower motor neurons, leading to muscle atrophy with spasticity and eventual death in 3-5 years after the disease onset. More than 50 mutations linked to ALS have been found in the gene TARDBP, encoding the protein TDP-43 that is the predominant component of neuronal inclusions in ALS. TDP-43 is an RNA binding protein with glycine-rich domains that binds to more than 6,000 RNAs in the human brain. However, ALS-related mutations do not appear to affect the function of these genes, indicating that a toxic gain-of-function may occur. We generated transgenic zebrafish lines expressing human TDP-43, either the wild-type form or the ALS-causative G348C mutation identified in a subset of ALS patients, with the transgene expression driven by an inducible heat shock promoter in order to bypass a potential early mortality. The expression of the mutant but not the wild-type human TDP-43 in zebrafish embryos induced a reduction of the locomotor activity in response to touch compared to controls and moderate axonopathy of the motor neurons of the spinal cord, with premature branching of the main axonal branch, recapitulating previous results obtained by mRNA injections. We used these lines to investigate transcriptomic changes due to the presence of mutant TDP-43 using RNA sequencing and have found 159 genes that are differentially expressed compared to control, with 67 genes up-regulated and 92 genes down-regulated. These transcriptomic changes are in line with recent transcriptomic data obtained in mouse models, indicating that these zebrafish transgenic lines are adequate to further study TDP-43-related ALS.

11.
JCI Insight ; 2(22)2017 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-29202456

RESUMO

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing, fatal disorder with no effective treatment. We used simple genetic models of ALS to screen phenotypically for potential therapeutic compounds. We screened libraries of compounds in C. elegans, validated hits in zebrafish, and tested the most potent molecule in mice and in a small clinical trial. We identified a class of neuroleptics that restored motility in C. elegans and in zebrafish, and the most potent was pimozide, which blocked T-type Ca2+ channels in these simple models and stabilized neuromuscular transmission in zebrafish and enhanced it in mice. Finally, a short randomized controlled trial of sporadic ALS subjects demonstrated stabilization of motility and evidence of target engagement at the neuromuscular junction. Simple genetic models are, thus, useful in identifying promising compounds for the treatment of ALS, such as neuroleptics, which may stabilize neuromuscular transmission and prolong survival in this disease.


Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Antipsicóticos/farmacocinética , Antipsicóticos/uso terapêutico , Doenças da Junção Neuromuscular/tratamento farmacológico , Animais , Caenorhabditis elegans , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio Tipo T/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Tolerância a Medicamentos , Feminino , Camundongos , Junção Neuromuscular/efeitos dos fármacos , Pimozida/farmacologia , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
13.
Am J Hum Genet ; 98(5): 1038-1046, 2016 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-27153400

RESUMO

Hereditary spastic paraplegia (HSP) is a genetically and clinically heterogeneous disease characterized by spasticity and weakness of the lower limbs with or without additional neurological symptoms. Although more than 70 genes and genetic loci have been implicated in HSP, many families remain genetically undiagnosed, suggesting that other genetic causes of HSP are still to be identified. HSP can be inherited in an autosomal-dominant, autosomal-recessive, or X-linked manner. In the current study, we performed whole-exome sequencing to analyze a total of nine affected individuals in three families with autosomal-recessive HSP. Rare homozygous and compound-heterozygous nonsense, missense, frameshift, and splice-site mutations in CAPN1 were identified in all affected individuals, and sequencing in additional family members confirmed the segregation of these mutations with the disease (spastic paraplegia 76 [SPG76]). CAPN1 encodes calpain 1, a protease that is widely present in the CNS. Calpain 1 is involved in synaptic plasticity, synaptic restructuring, and axon maturation and maintenance. Three models of calpain 1 deficiency were further studied. In Caenorhabditis elegans, loss of calpain 1 function resulted in neuronal and axonal dysfunction and degeneration. Similarly, loss-of-function of the Drosophila melanogaster ortholog calpain B caused locomotor defects and axonal anomalies. Knockdown of calpain 1a, a CAPN1 ortholog in Danio rerio, resulted in abnormal branchiomotor neuron migration and disorganized acetylated-tubulin axonal networks in the brain. The identification of mutations in CAPN1 in HSP expands our understanding of the disease causes and potential mechanisms.


