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1.
Neuropharmacology ; 197: 108722, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34273387

RESUMO

Although several ionic mechanisms are known to control rate and regularity of the slow pacemaker in dopamine (DA) neurons, the core mechanism of pacing is controversial. Here we tested the hypothesis that pacemaking of SNc DA neurons is enabled by an unconventional conductance. We found that 1-(2,4-xylyl)guanidinium (XG), an established blocker of gating pore currents, selectively inhibits pacemaking of DA neurons. The compound inhibited all slow pacemaking DA neurons that were tested, both in the substantia nigra pars compacta, and in the ventral tegmental area. Interestingly, bursting behavior was not affected by XG. Furthermore, the drug did not affect fast pacemaking of GABAergic neurons from substantia nigra pars reticulata neurons or slow pacemaking of noradrenergic neurons. In DA neurons, current-clamp analysis revealed that XG did not appear to affect ion channels involved in the action potential. Its inhibitory effect persisted during blockade of all ion channels previously suggested to contribute to pacemaking. RNA sequencing and voltage-clamp recordings yielded no evidence for a gating pore current to underlie the conductance. However, we could isolate a small subthreshold XG-sensitive current, which was carried by both Na+ and Cl- ions. Although the molecular target of XG remains to be defined, these observations represent a step towards understanding pacemaking in DA neurons.


Assuntos
Relógios Biológicos/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Guanidina/análogos & derivados , Guanidina/farmacologia , Mesencéfalo/efeitos dos fármacos , Animais , Neurônios GABAérgicos/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Norepinefrina/fisiologia , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Substância Negra/efeitos dos fármacos , Área Tegmentar Ventral/efeitos dos fármacos
2.
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
3.
Biomedicines ; 8(9)2020 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-32962139

RESUMO

Thiamine precursors, the most studied being benfotiamine (BFT), have protective effects in mouse models of neurodegenerative diseases. BFT decreased oxidative stress and inflammation, two major characteristics of neurodegenerative diseases, in a neuroblastoma cell line (Neuro2a) and an immortalized brain microglial cell line (BV2). Here, we tested the potential antioxidant and anti-inflammatory effects of the hitherto unexplored derivative O,S-dibenzoylthiamine (DBT) in these two cell lines. We show that DBT protects Neuro2a cells against paraquat (PQ) toxicity by counteracting oxidative stress at low concentrations and increases the synthesis of reduced glutathione and NADPH in a Nrf2-independent manner. In BV2 cells activated by lipopolysaccharides (LPS), DBT significantly decreased inflammation by suppressing translocation of NF-κB to the nucleus. Our results also demonstrate the superiority of DBT over thiamine and other thiamine precursors, including BFT, in all of the in vitro models. Finally, we show that the chronic administration of DBT arrested motor dysfunction in FUS transgenic mice, a model of amyotrophic lateral sclerosis, and it reduced depressive-like behavior in a mouse model of ultrasound-induced stress in which it normalized oxidative stress marker levels in the brain. Together, our data suggest that DBT may have therapeutic potential for brain pathology associated with oxidative stress and inflammation by novel, coenzyme-independent mechanisms.

4.
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.

5.
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.

6.
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
7.
Mol Cell Neurosci ; 82: 126-136, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28506637

RESUMO

Thiamine is essential for normal brain function and its deficiency causes metabolic impairment, specific lesions, oxidative damage and reduced adult hippocampal neurogenesis (AHN). Thiamine precursors with increased bioavailability, especially benfotiamine, exert neuroprotective effects not only for thiamine deficiency (TD), but also in mouse models of neurodegeneration. As it is known that AHN is impaired by stress in rodents, we exposed C57BL6/J mice to predator stress for 5 consecutive nights and studied the proliferation (number of Ki67-positive cells) and survival (number of BrdU-positive cells) of newborn immature neurons in the subgranular zone of the dentate gyrus. In stressed mice, the number of Ki67- and BrdU-positive cells was reduced compared to non-stressed animals. This reduction was prevented when the mice were treated (200mg/kg/day in drinking water for 20days) with thiamine or benfotiamine, that were recently found to prevent stress-induced behavioral changes and glycogen synthase kinase-3ß (GSK-3ß) upregulation in the CNS. Moreover, we show that thiamine and benfotiamine counteract stress-induced bodyweight loss and suppress stress-induced anxiety-like behavior. Both treatments induced a modest increase in the brain content of free thiamine while the level of thiamine diphosphate (ThDP) remained unchanged, suggesting that the beneficial effects observed are not linked to the role of this coenzyme in energy metabolism. Predator stress increased hippocampal protein carbonylation, an indicator of oxidative stress. This effect was antagonized by both thiamine and benfotiamine. Moreover, using cultured mouse neuroblastoma cells, we show that in particular benfotiamine protects against paraquat-induced oxidative stress. We therefore hypothesize that thiamine compounds may act by boosting anti-oxidant cellular defenses, by a mechanism that still remains to be unveiled. Our study demonstrates, for the first time, that thiamine and benfotiamine prevent stress-induced inhibition of hippocampal neurogenesis and accompanying physiological changes. The present data suggest that thiamine precursors with high bioavailability might be useful as a complementary therapy in several neuropsychiatric disorders.


