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1.
Sci Signal ; 15(734): eabg4982, 2022 05 17.
Artículo en Inglés | MEDLINE | ID: mdl-35580169

RESUMEN

The development of the neuromuscular junction (NMJ) requires dynamic trans-synaptic coordination orchestrated by secreted factors, including Wnt family morphogens. To investigate how these synaptic cues in NMJ development are transduced, particularly in the regulation of acetylcholine receptor (AChR) accumulation in the postsynaptic membrane, we explored the function of Van Gogh-like protein 2 (Vangl2), a core component of Wnt planar cell polarity signaling. We found that conditional, muscle-specific ablation of Vangl2 in mice reproduced the NMJ differentiation defects seen in mice with global Vangl2 deletion. These alterations persisted into adulthood and led to NMJ disassembly, impaired neurotransmission, and deficits in motor function. Vangl2 and the muscle-specific receptor tyrosine kinase MuSK were functionally associated in Wnt signaling in the muscle. Vangl2 bound to and promoted the signaling activity of MuSK in response to Wnt11. The loss of Vangl2 impaired RhoA activation in cultured mouse myotubes and caused dispersed, rather than clustered, organization of AChRs at the postsynaptic or muscle cell side of NMJs in vivo. Our results identify Vangl2 as a key player of the core complex of molecules shaping neuromuscular synapses and thus shed light on the molecular mechanisms underlying NMJ assembly.


Asunto(s)
Polaridad Celular , Proteínas del Tejido Nervioso/metabolismo , Proteínas Tirosina Quinasas , Animales , Ácidos Grasos Monoinsaturados , Ratones , Fibras Musculares Esqueléticas/metabolismo , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Sinapsis/genética , Sinapsis/metabolismo
2.
J Med Chem ; 63(21): 12682-12692, 2020 11 12.
Artículo en Inglés | MEDLINE | ID: mdl-33063995

RESUMEN

Venom peptides are promising drug leads, but their therapeutic use is often limited by stability and bioavailability issues. In this study, we designed cyclic analogues of α-conotoxin CIA, a potent muscle nicotinic acetylcholine receptor (nAChR) blocker with a significantly lower affinity at the neuronal α3ß2 subtype. Remarkably, all analogues retained the low nanomolar activity of native CIA toward muscle-type nAChRs but showed greatly improved resistance to degradation in human serum and, surprisingly, displayed up to 52-fold higher potency for the α3ß2 neuronal nAChR subtype (IC50 1.3 nM). Comparison of nuclear magnetic resonance-derived structures revealed some differences that might explain the gain of potency at α3ß2 nAChRs. All peptides were highly paralytic when injected into adult zebrafish and bath-applied to zebrafish larvae, suggesting barrier-crossing capabilities and efficient uptake. Finally, these cyclic CIA analogues were shown to be unique pharmacological tools to investigate the contribution of the presynaptic α3ß2 nAChR subtype to the train-of-four fade.


Asunto(s)
Ligandos , Músculos/metabolismo , Neuronas/metabolismo , Antagonistas Nicotínicos/química , Péptidos/química , Receptores Nicotínicos/metabolismo , Ponzoñas/metabolismo , Secuencia de Aminoácidos , Animales , Conotoxinas/química , Ciclización , Larva/efectos de los fármacos , Larva/fisiología , Locomoción/efectos de los fármacos , Ratones , Contracción Muscular/efectos de los fármacos , Antagonistas Nicotínicos/metabolismo , Antagonistas Nicotínicos/farmacología , Péptidos/metabolismo , Péptidos/farmacología , Unión Proteica , Estructura Terciaria de Proteína , Receptores Nicotínicos/química , Pez Cebra/crecimiento & desarrollo , Pez Cebra/fisiología
3.
FASEB J ; 33(5): 5823-5835, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30844310

