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1.
N Engl J Med ; 378(11): 1018-1028, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29539279

RESUMO

BACKGROUND: In juvenile myoclonic epilepsy, data are limited on the genetic basis of networks promoting convulsions with diffuse polyspikes on electroencephalography (EEG) and the subtle microscopic brain dysplasia called microdysgenesis. METHODS: Using Sanger sequencing, we sequenced the exomes of six members of a large family affected with juvenile myoclonic epilepsy and confirmed cosegregation in all 37 family members. We screened an additional 310 patients with this disorder for variants on DNA melting-curve analysis and targeted real-time DNA sequencing of the gene encoding intestinal-cell kinase ( ICK). We calculated Bayesian logarithm of the odds (LOD) scores for cosegregating variants, odds ratios in case-control associations, and allele frequencies in the Genome Aggregation Database. We performed functional tests of the effects of variants on mitosis, apoptosis, and radial neuroblast migration in vitro and conducted video-EEG studies in mice lacking a copy of Ick. RESULTS: A variant, K305T (c.914A→C), cosegregated with epilepsy or polyspikes on EEG in 12 members of the family affected with juvenile myoclonic epilepsy. We identified 21 pathogenic ICK variants in 22 of 310 additional patients (7%). Four strongly linked variants (K220E, K305T, A615T, and R632X) impaired mitosis, cell-cycle exit, and radial neuroblast migration while promoting apoptosis. Tonic-clonic convulsions and polyspikes on EEG resembling seizures in human juvenile myoclonic epilepsy occurred more often in knockout heterozygous mice than in wild-type mice (P=0.02) during light sleep with isoflurane anesthesia. CONCLUSIONS: Our data provide evidence that heterozygous variants in ICK caused juvenile myoclonic epilepsy in 7% of the patients included in our analysis. Variant ICK affects cell processes that help explain microdysgenesis and polyspike networks observed on EEG in juvenile myoclonic epilepsy. (Funded by the National Institutes of Health and others.).


Assuntos
Mutação , Epilepsia Mioclônica Juvenil/genética , Proteínas Serina-Treonina Quinases/genética , Adolescente , Animais , Teorema de Bayes , Estudos de Casos e Controles , Criança , Pré-Escolar , Cromossomos Humanos Par 6 , Modelos Animais de Doenças , Eletroencefalografia , Feminino , Heterozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Malformações do Desenvolvimento Cortical/genética , Camundongos , Camundongos Knockout , Epilepsia Mioclônica Juvenil/fisiopatologia , Análise de Sequência de DNA , Adulto Jovem
2.
Genet Med ; 19(2): 144-156, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27467453

RESUMO

PURPOSE: EFHC1 variants are the most common mutations in inherited myoclonic and grand mal clonic-tonic-clonic (CTC) convulsions of juvenile myoclonic epilepsy (JME). We reanalyzed 54 EFHC1 variants associated with epilepsy from 17 cohorts based on National Human Genome Research Institute (NHGRI) and American College of Medical Genetics and Genomics (ACMG) guidelines for interpretation of sequence variants. METHODS: We calculated Bayesian LOD scores for variants in coinheritance, unconditional exact tests and odds ratios (OR) in case-control associations, allele frequencies in genome databases, and predictions for conservation/pathogenicity. We reviewed whether variants damage EFHC1 functions, whether efhc1-/- KO mice recapitulate CTC convulsions and "microdysgenesis" neuropathology, and whether supernumerary synaptic and dendritic phenotypes can be rescued in the fly model when EFHC1 is overexpressed. We rated strengths of evidence and applied ACMG combinatorial criteria for classifying variants. RESULTS: Nine variants were classified as "pathogenic," 14 as "likely pathogenic," 9 as "benign," and 2 as "likely benign." Twenty variants of unknown significance had an insufficient number of ancestry-matched controls, but ORs exceeded 5 when compared with racial/ethnic-matched Exome Aggregation Consortium (ExAC) controls. CONCLUSIONS: NHGRI gene-level evidence and variant-level evidence establish EFHC1 as the first non-ion channel microtubule-associated protein whose mutations disturb R-type VDCC and TRPM2 calcium currents in overgrown synapses and dendrites within abnormally migrated dislocated neurons, thus explaining CTC convulsions and "microdysgenesis" neuropathology of JME.Genet Med 19 2, 144-156.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Epilepsia Mioclônica Juvenil/genética , Convulsões/genética , Animais , Dendritos/patologia , Exoma , Frequência do Gene , Humanos , Camundongos , Camundongos Knockout , Mutação , Epilepsia Mioclônica Juvenil/fisiopatologia , National Human Genome Research Institute (U.S.) , Neurônios/patologia , Linhagem , Polimorfismo de Nucleotídeo Único , Convulsões/fisiopatologia , Sinapses/patologia , Estados Unidos
3.
Hum Mol Genet ; 21(23): 5106-17, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-22926142