Assuntos
Axônios/patologia , Calpaína/genética , Predisposição Genética para Doença/genética , Neurônios Motores/patologia , Paraplegia Espástica Hereditária/genética , Adulto , Animais , Encéfalo/fisiologia , Caenorhabditis elegans/genética , Movimento Celular/genética , Modelos Animais de Doenças , Drosophila melanogaster/genética , Feminino , Humanos , Masculino , Neurônios Motores/citologia , Adulto Jovem , Peixe-Zebra/genética
14.
PLoS One ; 11(3): e0150188, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26930076

RESUMO

The methodology for site-directed editing of single nucleotides in the vertebrate genome is of considerable interest for research in biology and medicine. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 type II (Cas9) system has emerged as a simple and inexpensive tool for editing genomic loci of interest in a variety of animal models. In zebrafish, error-prone non-homologous end joining (NHEJ) has been used as a simple method to disrupt gene function. We sought to develop a method to easily create site-specific SNPs in the zebrafish genome. Here, we report simple methodologies for using CRISPR/Cas9-mediated homology directed repair using single-stranded oligodeoxynucleotide donor templates (ssODN) for site-directed single nucleotide editing, for the first time in two disease-related genes, tardbp and fus.


Assuntos
Esclerose Lateral Amiotrófica/genética , Sistemas CRISPR-Cas/genética , Proteínas de Ligação a DNA/genética , Mutação Puntual , Proteína FUS de Ligação a RNA/genética , Proteínas de Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Reparo do DNA , DNA de Cadeia Simples , Modelos Animais de Doenças , Técnicas de Introdução de Genes/métodos , Humanos , Oligodesoxirribonucleotídeos/genética , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos Testes , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Peixe-Zebra/genética
15.
J Med Genet ; 52(5): 303-11, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25650066

RESUMO

BACKGROUND: The heterogeneous group of 3-methylglutaconic aciduria disorders includes several inborn errors of metabolism that affect mitochondrial function through poorly understood mechanisms. We describe four newborn siblings, from a consanguineous family, who showed microcephaly, small birth weight, severe encephalopathy and 3-methylglutaconic aciduria. Their neurological examination was characterised by severe hypertonia and the induction of prolonged clonic movements of the four limbs upon minimal tactile stimulation. METHODS AND RESULTS: Using homozygosity mapping and exome sequencing, we identified a homozygous truncating mutation (p.I562Tfs*23) in CLPB segregating with the disease in this family. CLPB codes for a member of the family of ATPases associated with various cellular activities (AAA(+) proteins) whose function remains unknown. We found that CLPB expression is abolished in fibroblasts from the patients. To investigate the function of this gene, we interfered with the translation of the zebrafish clpb orthologue using an antisense morpholino. The clpb morphants showed an abnormal touch-evoked response with increased swim velocity and tail beat frequency. This motor phenotype is reminiscent of that observed in the patients and is suggestive of increased excitability in neuronal circuits. Interestingly, knocking down clpb reduced the number of inhibitory glycinergic interneurons and increased a population of excitatory glutamatergic neurons in the spinal cord. CONCLUSIONS: Altogether, our study suggests that disruption of CLPB causes a novel form of neonatal encephalopathy associated with 3-methylglutaconic aciduria.


Assuntos
Encefalopatias/genética , Endopeptidase Clp/genética , Estudos de Associação Genética , Erros Inatos do Metabolismo/genética , Microcefalia/genética , Animais , Encefalopatias/diagnóstico , Mapeamento Cromossômico , Consanguinidade , Análise Mutacional de DNA , Exoma , Técnicas de Silenciamento de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Recém-Nascido , Erros Inatos do Metabolismo/diagnóstico , Microcefalia/diagnóstico , Mutação , Linhagem , Fenótipo , Irmãos , Peixe-Zebra
16.
J Clin Invest ; 125(3): 1124-8, 2015 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-25642776

RESUMO

Idiopathic scoliosis (IS) is a spine deformity that affects approximately 3% of the population. The underlying causes of IS are not well understood, although there is clear evidence that there is a genetic component to the disease. Genetic mapping studies suggest high genetic heterogeneity, but no IS disease-causing gene has yet been identified. Here, genetic linkage analyses combined with exome sequencing identified a rare missense variant (p.A446T) in the centriolar protein gene POC5 that cosegregated with the disease in a large family with multiple members affected with IS. Subsequently, the p.A446T variant was found in an additional set of families with IS and in an additional 3 cases of IS. Moreover, POC5 variant p.A455P was present and linked to IS in one family and another rare POC5 variant (p.A429V) was identified in an additional 5 cases of IS. In a zebrafish model, expression of any of the 3 human IS-associated POC5 variant mRNAs resulted in spine deformity, without affecting other skeletal structures. Together, these findings indicate that mutations in the POC5 gene contribute to the occurrence of IS.