RESUMEN

Autism spectrum disorder (ASD) is characterized by early onset of behavioral and cognitive alterations. Low plasma levels of oxytocin (OT) have also been found in ASD patients; recently, a critical role for the enzyme CD38 in the regulation of OT release was demonstrated. CD38 is important in regulating several Ca2+-dependent pathways, but beyond its role in regulating OT secretion, it is not known whether a deficit in CD38 expression leads to functional modifications of the prefrontal cortex (PFC), a structure involved in social behavior. Here, we report that CD38-/- male mice show an abnormal cortex development, an excitation-inhibition balance shifted toward a higher excitation, and impaired synaptic plasticity in the PFC such as those observed in various mouse models of ASD. We also show that a lack of CD38 alters social behavior and emotional responses. Finally, examining neuromodulators known to control behavioral flexibility, we found elevated monoamine levels in the PFC of CD38-/- adult mice. Overall, our study unveiled major changes in PFC physiologic mechanisms and provides new evidence that the CD38-/- mouse could be a relevant model to study pathophysiological brain mechanisms of mental disorders such as ASD.-Martucci, L. L., Amar, M., Chaussenot, R., Benet, G., Bauer, O., de Zélicourt, A., Nosjean, A., Launay, J.-M., Callebert, J., Sebrié, C., Galione, A., Edeline, J.-M., de la Porte, S., Fossier, P., Granon, S., Vaillend, C., Cancela, J.-M., A multiscale analysis in CD38-/- mice unveils major prefrontal cortex dysfunctions.


Asunto(s)
ADP-Ribosil Ciclasa 1/genética , ADP-Ribosil Ciclasa 1/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Plasticidad Neuronal , Corteza Prefrontal/fisiopatología , Aminas/metabolismo , Animales , Ansiedad , Trastorno del Espectro Autista/genética , Conducta Animal , Tronco Encefálico , Calcio/metabolismo , Miedo , Regulación de la Expresión Génica , Genotipo , Imagen por Resonancia Magnética , Masculino , Aprendizaje por Laberinto , Megalencefalia/fisiopatología , Memoria , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxitocina/sangre , Polimorfismo de Nucleótido Simple , Reflejo de Sobresalto , Factores de Riesgo , Conducta Social
4.
Molecules ; 24(2)2019 Jan 09.
Artículo en Inglés | MEDLINE | ID: mdl-30634526

RESUMEN

The medical staff is often powerless to treat patients affected by drug abuse or misuse and poisoning. In the case of envenomation, the treatment of choice remains horse sera administration that poses a wealth of other medical conditions and threats. Previously, we have demonstrated that DNA-based aptamers represent powerful neutralizing tools for lethal animal toxins of venomous origin. Herein, we further pursued our investigations in order to understand whether all toxin-interacting aptamers possessed equivalent potencies to neutralize αC-conotoxin PrXA in vitro and in vivo. We confirmed the high lethality in mice produced by αC-conotoxin PrXA regardless of the mode of injection and further characterized myoclonus produced by the toxin. We used high-throughput patch-clamp technology to assess the effect of αC-conotoxin PrXA on ACh-mediated responses in TE671 cells, responses that are carried by muscle-type nicotinic receptors. We show that 2 out of 4 aptamers reduce the affinity of the toxin for its receptor, most likely by interfering with the pharmacophore. In vivo, more complex responses on myoclonus and mice lethality are observed depending on the type of aptamer and mode of administration (concomitant or differed). Concomitant administration always works better than differed administration indicating the stability of the complex in vivo. The most remarkable conclusion is that an aptamer that has no or a limited efficacy in vitro may nevertheless be functional in vivo probably owing to an impact on the biodistribution or pharmacokinetics of the toxin in vivo. Overall, the results highlight that a blind selection of aptamers against toxins leads to efficient neutralizing compounds in vivo regardless of the mode of action. This opens the door to the use of aptamer mixtures as substitutes to horse sera for the neutralization of life-threatening animal venoms, an important WHO concern in tropical areas.


