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1.
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
2.
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.

3.
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.

4.
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
5.
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
6.
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
7.
Behav Brain Res ; 262: 14-20, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24412349

RESUMO

Chronic food restriction (FR) and maintenance of low body weight have long been known to increase the rewarding and motor-activating effects of addictive drugs. However, the neurobiological mechanisms through which FR potentiates drug reward remain largely unknown. Melanin-concentrating hormone (MCH) signaling could be one of these mechanisms since this peptide is involved in energy homeostasis and modulates mesolimbic dopaminergic transmission. The purpose of the present study was to test this hypothesis by investigating the impact of FR on amphetamine reward in wild-type (WT) and knockout mice lacking the melanin-concentrating hormone receptor-1 (MCHR1-KO). The rewarding effects of amphetamine (0.75-2.25 mg/kg, i.p.) were measured with the conditioned place preference (CPP) technique. The food of the mice was restricted to maintain their body weight at 80-85% of their free-feeding (FF) weight throughout the entire CPP experiment. Locomotor activity of the animals was recorded during the conditioning sessions. Our results show that locomotion of all the food-restricted mice treated with saline or amphetamine increased over the sessions whatever the genotype. On the place preference test, the amplitude of CPP induced by 0.75 mg/kg amphetamine was higher in food restricted WT mice than in free-fed WT mice and food restricted MCHR1-KO mice. However, FR did not affect amphetamine reward in MCHR1-KO mice. The present results indicate that MCH signaling could be involved in the ability of FR to increase amphetamine-induced CPP.


Assuntos
Anfetamina/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Condicionamento Psicológico/efeitos dos fármacos , Privação de Alimentos , Receptores do Hormônio Hipofisário/genética , Recompensa , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos
8.
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
9.
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
10.
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
11.
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
12.
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
14.
Seizure ; 21(1): 32-9, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22000707

RESUMO

BACKGROUND: Given the continuous knowledge progression and the growing number of available antiepileptic drugs (AEDs), making appropriate treatment choices for patients with epilepsy is increasingly difficult. While published guidelines help for separate clinical aspects, patients with a combination of specific characteristics may escape proper guidance. This study aimed to determine the appropriateness of AEDs for particular clinical variables and to offer treatment recommendations for adult patients with epilepsy in a user-friendly format for practicing neurologists. METHODS: Using the RAND/UCLA Appropriateness Method, the appropriateness of AEDs as initial/second mono-therapy and combination therapy was assessed in relation to selected clinical variables by a Belgian panel of 13 experts in epilepsy. Panel recommendations for particular patient profiles were determined by the outcome of these separate ratings. RESULTS: The appropriateness outcome of individual AEDs was not substantially different between first and second mono-therapy; valproate was considered appropriate for all types of generalised and partial seizures. The outcome for combination therapy was highly dependent on the type of AED and seizures. With respect to co-morbidities and co-treatments, levetiracetam and pregabalin proved to have the least contra-indications. For the elderly and with respect to factors related to the female reproductive system the appropriateness of AEDs showed a more diffuse pattern. Although caution was deemed necessary for some combinations, the AEDs were never considered inappropriate regarding their drug interaction profile. CONCLUSIONS: The Epi-Scope(®) tool that displays appropriateness recommendations for highly specific, possibly complex cases, supports optimal treatment choices for adult patients with epilepsy in daily practice.


Assuntos
Anticonvulsivantes/uso terapêutico , Epilepsia/tratamento farmacológico , Sistemas Inteligentes , Adolescente , Adulto , Técnicas de Apoio para a Decisão , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Interface Usuário-Computador , Adulto Jovem
15.
PLoS One ; 5(10): e13616, 2010 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-21049048