RESUMO

Heterozygous mutations in Myoclonin1/EFHC1 cause juvenile myoclonic epilepsy (JME), the most common form of genetic generalized epilepsies, while homozygous F229L mutation is associated with primary intractable epilepsy in infancy. Heterozygous mutations in adolescent JME patients produce subtle malformations of cortical and subcortical architecture, whereas homozygous F229L mutation in infancy induces severe brain pathology and death. However, the underlying pathological mechanisms for these observations remain unknown. We had previously demonstrated that EFHC1 is a microtubule-associated protein (MAP) involved in cell division and radial migration during cerebral corticogenesis. Here, we show that JME mutations, including F229L, do not alter the ability of EFHC1 to colocalize with the centrosome and the mitotic spindle, but act in a dominant-negative manner to impair mitotic spindle organization. We also found that mutants EFHC1 expression disrupted radial and tangential migration by affecting the morphology of radial glia and migrating neurons. These results show how Myoclonin1/EFHC1 mutations disrupt brain development and potentially produce structural brain abnormalities on which epileptogenesis is established.


Assuntos
Encéfalo/embriologia , Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/genética , Mutação , Epilepsia Mioclônica Juvenil/embriologia , Epilepsia Mioclônica Juvenil/genética , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Ciclo Celular/genética , Linhagem Celular , Movimento Celular/genética , Proliferação de Células , Humanos , Espaço Intracelular/metabolismo , Camundongos , Neuroglia/metabolismo , Neurônios/metabolismo , Transporte Proteico , Ratos , Fuso Acromático/genética , Fuso Acromático/metabolismo , Células-Tronco/metabolismo
4.
J Neurol Neurosurg Psychiatry ; 85(4): 462-5, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24101679

RESUMO

BACKGROUND: Mutations in the proline-rich transmembrane protein 2 (PRRT2) gene have been identified in patients with benign (familial) infantile convulsions (B(F)IC), infantile convulsions with choreoathetosis (ICCA) and paroxysmal dyskinesias (PDs). However it remains unknown whether PRRT2 mutations are causal in other epilepsy syndromes. After we discovered a PRRT2 mutation in a large family with ICCA containing one individual with febrile seizures (FS) and one individual with West syndrome, we analysed PRRT2 in a heterogeneous cohort of patients with different types of infantile epilepsy. METHODS: We screened a cohort of 460 patients with B(F)IC or ICCA, fever related seizures or infantile epileptic encephalopathies. All patients were tested for point mutations using direct sequencing. RESULTS: We identified heterozygous mutations in 16 individuals: 10 familial and 6 sporadic cases. All patients were diagnosed with B(F)IC, ICCA or PD. We were not able to detect mutations in any of the other epilepsy syndromes. Several mutation carriers had learning disabilities and/or impaired fine motor skills later in life. CONCLUSIONS: PRRT2 mutations do not seem to be involved in the aetiology of FS or infantile epileptic encephalopathies. Therefore B(F)IC, ICCA and PD remain the core phenotypes associated with PRRT2 mutations. The presence of learning disabilities or neuropsychiatric problems in several mutation carriers calls for additional clinical studies addressing this developmental aspect in more detail.