Asunto(s)
Aptámeros de Nucleótidos/administración & dosificación , Conotoxinas/toxicidad , Mioclonía/prevención & control , Animales , Aptámeros de Nucleótidos/farmacología , Línea Celular , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Mioclonía/mortalidad , Receptores Nicotínicos/metabolismo , Técnica SELEX de Producción de Aptámeros
5.
Mol Neurobiol ; 56(4): 2670-2684, 2019 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-30051354

RESUMEN

In the Duchenne muscular dystrophy (DMD) syndrome, mutations affecting expression of Dp71, the main dystrophin isoform of the multipromoter dmd gene in brain, have been associated with intellectual disability and neuropsychiatric disturbances. Patients' profile suggests alterations in prefrontal cortex-dependent executive processes, but the specific dysfunctions due to Dp71 deficiency are unclear. Dp71 is involved in brain ion homeostasis, and its deficiency is expected to increase neuronal excitability, which might compromise the integrity of neuronal networks undertaking high-order cognitive functions. Here, we used electrophysiological (patch clamp) and behavioral techniques in a transgenic mouse that display a selective loss of Dp71 and no muscular dystrophy, to identify changes in prefrontal cortex excitatory/inhibitory (E/I) balance and putative executive dysfunctions. We found prefrontal cortex E/I balance is shifted toward enhanced excitation in Dp71-null mice. This is associated with a selective alteration of AMPA receptor-mediated glutamatergic transmission and reduced synaptic plasticity, while inhibitory transmission is unaffected. Moreover, Dp71-null mice display deficits in cognitive processes that depend on prefrontal cortex integrity, such as cognitive flexibility and sensitivity of spatial working memory to proactive interference. Our data suggest that impaired cortical E/I balance and executive dysfunctions contribute to the intellectual and behavioral disturbances associated with Dp71 deficiency in DMD, in line with current neurobehavioral models considering these functions as key pathophysiological factors in various neurodevelopmental disorders. These new insights in DMD neurobiology also suggest new directions for therapeutic developments targeting excitatory neurotransmission, as well as for guidance of academic environment in severely affected DMD children.


Asunto(s)
Distrofina/deficiencia , Función Ejecutiva/fisiología , Inhibición Neural/fisiología , Corteza Prefrontal/fisiopatología , Estimulación Acústica , Animales , Distrofina/metabolismo , Glutamatos/metabolismo , Memoria a Corto Plazo , Ratones Endogámicos C57BL , Potenciales Postsinápticos Miniatura/fisiología , Plasticidad Neuronal , Neuronas/metabolismo , Células Piramidales/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica
6.
Toxins (Basel) ; 10(3)2018 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-29495549

RESUMEN

Prorocentrolides are members of the cyclic imine phycotoxins family. Their chemical structure includes a 26-membered carbo-macrocycle and a 28-membered macrocyclic lactone arranged around a hexahydroisoquinoline that incorporates the characteristic cyclic imine group. Six prorocentrolides are already known. However, their mode of action remains undetermined. The aim of the present work was to explore whether prorocentrolide A acts on nicotinic acetylcholine receptors (nAChRs), using competition-binding assays and electrophysiological techniques. Prorocentrolide-A displaced [125I]α-bungarotoxin binding to Torpedo membranes, expressing the muscle-type (α12ß1γδ) nAChR, and in HEK-293 cells, expressing the chimeric chick neuronal α7-5HT3 nAChR. Functional studies revealed that prorocentrolide-A had no agonist action on nAChRs, but inhibited ACh-induced currents in Xenopus oocytes that had incorporated the muscle-type α12ß1γδ nAChR to their membranes, or that expressed the human α7 nAChR, as revealed by voltage-clamp recordings. Molecular docking calculations showed the absence of the characteristic hydrogen bond between the iminium group of prorocentrolide-A and the backbone carbonyl group of Trp147 in the receptor, explaining its weaker affinity as compared to all other cyclic imine toxins. In conclusion, this is the first study to show that prorocentrolide-A acts on both muscle and neuronal nAChRs, but with higher affinity on the muscle-type nAChR.