Assuntos
Hipocampo/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Tiamina Pirofosfato/farmacologia , Tiamina/análogos & derivados , Tiamina/metabolismo , Animais , Giro Denteado/efeitos dos fármacos , Giro Denteado/metabolismo , Modelos Animais de Doenças , Quinase 3 da Glicogênio Sintase/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/metabolismo , Hipocampo/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Tiamina/farmacologia
8.
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
9.
Metab Brain Dis ; 29(4): 1069-82, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24590690

RESUMO

Thiamine triphosphate (ThTP) was discovered over 60 years ago and it was long thought to be a specifically neuroactive compound. Its presence in most cell types, from bacteria to mammals, would suggest a more general role but this remains undefined. In contrast to thiamine diphosphate (ThDP), ThTP is not a coenzyme. In E. coli cells, ThTP is transiently produced in response to amino acid starvation, while in mammalian cells, it is constitutively produced at a low rate. Though it was long thought that ThTP was synthesized by a ThDP:ATP phosphotransferase, more recent studies indicate that it can be synthesized by two different enzymes: (1) adenylate kinase 1 in the cytosol and (2) FoF1-ATP synthase in brain mitochondria. Both mechanisms are conserved from bacteria to mammals. Thus ThTP synthesis does not seem to require a specific enzyme. In contrast, its hydrolysis is catalyzed, at least in mammalian tissues, by a very specific cytosolic thiamine triphosphatase (ThTPase), controlling the steady-state cellular concentration of ThTP. In some tissues where adenylate kinase activity is high and ThTPase is absent, ThTP accumulates, reaching ≥ 70% of total thiamine, with no obvious physiological consequences. In some animal tissues, ThTP was able to phosphorylate proteins, and activate a high-conductance anion channel in vitro. These observations raise the possibility that ThTP is part of a still uncharacterized cellular signaling pathway. On the other hand, its synthesis by a chemiosmotic mechanism in mitochondria and respiring bacteria might suggest a role in cellular energetics.


Assuntos
Tiamina Trifosfato/fisiologia , Trifosfato de Adenosina/metabolismo , Adenilato Quinase/metabolismo , Aminoácidos/metabolismo , Animais , Encéfalo/metabolismo , Citosol/metabolismo , Previsões , Humanos , Doença de Leigh/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Modelos Neurológicos , Especificidade de Órgãos , Fosforilação , Processamento de Proteína Pós-Traducional , ATPases Translocadoras de Prótons/metabolismo , Transdução de Sinais , Especificidade da Espécie , Tiamina Trifosfatase/metabolismo , Tiamina Trifosfato/biossíntese
10.
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
11.
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
12.
Biochim Biophys Acta ; 1830(10): 4513-23, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23707715

RESUMO

BACKGROUND: Thiamine triphosphate (ThTP) is present in most organisms and might be involved in intracellular signaling. In mammalian cells, the cytosolic ThTP level is controlled by a specific thiamine triphosphatase (ThTPase), belonging to the CYTH superfamily of proteins. CYTH proteins are present in all superkingdoms of life and act on various triphosphorylated substrates. METHODS: Using crystallography, mass spectrometry and mutational analysis, we identified the key structural determinants of the high specificity and catalytic efficiency of mammalian ThTPase. RESULTS: Triphosphate binding requires three conserved arginines while the catalytic mechanism relies on an unusual lysine-tyrosine dyad. By docking of the ThTP molecule in the active site, we found that Trp-53 should interact with the thiazole part of the substrate molecule, thus playing a key role in substrate recognition and specificity. Sea anemone and zebrafish CYTH proteins, which retain the corresponding Trp residue, are also specific ThTPases. Surprisingly, the whole chromosome region containing the ThTPase gene is lost in birds. CONCLUSIONS: The specificity for ThTP is linked to a stacking interaction between the thiazole heterocycle of thiamine and a tryptophan residue. The latter likely plays a key role in the secondary acquisition of ThTPase activity in early metazoan CYTH enzymes, in the lineage leading from cnidarians to mammals. GENERAL SIGNIFICANCE: We show that ThTPase activity is not restricted to mammals as previously thought but is an acquisition of early metazoans. This, and the identification of critically important residues, allows us to draw an evolutionary perspective of the CYTH family of proteins.