Assuntos
Proteínas de Transporte/genética , Escoliose/genética , Animais , Estudos de Casos e Controles , Análise Mutacional de DNA , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Desequilíbrio de Ligação , Masculino , Mutação de Sentido Incorreto , Linhagem , Polimorfismo de Nucleotídeo Único , Peixe-Zebra
17.
PLoS One ; 8(8): e72560, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23977317

RESUMO

SecA, an essential component of the Sec machinery, exists in a soluble and a membrane form in Escherichia coli. Previous studies have shown that the soluble SecA transforms into pore structures when it interacts with liposomes, and integrates into membranes containing SecYEG in two forms: SecAS and SecAM; the latter exemplified by two tryptic membrane-specific domains, an N-terminal domain (N39) and a middle M48 domain (M48). The formation of these lipid-specific domains was further investigated. The N39 and M48 domains are induced only when SecA interacts with anionic liposomes. Additionally, the N-terminus, not the C-terminus of SecA is required for inducing such conformational changes. Proteolytic treatment and sequence analyses showed that liposome-embedded SecA yields the same M48 and N39 domains as does the membrane-embedded SecA. Studies with chemical extraction and resistance to trypsin have also shown that these proteoliposome-embedded SecA fragments exhibit the same stability and characteristics as their membrane-embedded SecA equivalents. Furthermore, the cloned lipid-specific domains N39 and M48, but not N68 or C34, are able to form partial, but imperfect ring-like structures when they interact with phospholipids. These ring-like structures are characteristic of a SecA pore-structure, suggesting that these domains contribute part of the SecA-dependent protein-conducting channel. We, therefore, propose a model in which SecA alone is capable of forming a lipid-specific, asymmetric dimer that is able to function as a viable protein-conducting channel in the membrane, without any requirement for SecYEG.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Microscopia de Força Atômica , Fosfolipídeos/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Lipossomos/metabolismo , Dados de Sequência Molecular , Peptídeo Hidrolases/metabolismo , Estabilidade Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteólise , Canais de Translocação SEC , Proteínas SecA , Solubilidade
18.
PLoS Genet ; 9(1): e1003124, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23300475

RESUMO

Hereditary sensory and autonomic neuropathy type 2 (HSNAII) is a rare pathology characterized by an early onset of severe sensory loss (all modalities) in the distal limbs. It is due to autosomal recessive mutations confined to exon "HSN2" of the WNK1 (with-no-lysine protein kinase 1) serine-threonine kinase. While this kinase is well studied in the kidneys, little is known about its role in the nervous system. We hypothesized that the truncating mutations present in the neural-specific HSN2 exon lead to a loss-of-function of the WNK1 kinase, impairing development of the peripheral sensory system. To investigate the mechanisms by which the loss of WNK1/HSN2 isoform function causes HSANII, we used the embryonic zebrafish model and observed strong expression of WNK1/HSN2 in neuromasts of the peripheral lateral line (PLL) system by immunohistochemistry. Knocking down wnk1/hsn2 in embryos using antisense morpholino oligonucleotides led to improper PLL development. We then investigated the reported interaction between the WNK1 kinase and neuronal potassium chloride cotransporter KCC2, as this transporter is a target of WNK1 phosphorylation. In situ hybridization revealed kcc2 expression in mature neuromasts of the PLL and semi-quantitative RT-PCR of wnk1/hsn2 knockdown embryos showed an increased expression of kcc2 mRNA. Furthermore, overexpression of human KCC2 mRNA in embryos replicated the wnk1/hsn2 knockdown phenotype. We validated these results by obtaining double knockdown embryos, both for wnk1/hsn2 and kcc2, which alleviated the PLL defects. Interestingly, overexpression of inactive mutant KCC2-C568A, which does not extrude ions, allowed a phenocopy of the PLL defects. These results suggest a pathway in which WNK1/HSN2 interacts with KCC2, producing a novel regulation of its transcription independent of KCC2's activation, where a loss-of-function mutation in WNK1 induces an overexpression of KCC2 and hinders proper peripheral sensory nerve development, a hallmark of HSANII.