Asunto(s)
Dinoflagelados , Piranos/farmacología , Receptores Nicotínicos/fisiología , Animales , Femenino , Células HEK293 , Humanos , Japón , Simulación del Acoplamiento Molecular , Músculo Esquelético , Neuronas , Oocitos/efectos de los fármacos , Oocitos/fisiología , Xenopus
7.
Neuropharmacology ; 133: 404-414, 2018 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-29474819

RESUMEN

The Chinese bird spider huwentoxin-IV (HwTx-IV) is well-known to be a highly potent blocker of NaV1.7 subtype of voltage-gated sodium (NaV) channels, a genetically validated analgesic target, and thus promising as a potential lead molecule for the development of novel pain therapeutics. In the present study, the interaction between HwTx-IV and NaV1.6 channel subtype was investigated using multiscale (from in vivo to individual cell) functional approaches. HwTx-IV was approximatively 2 times more efficient than tetrodotoxin (TTX) to inhibit the compound muscle action potential recorded from the mouse skeletal neuromuscular system in vivo, and 30 times more effective to inhibit nerve-evoked than directly-elicited muscle contractile force of isolated mouse hemidiaphragms. These results strongly suggest that the inhibition of nerve-evoked skeletal muscle functioning, produced by HwTx-IV, resulted from a toxin-induced preferential blockade of NaV1.6, compared to NaV1.4, channel subtype. This was confirmed by whole-cell automated patch-clamp experiments performed on human embryonic kidney (HEK)-293 cells overexpressing hNaV1.1-1.8 channel subtypes. HwTx-IV was also approximatively 850 times more efficient to inhibit TTX-sensitive than TTX-resistant sodium currents recorded from mouse dorsal root ganglia neurons. Finally, based on our data, we predict that blockade of the NaV1.6 channel subtype was involved in the in vivo toxicity of HwTx-IV, although this toxicity was more than 2 times lower than that of TTX. In conclusion, our results provide detailed information regarding the effects of HwTx-IV and allow a better understanding of the side-effect mechanisms involved in vivo and of channel subtype interactions resulting from the toxin activity.


Asunto(s)
Potenciales de Acción/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Unión Neuromuscular/efectos de los fármacos , Bloqueadores de los Canales de Sodio/farmacología , Venenos de Araña/farmacología , Animales , Células Cultivadas , Cricetulus , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Femenino , Ganglios Espinales/citología , Humanos , Técnicas In Vitro , Ratones , Contracción Muscular/efectos de los fármacos , Contracción Muscular/genética , Canal de Sodio Activado por Voltaje NAV1.6/genética , Unión Neuromuscular/fisiología , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Canales de Sodio/genética , Canales de Sodio/metabolismo , Estadísticas no Paramétricas , Tetrodotoxina/farmacología , Transfección
8.
Sci Rep ; 7(1): 7202, 2017 08 03.
Artículo en Inglés | MEDLINE | ID: mdl-28775316

RESUMEN

Medical means to save the life of human patients affected by drug abuse, envenomation or critical poisoning are currently limited. While the compounds at risks are most often well identified, particularly for bioterrorism, chemical intervention to counteract the toxic effects of the ingested/injected compound(s) is restricted to the use of antibodies. Herein, we illustrate that DNA aptamers, targeted to block the pharmacophore of a poisonous compound, represent a fast-acting and reliable method of neutralization in vivo that possesses efficient and long-lasting life-saving properties. For this proof of concept, we used one putative bioweapon, αC-conotoxin PrXA, a marine snail ultrafast-killing paralytic toxin, to identify peptide-binding DNA aptamers. We illustrate that they can efficiently neutralize the toxin-induced (i) displacement of [125I]-α-bungarotoxin binding onto nicotinic receptors, (ii) inhibition of diaphragm muscle contraction, and (iii) lethality in mice. Our results demonstrate the preclinical value of DNA aptamers as fast-acting, safe and cheap antidotes to lethal toxins at risk of misuse in bioterrorism and offer hope for an alternative method than donor sera to treat cases of envenomation.