RESUMO

BACKGROUND: Thiamine (vitamin B1) is an essential molecule for all life forms because thiamine diphosphate (ThDP) is an indispensable cofactor for oxidative energy metabolism. The less abundant thiamine monophosphate (ThMP), thiamine triphosphate (ThTP) and adenosine thiamine triphosphate (AThTP), present in many organisms, may have still unidentified physiological functions. Diseases linked to thiamine deficiency (polyneuritis, Wernicke-Korsakoff syndrome) remain frequent among alcohol abusers and other risk populations. This is the first comprehensive study on the distribution of thiamine derivatives in human biopsies, body fluids and cell lines. METHODOLOGY AND PRINCIPAL FINDINGS: Thiamine derivatives were determined by HPLC. In human tissues, the total thiamine content is lower than in other animal species. ThDP is the major thiamine compound and tissue levels decrease at high age. In semen, ThDP content correlates with the concentration of spermatozoa but not with their motility. The proportion of ThTP is higher in humans than in rodents, probably because of a lower 25-kDa ThTPase activity. The expression and activity of this enzyme seems to correlate with the degree of cell differentiation. ThTP was present in nearly all brain and muscle samples and in ∼60% of other tissue samples, in particular fetal tissue and cultured cells. A low ([ThTP]+[ThMP])/([Thiamine]+[ThMP]) ratio was found in cardiovascular tissues of patients with cardiac insufficiency. AThTP was detected only sporadically in adult tissues but was found more consistently in fetal tissues and cell lines. CONCLUSIONS AND SIGNIFICANCE: The high sensitivity of humans to thiamine deficiency is probably linked to low circulating thiamine concentrations and low ThDP tissue contents. ThTP levels are relatively high in many human tissues, as a result of low expression of the 25-kDa ThTPase. Another novel finding is the presence of ThTP and AThTP in poorly differentiated fast-growing cells, suggesting a hitherto unsuspected link between these compounds and cell division or differentiation.


Assuntos
Líquidos Corporais/metabolismo , Tiamina/metabolismo , Animais , Biópsia , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Humanos , Camundongos , Oxirredução , Fosforilação , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
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
17.
Epilepsy Res ; 88(2-3): 196-207, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20015616

RESUMO

EFHC1, a gene mutated in juvenile myoclonic epilepsy, encodes EFHC1, a protein with three DM10 domains and one EF-hand motif. We recently demonstrated that this molecule is a microtubule-associated protein (MAP) implicated in neuronal migration. Because some controversies persist about the precise localization in the CNS, we studied the neuroanatomical distribution of EFHC1 in mature and developing mouse brain. In the adult, low mRNA expression was detected in several brain structures such as cortex, striatum, hippocampus and cerebellum. At E16, EFHC1 mRNA was shown to be expressed in cortex and not only in cells lining ventricles. Using a purified polyclonal antibody, EFHC1 staining was observed in all cortical layers, in piriform cortex, in hippocampus and in Purkinje cells of cerebellum. In the cortex, the majority of EFHC1 positive cells correspond to neurons, however some glial cells were also stained. In agreement with a previous study, we demonstrated strong EFHC1 expression in cilia of ependymal cells lining cerebral ventricles. Moreover, at E16, the protein was observed at the borders of brain ventricles, in choroid plexus, but also, although to a lesser extent, in piriform and neocortex. In these latter structures, the pattern of expression seems to correspond to the extensions of the radial glia fibers as demonstrated by BLBP immunostaining. Finally, we confirmed that EFHC1 was also expressed and co-localized with the mitotic spindle of neural stem cells. These results confirm that EFHC1 is a protein with a likely low expression level in mouse brain but detectable both in adult and embryonic brain supporting our previous data and hypothesis that EFHC1 could play an important role during brain development. As discussed, this opens the door to a new conceptual approach viewing some idiopathic generalized epilepsies as developmental diseases instead of classical channelopathies.


Assuntos
Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Animais , Proteínas de Ligação ao Cálcio/genética , Ciclo Celular/genética , Movimento Celular/genética , Células Cultivadas , Imuno-Histoquímica , Camundongos , Mitose/genética , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
Nat Neurosci ; 12(10): 1266-74, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19734894

RESUMO

Mutations in the EFHC1 gene are linked to juvenile myoclonic epilepsy (JME), one of the most frequent forms of idiopathic generalized epilepsies. JME is associated with subtle alterations of cortical and subcortical architecture, but the underlying pathological mechanism remains unknown. We found that EFHC1 is a microtubule-associated protein involved in the regulation of cell division. In vitro, EFHC1 loss of function disrupted mitotic spindle organization, impaired M phase progression, induced microtubule bundling and increased apoptosis. EFHC1 impairment in the rat developing neocortex by ex vivo and in utero electroporation caused a marked disruption of radial migration. We found that this effect was a result of cortical progenitors failing to exit the cell cycle and defects in the radial glia scaffold organization and in the locomotion of postmitotic neurons. Therefore, we propose that EFHC1 is a regulator of cell division and neuronal migration during cortical development and that disruption of its functions leads to JME.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Divisão Celular/fisiologia , Córtex Cerebral , Microtúbulos/metabolismo , Neurônios/fisiologia , Análise de Variância , Animais , Animais Geneticamente Modificados , Apoptose/fisiologia , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Transformada , Movimento Celular/fisiologia , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Eletroporação/métodos , Embrião de Mamíferos , Células-Tronco Embrionárias/fisiologia , Feminino , Citometria de Fluxo/métodos , Proteínas de Fluorescência Verde/genética , Humanos , Imunoprecipitação/métodos , Marcação In Situ das Extremidades Cortadas/métodos , Mutação , Neuroglia/metabolismo , Gravidez , Ligação Proteica , Interferência de RNA/fisiologia , Ratos , Ratos Wistar , Transfecção/métodos
19.
Peptides ; 30(11): 2076-80, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19450627