Assuntos
Neuropatias Hereditárias Sensoriais e Autônomas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Sistema Nervoso Periférico , Proteínas Serina-Treonina Quinases/genética , Simportadores , Peixe-Zebra , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Neuropatias Hereditárias Sensoriais e Autônomas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Antígenos de Histocompatibilidade Menor , Morfolinos , Mutação , Neurônios/metabolismo , Sistema Nervoso Periférico/crescimento & desenvolvimento , Sistema Nervoso Periférico/metabolismo , Fenótipo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Simportadores/genética , Simportadores/metabolismo , Ativação Transcricional , Proteína Quinase 1 Deficiente de Lisina WNK , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Cotransportadores de K e Cl-
19.
PLoS Genet ; 7(8): e1002214, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21829392

RESUMO

Mutations in the SOD1 and TARDBP genes have been commonly identified in Amyotrophic Lateral Sclerosis (ALS). Recently, mutations in the Fused in sarcoma gene (FUS) were identified in familial (FALS) ALS cases and sporadic (SALS) patients. Similarly to TDP-43 (coded by TARDBP gene), FUS is an RNA binding protein. Using the zebrafish (Danio rerio), we examined the consequences of expressing human wild-type (WT) FUS and three ALS-related mutations, as well as their interactions with TARDBP and SOD1. Knockdown of zebrafish Fus yielded a motor phenotype that could be rescued upon co-expression of wild-type human FUS. In contrast, the two most frequent ALS-related FUS mutations, R521H and R521C, unlike S57Δ, failed to rescue the knockdown phenotype, indicating loss of function. The R521H mutation caused a toxic gain of function when expressed alone, similar to the phenotype observed upon knockdown of zebrafish Fus. This phenotype was not aggravated by co-expression of both mutant human TARDBP (G348C) and FUS (R521H) or by knockdown of both zebrafish Tardbp and Fus, consistent with a common pathogenic mechanism. We also observed that WT FUS rescued the Tardbp knockdown phenotype, but not vice versa, suggesting that TARDBP acts upstream of FUS in this pathway. In addition we observed that WT SOD1 failed to rescue the phenotype observed upon overexpression of mutant TARDBP or FUS or upon knockdown of Tardbp or Fus; similarly, WT TARDBP or FUS also failed to rescue the phenotype induced by mutant SOD1 (G93A). Finally, overexpression of mutant SOD1 exacerbated the motor phenotype caused by overexpression of mutant FUS. Together our results indicate that TARDBP and FUS act in a pathogenic pathway that is independent of SOD1.


Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas de Ligação a DNA/genética , Modelos Genéticos , Proteína FUS de Ligação a RNA/genética , Superóxido Dismutase/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Epistasia Genética , Humanos , Atividade Motora/genética , Mutação/genética , Fenótipo , Proteína FUS de Ligação a RNA/metabolismo , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
20.
J Neurosci ; 30(26): 8871-81, 2010 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-20592209

RESUMO

Neurons respond homeostatically to chronic changes in network activity with compensatory changes such as a uniform alteration in the size of miniature postsynaptic current (mPSC) amplitudes termed synaptic scaling. However, little is known about the impact of synaptic scaling on the function of neural networks in vivo. We used the embryonic zebrafish to address the effect of synaptic scaling on the neural network underlying locomotion. Activity was decreased during development by TTX injection to block action potentials or CNQX injection to block glutamatergic transmission. Alternatively TNFalpha was chronically applied. Recordings from spinal neurons showed that glutamatergic mPSCs scaled up approximately 25% after activity reduction and fortuitously scaled down approximately 20% after TNFalpha treatment, and were unchanged following blockade of neuromuscular activity alone with alpha-bungarotoxin. Regardless of the direction of scaling, immediately following reversal of treatment no chronic effect was distinguishable in motoneuron activity patterns or in swimming behavior. We also acutely induced a similar increase of glutamatergic mPSC amplitudes using cyclothiazide to reduce AMPA receptor desensitization or decrease of glutamatergic mPSC amplitudes using a low concentration of CNQX to partially block AMPA receptors. Though the strength of the motor output was altered, neither chronic nor acute treatments disrupted the patterning of synaptic activity or swimming. Our results show, for the first time, that scaling of glutamatergic synapses can be induced in vivo in the zebrafish and that synaptic patterning is less plastic than synaptic strength during development.


Assuntos
Neurônios Motores/fisiologia , Natação/fisiologia , Sinapses/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Ácido Glutâmico/metabolismo , Neurônios Motores/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/embriologia , Músculo Esquelético/fisiologia , Vias Neurais/efeitos dos fármacos , Vias Neurais/embriologia , Vias Neurais/fisiologia , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Receptores de AMPA/metabolismo , Sinapses/efeitos dos fármacos , Potenciais Sinápticos/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Peixe-Zebra
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...