Asunto(s)
Oligonucleótidos/administración & dosificación , Péptidos/antagonistas & inhibidores , Toxinas Biológicas/antagonistas & inhibidores , Animales , Aptámeros de Nucleótidos/administración & dosificación , Conotoxinas/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Femenino , Dosificación Letal Mediana , Masculino , Ratones , Pruebas de Neutralización , Péptidos/toxicidad , Toxinas Biológicas/toxicidad
9.
Toxicon ; 130: 1-10, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28238803

RESUMEN

Animal toxins are peptides that often bind with remarkable affinity and selectivity to membrane receptors such as nicotinic acetylcholine receptors (nAChRs). The latter are, for example, targeted by α-conotoxins, a family of peptide toxins produced by venomous cone snails. nAChRs are implicated in numerous physiological processes explaining why the design of new pharmacological tools and the discovery of potential innovative drugs targeting these receptor channels appear so important. This work describes a methodology developed to discover new ligands of nAChRs from complex mixtures of peptides. The methodology was set up by the incubation of Torpedo marmorata electrocyte membranes rich in nAChRs with BSA tryptic digests (>100 peptides) doped by small amounts of known nAChRs ligands (α-conotoxins). Peptides that bind to the receptors were purified and analyzed by MALDI-TOF/TOF mass spectrometry which revealed an enrichment of α-conotoxins in membrane-containing fractions. This result exhibits the binding of α-conotoxins to nAChRs. Negative controls were performed to demonstrate the specificity of the binding. The usefulness and the power of the methodology were also investigated for a discovery issue. The workflow was then applied to the screening of Conus ermineus crude venom, aiming at characterizing new nAChRs ligands from this venom, which has not been extensively investigated to date. The methodology validated our experiments by allowing us to bind two α-conotoxins (α-EI and α-EIIA) which have already been described as nAChRs ligands. Moreover, a new conotoxin, never described to date, was also captured, identified and sequenced from this venom. Classical pharmacology tests by radioligand binding using a synthetic homologue of the toxin confirm the activity of the new peptide, called α-EIIB. The Ki value of this peptide for Torpedo nicotinic receptors was measured at 2.2 ± 0.7 nM.


Asunto(s)
Conotoxinas/aislamiento & purificación , Receptores Nicotínicos/química , Animales , Conotoxinas/química , Caracol Conus/química , Ligandos , Espectrometría de Masas/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos
10.
PLoS One ; 11(3): e0151233, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27003418

RESUMEN

N-methyl-D-aspartate receptors (NMDARs) play a central role in synaptic plasticity. Their activation requires the binding of both glutamate and d-serine or glycine as co-agonist. The prevalence of either co-agonist on NMDA-receptor function differs between brain regions and remains undetermined in the visual cortex (VC) at the critical period of postnatal development. Here, we therefore investigated the regulatory role that d-serine and/or glycine may exert on NMDARs function and on synaptic plasticity in the rat VC layer 5 pyramidal neurons of young rats. Using selective enzymatic depletion of d-serine or glycine, we demonstrate that d-serine and not glycine is the endogenous co-agonist of synaptic NMDARs required for the induction and expression of Long Term Potentiation (LTP) at both excitatory and inhibitory synapses. Glycine on the other hand is not involved in synaptic efficacy per se but regulates excitatory and inhibitory neurotransmission by activating strychnine-sensitive glycine receptors, then producing a shunting inhibition that controls neuronal gain and results in a depression of synaptic inputs at the somatic level after dendritic integration. In conclusion, we describe for the first time that in the VC both D-serine and glycine differentially regulate somatic depolarization through the activation of distinct synaptic and extrasynaptic receptors.