Assuntos
Epilepsia/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Mutação Puntual/genética , Epilepsia/complicações , Epilepsia/diagnóstico , Feminino , Humanos , Deficiências da Aprendizagem/complicações , Deficiências da Aprendizagem/genética , Masculino , Transtornos das Habilidades Motoras/complicações , Transtornos das Habilidades Motoras/genética , Linhagem , Fenótipo
5.
Epilepsia ; 55(8): 1170-86, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24965021

RESUMO

New genetic investigation techniques, including next-generation sequencing, epigenetic profiling, cell lineage mapping, targeted genetic manipulation of specific neuronal cell types, stem cell reprogramming, and optogenetic manipulations within epileptic networks are progressively unraveling the mysteries of epileptogenesis and ictogenesis. These techniques have opened new avenues to discover the molecular basis of epileptogenesis and to study the physiologic effects of mutations in epilepsy-associated genes on a multilayer level, from cells to circuits. This manuscript reviews recently published applications of these new genetic technologies in the study of epilepsy, as well as work presented by the authors at the genetic session of the XII Workshop on the Neurobiology of Epilepsy (WONOEP 2013) in Quebec, Canada. Next-generation sequencing is providing investigators with an unbiased means to assess the molecular causes of sporadic forms of epilepsy and has revealed the complexity and genetic heterogeneity of sporadic epilepsy disorders. To assess the functional impact of mutations in these newly identified genes on specific neuronal cell types during brain development, new modeling strategies in animals, including conditional genetics in mice and in utero knock-down approaches, are enabling functional validation with exquisite cell-type and temporal specificity. In addition, optogenetics, using cell-type-specific Cre recombinase driver lines, is enabling investigators to dissect networks involved in epilepsy. In addition, genetically encoded cell-type labeling is providing new means to assess the role of the nonneuronal components of epileptic networks such as glial cells. Furthermore, beyond its role in revealing coding variants involved in epileptogenesis, next-generation sequencing can be used to assess the epigenetic modifications that lead to sustained network hyperexcitability in epilepsy, including methylation changes in gene promoters and noncoding ribonucleic acid (RNA) involved in modifying gene expression following seizures. In addition, genetically based bioluminescent reporters are providing new opportunities to assess neuronal activity and neurotransmitter levels both in vitro and in vivo in the context of epilepsy. Finally, genetically rederived neurons generated from patient induced pluripotent stem cells and genetically modified zebrafish have become high-throughput means to investigate disease mechanisms and potential new therapies. Genetics has changed the field of epilepsy research considerably, and is paving the way for better diagnosis and therapies for patients with epilepsy.


Assuntos
Educação/métodos , Epigênese Genética/genética , Epilepsia/diagnóstico , Epilepsia/genética , Hibridização Genética/genética , Animais , Humanos , MicroRNAs/genética
6.
Epilepsy Behav ; 28 Suppl 1: S58-60, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23756481

RESUMO

Juvenile Myoclonic Epilepsy (JME) accounts for almost 12% of all epilepsies and is one of the most frequent forms of genetic generalized epilepsies. Genetic studies have revealed that mutations in EFHC1 (EF-hand containing one) account for 3 to 9% of all cases around the world. This gene encodes a protein that is not an ion channel, and several studies have tried to find its cellular role. In this article, we review the various functions that have been proposed for this protein. Interestingly, all of them could affect brain development at different steps, suggesting that the developmental assembly of neural circuits may play a prominent role in JME.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Deficiências do Desenvolvimento/complicações , Deficiências do Desenvolvimento/genética , Epilepsia Mioclônica Juvenil/complicações , Epilepsia Mioclônica Juvenil/genética , Animais , Deficiências do Desenvolvimento/epidemiologia , Predisposição Genética para Doença , Humanos , Epilepsia Mioclônica Juvenil/epidemiologia
7.
Epilepsy Behav ; 28 Suppl 1: S87-90, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23756490

RESUMO

An international workshop on juvenile myoclonic epilepsy (JME) was conducted in Avignon, France in May 2011. During that workshop, a group of 45 experts on JME, together with one of the founding fathers of the syndrome of JME ("Janz syndrome"), Prof. Dr. Dieter Janz from Berlin, reached a consensus on diagnostic criteria and management of JME. The international experts on JME proposed two sets of criteria, which will be helpful for both clinical and scientific purposes. Class I criteria encompass myoclonic jerks without loss of consciousness exclusively occurring on or after awakening and associated with typical generalized epileptiform EEG abnormalities, with an age of onset between 10 and 25. Class II criteria allow the inclusion of myoclonic jerks predominantly occurring after awakening, generalized epileptiform EEG abnormalities with or without concomitant myoclonic jerks, and a greater time window for age at onset (6-25years). For both sets of criteria, patients should have a clear history of myoclonic jerks predominantly occurring after awakening and an EEG with generalized epileptiform discharges supporting a diagnosis of idiopathic generalized epilepsy. Patients with JME require special management because their epilepsy starts in the vulnerable period of adolescence and, accordingly, they have lifestyle issues that typically increase the likelihood of seizures (sleep deprivation, exposure to stroboscopic flashes in discos, alcohol intake, etc.) with poor adherence to antiepileptic drugs (AEDs). Results of an inventory of the different clinical management strategies are given. This article is part of a supplemental special issue entitled Juvenile Myoclonic Epilepsy: What is it Really?