Assuntos
Tiamina Trifosfatase/metabolismo , Sequência de Aminoácidos , Animais , Biocatálise , Dicroísmo Circular , Cristalografia por Raios X , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato , Tiamina Trifosfatase/química
13.
Nat Neurosci ; 16(7): 845-7, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23708141
14.
Sci Rep ; 3: 1071, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23323214

RESUMO

In E. coli, thiamine triphosphate (ThTP), a putative signaling molecule, transiently accumulates in response to amino acid starvation. This accumulation requires the presence of an energy substrate yielding pyruvate. Here we show that in intact bacteria ThTP is synthesized from free thiamine diphosphate (ThDP) and P(i), the reaction being energized by the proton-motive force (Δp) generated by the respiratory chain. ThTP production is suppressed in strains carrying mutations in F(1) or a deletion of the atp operon. Transformation with a plasmid encoding the whole atp operon fully restored ThTP production, highlighting the requirement for F(o)F(1)-ATP synthase in ThTP synthesis. Our results show that, under specific conditions of nutritional downshift, F(o)F(1)-ATP synthase catalyzes the synthesis of ThTP, rather than ATP, through a highly regulated process requiring pyruvate oxidation. Moreover, this chemiosmotic mechanism for ThTP production is conserved from E. coli to mammalian brain mitochondria.


Assuntos
Escherichia coli/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Tiamina Trifosfato/biossíntese , Ciclo do Ácido Cítrico , Mutação , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/isolamento & purificação , Ácido Pirúvico/química , Ácido Pirúvico/metabolismo
15.
PLoS One ; 7(9): e43879, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22984449

RESUMO

BACKGROUND: We recently characterized a specific inorganic triphosphatase (PPPase) from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPP(i)) is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPP(i) but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility. CONCLUSIONS AND GENERAL SIGNIFICANCE: We show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPP(i) in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPP(i), which could be cytotoxic because of its high affinity for Ca(2+), thereby interfering with Ca(2+) signaling.


Assuntos
Bactérias/enzimologia , Compostos Inorgânicos/metabolismo , Mamíferos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Animais , Biocatálise , Sobrevivência Celular , Eletroforese Capilar , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Hidrólise , Pirofosfatase Inorgânica/metabolismo , Dados de Sequência Molecular , Nitrosomonas europaea/enzimologia , Fosfoproteínas Fosfatases/química , Polifosfatos/isolamento & purificação , Polifosfatos/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Frações Subcelulares/enzimologia , Especificidade por Substrato
16.
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
17.
Eur J Neurosci ; 36(8): 3126-33, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22775118

RESUMO

The orexigenic neuropeptide melanin-concentrating hormone (MCH) is well positioned to play a key role in connecting brain reward and homeostatic systems due to its synthesis in hypothalamic circuitry and receptor expression throughout the cortico-striatal reward circuit. Here we examined whether targeted-deletion of the MCH receptor (MCH-1R) in gene-targeted heterozygote and knockout mice (KO), or systemic treatment with pharmacological agents designed to antagonise MCH-1R in C57BL/6J mice would disrupt two putative consequences of reward learning that rely on different neural circuitries: conditioned reinforcement (CRf) and Pavlovian-instrumental transfer (PIT). Mice were trained to discriminate between presentations of a reward-paired cue (CS+) and an unpaired CS-. Following normal acquisition of the Pavlovian discrimination in all mice, we assessed the capacity for the CS+ to act as a reinforcer for new nose-poke learning (CRf). Pharmacological disruption in control mice and genetic deletion in KO mice impaired CRf test performance, suggesting MCH-1R is necessary for initiating and maintaining behaviors that are under the control of conditioned reinforcers. To examine a dissociable form of reward learning (PIT), a naïve group of mice were trained in separate Pavlovian and instrumental lever training sessions followed by the PIT test. For all mice the CS+ was capable of augmenting ongoing lever responding relative to CS- periods. These results suggest a role for MCH in guiding behavior based on the conditioned reinforcing value of a cue, but not on its incentive motivational value.