Asunto(s)
Glicina/metabolismo , Serina/metabolismo , Transmisión Sináptica/fisiología , Corteza Visual/metabolismo , Animales , Ácido Glutámico/metabolismo , Potenciación a Largo Plazo/fisiología , Masculino , Plasticidad Neuronal/fisiología , Células Piramidales/metabolismo , Células Piramidales/fisiología , Ratas , Ratas Wistar , Receptores de Glicina/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismo
11.
Toxicol Sci ; 147(1): 156-67, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26063663

RESUMEN

Spirolides are a large family of lipophilic marine toxins produced by dinoflagellates that have been detected in contaminated shellfish. Among them, 13,19-didesmethyl and 13-desmethyl spirolide C phycotoxins are widely distributed and their mode of action needs to be clearly defined. In order to further characterize the pharmacological profiles of these phycotoxins on various nicotinic acetylcholine receptor (nAChR) subtypes and to examine whether they act on muscarinic receptors (mAChRs), functional electrophysiological studies and competition binding experiments have been performed. While 13-desmethyl spirolide C interacted efficiently with sub-nanomolar affinities and low selectivity with muscular and neuronal nAChRs, 13,19-didesmethyl spirolide C was more selective of muscular and homopentameric α7 receptors and recognized only weakly neuronal heteropentameric receptors, especially the α4ß2 subtype. Thus, the presence of an additional methyl group on the tetrahydropyran ring significantly modified the pharmacological profile of 13-desmethyl spirolide C by notably increasing its affinity on certain neuronal nAChRs. Structural explanations of this selectivity difference are proposed, based on molecular docking experiments modeling different spirolide-receptor complexes. In addition, the 2 spirolides interacted only with low micromolar affinities with the 5 mAChRs, highlighting that the toxicity of the spirolide C analogs is mainly due to their high inhibition potency on various peripheral and central nAChRs and not to their low ability to interact with mAChR subtypes.


Asunto(s)
Toxinas Marinas/toxicidad , Síndromes de Neurotoxicidad/metabolismo , Receptores Muscarínicos/efectos de los fármacos , Receptores Nicotínicos/efectos de los fármacos , Compuestos de Espiro/toxicidad , Animales , Células CHO , Cricetinae , Cricetulus , Humanos , Contracción Isométrica/efectos de los fármacos , Ratones , Simulación del Acoplamiento Molecular , Células Musculares/efectos de los fármacos , Unión Neuromuscular/efectos de los fármacos , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Técnicas de Placa-Clamp , Compuestos de Espiro/química , Relación Estructura-Actividad , Xenopus , Receptor Nicotínico de Acetilcolina alfa 7/efectos de los fármacos
12.
J Cell Biol ; 203(2): 283-98, 2013 Oct 28.
Artículo en Inglés | MEDLINE | ID: mdl-24165939

RESUMEN

Several studies have suggested that the V0 domain of the vacuolar-type H(+)-adenosine triphosphatase (V-ATPase) is directly implicated in secretory vesicle exocytosis through a role in membrane fusion. We report in this paper that there was a rapid decrease in neurotransmitter release after acute photoinactivation of the V0 a1-I subunit in neuronal pairs. Likewise, inactivation of the V0 a1-I subunit in chromaffin cells resulted in a decreased frequency and prolonged kinetics of amperometric spikes induced by depolarization, with shortening of the fusion pore open time. Dissipation of the granular pH gradient was associated with an inhibition of exocytosis and correlated with the V1-V0 association status in secretory granules. We thus conclude that V0 serves as a sensor of intragranular pH that controls exocytosis and synaptic transmission via the reversible dissociation of V1 at acidic pH. Hence, the V-ATPase membrane domain would allow the exocytotic machinery to discriminate fully loaded and acidified vesicles from vesicles undergoing neurotransmitter reloading.