Assuntos
Consenso , Gerenciamento Clínico , Epilepsia Mioclônica Juvenil/diagnóstico , Epilepsia Mioclônica Juvenil/terapia , Humanos , Cooperação Internacional
8.
Planta Med ; 79(5): 334-7, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23457020

RESUMO

In the course of our investigations on Umutambasha in order to identify its convulsant principles, small quantities of monofluoroacetate were observed in stem bark, leaves, and fruits of this plant newly identified as Dichapetalum michelsonii Hauman. Conclusive evidence for a monofluoroacetate presence came from its isolation from the freeze-dried extract of stem bark. Three free unusual amino acids, named N-methyl-α-alanine, N-methyl-ß-alanine, and 2,7-diaminooctan-1,8-dioic acid, described for the first time in a plant, and known trigonelline were also isolated from the stem bark of D. michelsonii. Structure elucidations were mainly achieved by spectroscopic methods (1H-NMR, 2D-NMR, MS) and by comparison with authentic references. These unusual amino acids were detected by a fast, reliable TLC analysis in all our batches of Umutambasha, suggesting that they could be used for identification purposes in case of human or livestock intoxications. Finally, EEG recordings and behavioural observations performed in mice suggested that the convulsive patterns produced by Umutambasha are the consequence of monofluoroacetate presence in D. michelsonii.


Assuntos
Aminoácidos/análise , Fluoracetatos/análise , Magnoliopsida/química , Árvores/química , Animais , Magnoliopsida/toxicidade , Camundongos , Ruanda , Testes de Toxicidade , Árvores/toxicidade
9.
Mol Genet Genomic Med ; 9(2): e1588, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33507632

RESUMO

BACKGROUND: Myotonia congenita (MC) is a common channelopathy affecting skeletal muscle and which is due to pathogenic variants within the CLCN1 gene. Various alterations in the function of the channel have been reported and we here illustrate a novel one. METHODS: A patient presenting the symptoms of myotonia congenita was shown to bear a new heterozygous missense variant in exon 9 of the CLCN1 gene (c.1010 T > G, p.(Phe337Cys)). Confocal imaging and patch clamp recordings of transiently transfected HEK293 cells were used to functionally analyze the effect of this variant on channel properties. RESULTS: Confocal imaging showed that the F337C mutant incorporated as well as the WT channel into the plasma membrane. However, in patch clamp, we observed a smaller conductance for F337C at -80 mV. We also found a marked reduction of the fast gating component in the mutant channels, as well as an overall reduced voltage dependence. CONCLUSION: To our knowledge, this is the first report of a mixed alteration in the biophysical properties of hClC-1 consisting of a reduced conductance at resting potential and an almost abolished voltage dependence.


Assuntos
Canais de Cloreto/genética , Mutação de Sentido Incorreto , Miotonia Congênita/genética , Potenciais de Ação , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Canais de Cloreto/metabolismo , Células HEK293 , Humanos , Ativação do Canal Iônico , Miotonia Congênita/metabolismo , Transporte Proteico
10.
J Neurophysiol ; 104(3): 1417-25, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20592115

RESUMO

The hypothalamic neuropeptide melanin-concentrating hormone (MCH) plays important roles in energy homeostasis, anxiety, and sleep regulation. Since the MCH receptor-1 (MCH-R1), the only functional receptor that mediates MCH functions in rodents, facilitates behavioral performance in hippocampus-dependent learning tasks, we investigated whether glutamatergic transmission in CA1 pyramidal cells could be modulated in mice lacking the MCH-R1 gene (MCH-R1(-/-)). We found that both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartate (NMDA) receptor-mediated transmissions were diminished in the mutant mice compared with their controls. This deficit was explained, at least in part, by a postsynaptic down-regulation of these receptors since the amplitude of miniature excitatory postsynaptic currents and the NMDA/AMPA ratio were decreased. Long-term synaptic potentiation (LTP) was also impaired in MCH-R1(-/-) mice. This was due to an altered induction, rather than an impaired, expression because repeating the induction stimulus restored LTP to a normal magnitude. In addition, long-term synaptic depression was strongly diminished in MCH-R1(-/-) mice. These results suggest that MCH exerts a facilitatory effect on CA1 glutamatergic synaptic transmission and long-term synaptic plasticity. Recently, it has been shown that MCH neurons fire exclusively during sleep and mainly during rapid eye movement sleep. Thus these findings provide a mechanism by which sleep might facilitate memory consolidation.