Assuntos
Condicionamento Clássico , Aprendizagem , Receptores do Hormônio Hipofisário/fisiologia , Recompensa , Animais , Deleção de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Oligopeptídeos/farmacologia , Receptores do Hormônio Hipofisário/antagonistas & inibidores , Receptores do Hormônio Hipofisário/genética
18.
J Biol Chem ; 286(39): 34023-35, 2011 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-21840996

RESUMO

The CYTH superfamily of proteins is named after its two founding members, the CyaB adenylyl cyclase from Aeromonas hydrophila and the human 25-kDa thiamine triphosphatase. Because these proteins often form a closed ß-barrel, they are also referred to as triphosphate tunnel metalloenzymes (TTM). Functionally, they are characterized by their ability to bind triphosphorylated substrates and divalent metal ions. These proteins exist in most organisms and catalyze different reactions depending on their origin. Here we investigate structural and catalytic properties of the recombinant TTM protein from Nitrosomonas europaea (NeuTTM), a 19-kDa protein. Crystallographic data show that it crystallizes as a dimer and that, in contrast to other TTM proteins, it has an open ß-barrel structure. We demonstrate that NeuTTM is a highly specific inorganic triphosphatase, hydrolyzing tripolyphosphate (PPP(i)) with high catalytic efficiency in the presence of Mg(2+). These data are supported by native mass spectrometry analysis showing that the enzyme binds PPP(i) (and Mg-PPP(i)) with high affinity (K(d) < 1.5 µm), whereas it has a low affinity for ATP or thiamine triphosphate. In contrast to Aeromonas and Yersinia CyaB proteins, NeuTTM has no adenylyl cyclase activity, but it shares several properties with other enzymes of the CYTH superfamily, e.g. heat stability, alkaline pH optimum, and inhibition by Ca(2+) and Zn(2+) ions. We suggest a catalytic mechanism involving a catalytic dyad formed by Lys-52 and Tyr-28. The present data provide the first characterization of a new type of phosphohydrolase (unrelated to pyrophosphatases or exopolyphosphatases), able to hydrolyze inorganic triphosphate with high specificity.


Assuntos
Proteínas de Bactérias/química , Hidrolases/química , Metaloproteínas/química , Nitrosomonas europaea/enzimologia , Proteínas de Bactérias/genética , Catálise , Hidrolases/genética , Metaloproteínas/genética , Nitrosomonas europaea/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Relação Estrutura-Atividade
19.
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
20.
BMC Dev Biol ; 10: 95, 2010 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-20815887

RESUMO

BACKGROUND: Conditional gene activation is an efficient strategy for studying gene function in genetically modified animals. Among the presently available gene switches, the tetracycline-regulated system has attracted considerable interest because of its unique potential for reversible and adjustable gene regulation. RESULTS: To investigate whether the ubiquitously expressed Gt(ROSA)26Sor locus enables uniform DOX-controlled gene expression, we inserted the improved tetracycline-regulated transcription activator iM2 together with an iM2 dependent GFP gene into the Gt(ROSA)26Sor locus, using gene targeting to generate ROSA26-iM2-GFP (R26t1Δ) mice. Despite the presence of ROSA26 promoter driven iM2, R26t1Δ mice showed very sparse DOX-activated expression of different iM2-responsive reporter genes in the brain, mosaic expression in peripheral tissues and more prominent expression in erythroid, myeloid and lymphoid lineages, in hematopoietic stem and progenitor cells and in olfactory neurons. CONCLUSIONS: The finding that gene regulation by the DOX-activated transcriptional factor iM2 in the Gt(ROSA)26Sor locus has its limitations is of importance for future experimental strategies involving transgene activation from the endogenous ROSA26 promoter. Furthermore, our ROSA26-iM2 knock-in mouse model (R26t1Δ) represents a useful tool for implementing gene function in vivo especially under circumstances requiring the side-by-side comparison of gene manipulated and wild type cells. Since the ROSA26-iM2 mouse allows mosaic gene activation in peripheral tissues and haematopoietic cells, this model will be very useful for uncovering previously unknown or unsuspected phenotypes.


Assuntos
Técnicas de Introdução de Genes/métodos , Técnicas Genéticas , Transgenes , Animais , Doxiciclina , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Transgênicos , Tetraciclina
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