Asunto(s)
Exocitosis , Neuronas/enzimología , Vesículas Secretoras/enzimología , Transmisión Sináptica , Vesículas Sinápticas/enzimología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Catecolaminas/metabolismo , Bovinos , Células Cromafines/enzimología , Células Cromafines/metabolismo , Exocitosis/efectos de los fármacos , Exocitosis/efectos de la radiación , Concentración de Iones de Hidrógeno , Cinética , Luz , Fusión de Membrana , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/efectos de la radiación , Células PC12 , Estructura Terciaria de Proteína , Interferencia de ARN , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/metabolismo , Vesículas Secretoras/efectos de la radiación , Potenciales Sinápticos , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/efectos de la radiación , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/efectos de la radiación , Transfección , ATPasas de Translocación de Protón Vacuolares/genética
13.
J Biol Chem ; 287(36): 30084-96, 2012 Aug 31.
Artículo en Inglés | MEDLINE | ID: mdl-22815483

RESUMEN

p21-activated kinase 1 (PAK1) and PAK3 belong to group I of the PAK family and control cell movement and division. They also regulate dendritic spine formation and maturation in the brain, and play a role in synaptic transmission and synaptic plasticity. PAK3, in particular, is known for its implication in X-linked intellectual disability. The pak3 gene is expressed in neurons as a GTPase-regulated PAK3a protein and also as three splice variants which display constitutive kinase activity. PAK1 regulation is based on its homodimerization, forming an inactive complex. Here, we analyze the PAK3 capacity to dimerize and show that although PAK3a is able to homodimerize, it is more likely to form heterodimeric complexes with PAK1. We further show that two intellectual disability mutations impair dimerization with PAK1. The b and c inserts present in the regulatory domain of PAK3 splice variants decrease the dimerization but retain the capacity to form heterodimers with PAK1. PAK1 and PAK3 are co-expressed in neurons, are colocalized within dendritic spines, co-purify with post-synaptic densities, and co-immunoprecipitate in brain lysates. Using kinase assays, we demonstrate that PAK1 inhibits the activity of PAK3a but not of the splice variant PAK3b in a trans-regulatory manner. Altogether, these results show that PAK3 and PAK1 signaling may be coordinated by heterodimerization.


Asunto(s)
Espinas Dendríticas/enzimología , Proteínas del Tejido Nervioso/metabolismo , Densidad Postsináptica/enzimología , Multimerización de Proteína , Quinasas p21 Activadas/metabolismo , Empalme Alternativo/genética , Animales , Activación Enzimática/genética , Regulación Enzimológica de la Expresión Génica/genética , Enfermedades Genéticas Ligadas al Cromosoma X/enzimología , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Células HeLa , Humanos , Discapacidad Intelectual/enzimología , Discapacidad Intelectual/genética , Ratones , Mutación , Proteínas del Tejido Nervioso/genética , Estructura Terciaria de Proteína , Transducción de Señal/genética , Quinasas p21 Activadas/genética
14.
J Biol Chem ; 286(46): 40044-59, 2011 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-21949127

RESUMEN

Mutations in the p21-activated kinase 3 gene (pak3) are responsible for nonsyndromic forms of mental retardation. Expression of mutated PAK3 proteins in hippocampal neurons induces abnormal dendritic spine morphology and long term potentiation anomalies, whereas pak3 gene invalidation leads to cognitive impairments. How PAK3 regulates synaptic plasticity is still largely unknown. To better understand how PAK3 affects neuronal synaptic plasticity, we focused on its interaction with the Nck adaptors that play a crucial role in PAK signaling. We report here that PAK3 interacts preferentially with Nck2/Grb4 in brain extracts and in transfected cells. This interaction is independent of PAK3 kinase activity. Selective uncoupling of the Nck2 interactions in acute cortical slices using an interfering peptide leads to a rapid increase in evoked transmission to pyramidal neurons. The P12A mutation in the PAK3 protein strongly decreases the interaction with Nck2 but only slightly with Nck1. In transfected hippocampal cultures, expression of the P12A-mutated protein has no effect on spine morphogenesis or synaptic density. The PAK3-P12A mutant does not affect synaptic transmission, whereas the expression of the wild-type PAK3 protein decreases the amplitude of spontaneous miniature excitatory currents. Altogether, these data show that PAK3 down-regulates synaptic transmission through its interaction with Nck2.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Oncogénicas/metabolismo , Transmisión Sináptica/fisiología , Quinasas p21 Activadas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Sustitución de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Mutación Missense , Proteínas Oncogénicas/genética , Quinasas p21 Activadas/genética
15.
Cereb Cortex ; 20(2): 456-67, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19520765