Assuntos
Ácido Glutâmico/fisiologia , Hipocampo/fisiologia , Plasticidade Neuronal/fisiologia , Receptores de Somatostatina/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Somatostatina/deficiência , Fatores de Tempo
11.
Front Cell Neurosci ; 13: 433, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31611775

RESUMO

Juvenile myoclonic epilepsy (JME), a lifelong disorder that starts during adolescence, is the most common of genetic generalized epilepsy syndromes. JME is characterized by awakening myoclonic jerks and myoclonic-tonic-clonic (m-t-c) grand mal convulsions. Unfortunately, one third of JME patients have drug refractory m-t-c convulsions and these recur in 70-80% who attempt to stop antiepileptic drugs (AEDs). Behavioral studies documented impulsivity, but also impairment of executive functions relying on organization and feedback, which points to prefrontal lobe dysfunction. Quantitative voxel-based morphometry (VBM) revealed abnormalities of gray matter (GM) volumes in cortical (frontal and parietal) and subcortical structures (thalamus, putamen, and hippocampus). Proton magnetic resonance spectroscopy (MRS) found evidence of dysfunction of thalamic neurons. White matter (WM) integrity was disrupted in corpus callosum and frontal WM tracts. Magnetic resonance imaging (MRI) further unveiled anomalies in both GM and WM structures that were already present at the time of seizure onset. Aberrant growth trajectories of brain development occurred during the first 2 years of JME diagnosis. Because of genetic origin, disease causing variants were sought, first by positional cloning, and most recently, by next generation sequencing. To date, only six genes harboring pathogenic variants (GABRA1, GABRD, EFHC1, BRD2, CASR, and ICK) with Mendelian and complex inheritance and covering a limited proportion of the world population, are considered as major susceptibility alleles for JME. Evidence on the cellular role, developmental and cell-type expression profiles of these six diverse JME genes, point to their pathogenic variants driving the first steps of brain development when cell division, expansion, axial, and tangential migration of progenitor cells (including interneuron cortical progenitors) sculpture subtle alterations in brain networks and microcircuits during development. These alterations may explain "microdysgenesis" neuropathology, impulsivity, executive dysfunctions, EEG polyspike waves, and awakening m-t-c convulsions observed in JME patients.

12.
Eur J Neurosci ; 27(7): 1793-800, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18380672

RESUMO

Growing amounts of data indicate involvement of the posterior hypothalamus in the regulation of sleep, especially paradoxical sleep (PS). Accordingly, we previously showed that the melanin-concentrating hormone (MCH)-producing neurons of the rat hypothalamus are selectively activated during a PS rebound. In addition, intracerebroventricular infusion of MCH increases total sleep duration, suggesting a new role for MCH in sleep regulation. To determine whether activation of the MCH system promotes sleep, we studied spontaneous sleep and its homeostatic regulation in mice with deletion of the MCH-receptor 1 gene (MCH-R1-/- vs. MCH-R1+/+) and their behavioural response to modafinil, a powerful antinarcoleptic drug. Here, we show that the lack of functional MCH-R1 results in a hypersomniac-like phenotype, both in basal conditions and after total sleep deprivation, compared to wild-type mice. Further, we found that modafinil was less potent at inducing wakefulness in MCH-R1-/- than in MCH-R1+/+ mice. We report for the first time that animals with genetically inactivated MCH signaling exhibit altered vigilance state architecture and sleep homeostasis. This study also suggests that the MCH system may modulate central pathways involved in the wake-promoting effect of modafinil.