RESUMO

To date, melanin-concentrating hormone (MCH) has been generally considered as peptide acting almost exclusively in the central nervous system. In the present paper, we revise the experimental evidence, demonstrating that MCH and its receptors are expressed by cells of the immune system and directly influence the response of these cells in some circumstances. This therefore supports the idea that, as with other peptides, MCH could be considered as a modulator of the immune system. Moreover, we suggest that this could have important implications in several immune-mediated disorders and affirm that there is a clear need for further investigation.


Assuntos
Hormônios Hipotalâmicos/imunologia , Sistema Imunitário/metabolismo , Melaninas/imunologia , Hormônios Hipofisários/imunologia , Animais , Citocinas/metabolismo , Humanos , Hormônios Hipotalâmicos/genética , Hormônios Hipotalâmicos/metabolismo , Sistema Imunitário/imunologia , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Linfócitos/imunologia , Linfócitos/metabolismo , Melaninas/genética , Melaninas/metabolismo , Hormônios Hipofisários/genética , Hormônios Hipofisários/metabolismo , Receptores do Hormônio Hipofisário/genética , Receptores do Hormônio Hipofisário/imunologia , Receptores do Hormônio Hipofisário/metabolismo
20.
Psychopharmacology (Berl) ; 202(4): 673-87, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18843481

RESUMO

RATIONALE: Previous studies have shown that intraperitoneal injections of thioperamide, an imidazole-based H3 receptor inverse agonist that enhances histamine release in the brain, potentiate cocaine-induced hyperlocomotion. The present study examined the involvement of the histaminergic system in these effects of thioperamide in mice. MATERIALS AND METHODS: We investigated whether immepip, a selective H3 agonist, could reverse the potentiating effects of thioperamide. Moreover, the non-imidazole H3 inverse agonist A-331440 was tested on the locomotor effects of cocaine. Using high-performance liquid chromatography with ultraviolet detection, cocaine plasma concentrations were measured to study potential drug-drug interactions between thioperamide and cocaine. Finally, thioperamide was tested on the locomotor effects of cocaine in histamine-deficient knockout mice in order to determine the contribution of histamine to the modulating effects of thioperamide. RESULTS: Thioperamide potentiated cocaine-induced hyperlocomotion in normal mice, and to a higher extent, in histamine-deficient knockout mice. A-331440 only slightly affected the locomotor effects of cocaine. Immepip did not alter cocaine-induced hyperactivity but significantly reduced the potentiating actions of thioperamide on cocaine's effects. Finally, plasma cocaine concentrations were more elevated in mice treated with thioperamide than in mice that received cocaine alone. CONCLUSIONS: The present results indicate that histamine released by thioperamide through the blockade of H3 autoreceptors is not involved in the ability of this compound to potentiate cocaine induced-hyperactivity. Our data suggest that thioperamide, at least at 10 mg/kg, increases cocaine-induced locomotion through the combination of pharmacokinetic effects and the blockade of H3 receptors located on non-histaminergic neurons.


Assuntos
Cocaína/antagonistas & inibidores , Cocaína/farmacologia , Agonistas dos Receptores Histamínicos/farmacologia , Histamina/fisiologia , Atividade Motora/efeitos dos fármacos , Piperidinas/farmacologia , Animais , Área Sob a Curva , Compostos de Bifenilo/farmacologia , Cocaína/farmacocinética , Relação Dose-Resposta a Droga , Interações Medicamentosas , Agonistas dos Receptores Histamínicos/farmacocinética , Liberação de Histamina/efeitos dos fármacos , Imidazóis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitrilas/farmacologia , Piperidinas/farmacocinética , Pirrolidinas/farmacologia , Receptores Histamínicos H3/efeitos dos fármacos , Receptores Histamínicos H3/genética , Receptores Histamínicos H3/fisiologia
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