Assuntos
Receptores de Somatostatina/fisiologia , Sono/fisiologia , Animais , Homeostase/genética , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Receptores de Somatostatina/antagonistas & inibidores , Receptores de Somatostatina/deficiência , Receptores de Somatostatina/genética , Sono/genética , Vigília/genética
13.
Pharmacol Biochem Behav ; 88(4): 446-55, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17996928

RESUMO

The present study aimed to test the hypothesis that mice lacking the MCHR1 receptor (Melanin-Concentrating Hormone Receptor-1) present an elevated vulnerability towards the neurobehavioural effects of D-amphetamine, presumably due to previously established up-regulations of dopamine D1 receptors in these mice. We examined the psychomotor effects of five once-daily injections of 1.5 and 3 mg/kg D-amphetamine (i.p.) or ten once-daily injections of 2.25 mg/kg D-amphetamine in knockout (KO) mice lacking the MCHR1 receptor. The first injection of D-amphetamine induced a greater psychomotor response amongst the KO mice at 2.25 and 3.0 mg/kg. On all subsequent d-amphetamine injections, KO mice still showed greater levels of psychomotor activity than the WT mice, but with no between-genotype difference in the rate of development of sensitization (similar slopes of the curves). Furthermore, 24 h after the last injection of 2.25 mg/kg D-amphetamine both genotypes exhibited a significant post-sensitization conditioned activity. Thus, MCHR1 receptors are likely not deeply involved in the mechanisms of induction of sensitization and related conditioned activity induced by D-amphetamine, albeit our results confirm a contribution of these receptors to the mechanisms of the acute effects of that drug, possibly via an inhibitory action on the dopaminergic mesolimbic system. Our results do not support the hypothesis of a functional contribution of MCHR1 receptors to the addictive effects of D-amphetamine.


Assuntos
Estimulantes do Sistema Nervoso Central/farmacologia , Condicionamento Operante/efeitos dos fármacos , Dextroanfetamina/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Desempenho Psicomotor/efeitos dos fármacos , Receptores de Somatostatina/genética , Animais , Interpretação Estatística de Dados , Dopamina/fisiologia , Relação Dose-Resposta a Droga , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
14.
Front Mol Neurosci ; 11: 234, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30042658

RESUMO

The building of the brain is a multistep process that requires the coordinate expression of thousands of genes and an intense nucleocytoplasmic transport of RNA and proteins. This transport is mediated by karyopherins that comprise importins and exportins. Here, we investigated the role of the ß-importin, importin-8 (IPO8) during mouse cerebral corticogenesis as several of its cargoes have been shown to be essential during this process. First, we showed that Ipo8 mRNA is expressed in mouse brain at various embryonic ages with a clear signal in the sub-ventricular/ventricular zone (SVZ/VZ), the cerebral cortical plate (CP) and the ganglionic eminences. We found that acute knockdown of IPO8 in cortical progenitors reduced both their proliferation and cell cycle exit leading to the increase in apical progenitor pool without influencing the number of basal progenitors (BPs). Projection neurons ultimately reached their appropriate cerebral cortical layer, but their dendritogenesis was specifically affected, resulting in neurons with reduced dendrite complexity. IPO8 knockdown also slowed the migration of cortical interneurons. Together, our data demonstrate that IPO8 contribute to the coordination of several critical steps of cerebral cortex development. These results suggest that the impairment of IPO8 function might be associated with some diseases of neuronal migration defects.

15.
Regul Pept ; 143(1-3): 104-8, 2007 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-17537530

RESUMO

The mRNA encoding prepro-Melanin concentrating hormone (ppMCH) is mainly expressed in the central nervous system but has also been detected at lower amount in many peripheral tissues including spleen and thymus. At the peptide level however, several forms of the precursor can be detected in these tissues and are sometimes expressed at similar levels compared to brain. In the present work, we have studied the in vitro action of a wide range of concentration (1 nM to 1 microM) of the different peptides encoded by ppMCH i.e. neuropeptide glycine-glutamic acid (NGE), neuropeptide glutamic acid-isoleucine (NEI), Melanin concentrating hormone (MCH) and the dipeptide NEI-MCH on peripheral blood mononuclear cells (PBMC) proliferation and cytokine production following anti-CD3 stimulation. Among them only MCH decreased PBMC proliferation with a maximal effect of 35% at 100 nM. Moreover as demonstrated by using ELISA, MCH significantly decreases IL-2 production by 25% but not IL-4, INF-gamma or TNF-alpha expression. Interestingly, exogenous IL-2 decreases significantly MCH-mediated inhibition, suggesting that it is an important downstream mediator of MCH action. Finally, we showed that after 7 to 9 days of incubation, MCH also inhibits proliferation of non-stimulated PBMC. Altogether, these data demonstrate that fully mature MCH modulates proliferation of anti-CD3 stimulated PBMC partially through regulation of IL-2 production.


Assuntos
Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Hormônios Hipotalâmicos/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Melaninas/farmacologia , Hormônios Hipofisários/farmacologia , Células Cultivadas , Humanos , Interferon gama/metabolismo , Interleucina-2/metabolismo , Interleucina-2/farmacologia , Interleucina-4/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Fragmentos de Peptídeos/farmacologia , Fator de Necrose Tumoral alfa/metabolismo
16.
Toxicon ; 49(8): 1109-19, 2007 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-17395230

RESUMO

This study was designed to document convulsant and neurotoxic properties of extracts of a tropical tree, Magnistipula butayei subsp. Montana, and to investigate the involvement of the glutamatergic system in these effects. Continuous behavioral observations and electroencephalographic (EEG) records were obtained after per os administration of an aqueous extract of Magnistipula (MBMAE) in rats. MBMAE (800 mg/kg) induced behavioral changes resembling motor limbic seizures: staring and head tremor, automatisms, forelimb clonic movements and violent tonic-clonic seizures leading to death in all animals. Concomitantly, important seizure activity that gradually evolved to epileptiform activity was recorded on the EEG. Moreover, c-Fos immunohistochemistry has revealed an increased c-Fos expression in the dentate gyrus and in piriform, peri- and entorhinal cortices 2 and 4h after treatment. This expression pattern suggested that the mechanism of action for the MBMAE is similar to that observed in glutamate-induced models of epilepsy. The MBMAE increased cell death also in hippocampal cell cultures. Furthermore, the build-up of convulsive activity and epileptic discharges induced by MBMAE in rat were abolished by MK-801, an NMDA receptor antagonist. Our study suggests that MBMAE contains a potent toxin, with a powerful neurotoxic activity in rat, and corresponding to a new natural component(s) that act as an NMDA-mediated convulsant molecule.


Assuntos
Chrysobalanaceae/química , Convulsivantes/toxicidade , Epilepsia Tônico-Clônica/induzido quimicamente , Neurotoxinas/toxicidade , Extratos Vegetais/toxicidade , Análise de Variância , Animais , Convulsivantes/análise , Maleato de Dizocilpina/farmacologia , Eletroencefalografia/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Imuno-Histoquímica , Masculino , Neurotoxinas/análise , Extratos Vegetais/análise , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores
17.
Biochim Biophys Acta ; 1725(1): 93-102, 2005 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-16000236

RESUMO

Thiamine triphosphate (ThTP) is found in most organisms and may be an intracellular signal molecule produced in response to stress. We have recently cloned the cDNA coding for a highly specific mammalian 25-kDa thiamine triphosphatase. The enzyme was active in all mammalian species studied except pig, although the corresponding mRNA was present. In order to determine whether the very low ThTPase activity in pig tissues is due to the absence of the protein or to a lack of catalytic efficiency, we expressed human and pig ThTPase in E. coli as GST fusion proteins. The purified recombinant pig GST-ThTPase was found to be 2-3 orders of magnitude less active than human GST-ThTPase. Using site-directed mutagenesis, we show that, in particular, the change of Glu85 to lysine is responsible for decreased solubility and catalytic activity of the pig enzyme. Immunohistochemical studies revealed a distribution of the protein in pig brain very similar to the one reported in rodent brain. Thus, our results suggest that a 25-kDa protein homologous to hThTPase but practically devoid of enzyme activity is expressed in pig tissues. This raises the possibility that this protein may play a physiological role other than ThTP hydrolysis.


Assuntos
Suínos , Tiamina Trifosfatase/química , Tiamina Trifosfatase/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/enzimologia , Catálise , Clonagem Molecular , Escherichia coli/genética , Humanos , Imuno-Histoquímica , Cinética , Dados de Sequência Molecular , Peso Molecular , Mutagênese Sítio-Dirigida , Homologia de Sequência de Aminoácidos , Tiamina Trifosfatase/genética
18.
Behav Brain Res ; 173(1): 94-103, 2006 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-16839618

RESUMO

The present study aimed at characterizing the acute and intermittent psychomotor responsiveness to cocaine in mice lacking the MCHR1 receptor, which is thought to modulate the mesocorticolimbic sytem functioning [Smith DG, Tzavara ET, Shaw J, Luecke S, Wade M, Davis R, et al. Mesolimbic dopamine super-sensitivity in melanin-concentrating hormone-1 receptor deficient mice. J Neurosci 2005;25:914-22]. On a first free-drug session, MCHR1-deficient mice exhibited significantly higher levels of locomotor activity elicited by the novelty of the test chambers than their wild-type counterparts. On the following day session, a first injection of 6 or 12 mg/kg cocaine induced comparable dose-related psychomotor activations in both genotypes, without significant difference in the relative increase in locomotion. Over the following eight once-daily test sessions, the slight psychomotor increase induced by 6 mg/kg was equivalent in both genotypes and constant over the sessions. At 12 mg/kg, cocaine induced a clear-cut incremental responsiveness to cocaine in both genotypes on the three first sessions; on the following sessions, only the wild-types displayed an incremental responsiveness until the last session, a sensitized effect that was confirmed for the wild-types but not for the knockouts on a subsequent sensitization test (cocaine challenge). Finally, the knockouts did not exhibit any sign of cocaine-conditioning (saline challenge), contrarily to the wild-types. It is speculated that MCHR1 may contribute to the neurobiological mechanisms of conditioned cocaine-induced psychomotor effects, possibly to those underpinning sensitization, and to a lesser extent to those sub-serving acute pharmacological cocaine action.


Assuntos
Comportamento Animal/efeitos dos fármacos , Cocaína/administração & dosagem , Condicionamento Clássico/efeitos dos fármacos , Inibidores da Captação de Dopamina/administração & dosagem , Desempenho Psicomotor/efeitos dos fármacos , Receptores de Somatostatina/metabolismo , Análise de Variância , Animais , Comportamento Animal/fisiologia , Cocaína/farmacologia , Condicionamento Clássico/fisiologia , Inibidores da Captação de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Esquema de Medicação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Desempenho Psicomotor/fisiologia , Receptores de Somatostatina/genética
19.
Biochim Biophys Acta ; 1678(1): 1-6, 2004 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-15093132

RESUMO

The gene encoding the mouse melanin-concentrating hormone receptor 1 was isolated and its structural organization and flanking regions were characterized. The 3' flanking region is marked by the presence of two polyadenylation signals but used with different frequencies. RNase protection and 5' rapid amplification of cDNA ends (RACE) identified multiple transcription initiation sites between -150 and -203 bp upstream of the ATG initiation codon. Functional analysis of deletion mutants reveals a cell independent transcriptional activity localized between nucleotide -305 and -589. The proximal 1.5 kb region does not possess consensus TATA or CAAT boxes but has several consensus sequences for regulatory elements including USF, GATA, AP1, AP4, MyoD, GKLF and Ikaros that could explain the broad expression of the receptor.


Assuntos
Receptores de Somatostatina/genética , Região 3'-Flanqueadora , Região 5'-Flanqueadora , Animais , Sequência de Bases , Códon de Iniciação , Sequência Consenso , Fator 4 Semelhante a Kruppel , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Sítio de Iniciação de Transcrição
20.
Biochim Biophys Acta ; 1592(2): 117-21, 2002 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-12379473

RESUMO

Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues and it may act as a phosphate donor for the phosphorylation of proteins, suggesting a potential role in cell signaling. Two mechanisms have been proposed for the enzymatic synthesis of ThTP. A thiamine diphosphate (ThDP) kinase (ThDP+ATP if ThTP+ADP) has been purified from brewer's yeast and shown to exist in rat liver. However, other data suggest that, at least in skeletal muscle, adenylate kinase 1 (AK1) is responsible for ThTP synthesis. In this study, we show that AK1 knockout mice have normal ThTP levels in skeletal muscle, heart, brain, liver and kidney, demonstrating that AK1 is not responsible for ThTP synthesis in those tissues. We predict that the high ThTP content of particular tissues like the Electrophorus electricus electric organ, or pig and chicken skeletal muscle is more tightly correlated with high ThDP kinase activity or low soluble ThTPase activity than with non-stringent substrate specificity and high activity of adenylate kinase.


Assuntos
Adenilato Quinase/deficiência , Isoenzimas/deficiência , Tiamina Trifosfato/metabolismo , Animais , Encéfalo/metabolismo , Rim/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Tiamina Trifosfato/análise , Tiamina Trifosfato/biossíntese
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