RESUMEN

Fundamental brain functions depend on a balance between excitation (E) and inhibition (I) that is highly adjusted to a 20-80% set point in layer 5 pyramidal neurons (L5PNs) of rat visual cortex. Dysregulations of both the E-I balance and the serotonergic system in neocortical networks lead to serious neuronal diseases including depression, schizophrenia, and epilepsy. However, no link between the activation of neuronal 5-hydroxytryptamine receptors (5-HTRs) and the cortical E-I balance has yet been reported. Here we used a combination of patch-clamp recordings of composite stimulus-locked responses in L5PN following local electrical stimulations in either layer 2/3 or 6, simultaneous measurement of excitatory and inhibitory conductance dynamics, together with selective pharmacological targeting and single-cell reverse transcriptase-polymerase chain reaction. We show that cortical serotonin shifts the E-I balance in favor of more E and we reveal fine and differential modulations of the E-I balance between 5-HTR subtypes, in relation to whether layer 2/3 or 6 was stimulated and in concordance with the specific expression pattern of these subtypes in pyramidal cells and deep interneurons. This first evidence for the functional segregation of 5-HTR subtypes sheds new light on their coherent functioning in polysynaptic sensory circuits.


Asunto(s)
Axones/metabolismo , Potenciales Postsinápticos Excitadores/fisiología , Potenciales Postsinápticos Inhibidores/fisiología , Red Nerviosa/metabolismo , Serotonina/metabolismo , Corteza Visual/metabolismo , Animales , Axones/ultraestructura , Estimulación Eléctrica , Electrofisiología , Interneuronas/citología , Interneuronas/metabolismo , Red Nerviosa/citología , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Células Piramidales/citología , Células Piramidales/metabolismo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores de Serotonina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transmisión Sináptica/fisiología , Corteza Visual/citología , Percepción Visual/fisiología
16.
Cereb Cortex ; 19(10): 2411-27, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19176636

RESUMEN

This study aims to clarify how endogenous release of cortical acetylcholine (ACh) modulates the balance between excitation and inhibition evoked in visual cortex. We show that electrical stimulation in layer 1 produced a significant release of ACh measured intracortically by chemoluminescence and evoked a composite synaptic response recorded intracellularly in layer 5 pyramidal neurons of rat visual cortex. The pharmacological specificity of the ACh neuromodulation was determined from the continuous whole-cell voltage clamp measurement of stimulation-locked changes of the input conductance during the application of cholinergic agonists and antagonists. Blockade of glutamatergic and gamma-aminobutyric acid (GABAergic) receptors suppressed the evoked response, indicating that stimulation-induced release of ACh does not directly activate a cholinergic synaptic conductance in recorded neurons. Comparison of cytisine and mecamylamine effects on nicotinic receptors showed that excitation is enhanced by endogenous evoked release of ACh through the presynaptic activation of alpha(*)beta4 receptors located on glutamatergic fibers. DHbetaE, the selective alpha4beta2 nicotinic receptor antagonist, induced a depression of inhibition. Endogenous ACh could also enhance inhibition by acting directly on GABAergic interneurons, presynaptic to the recorded cell. We conclude that endogenous-released ACh amplifies the dominance of the inhibitory drive and thus decreases the excitability and sensory responsiveness of layer 5 pyramidal neurons.


Asunto(s)
Acetilcolina/metabolismo , Corteza Cerebral/metabolismo , Neuronas/fisiología , Receptores Muscarínicos/metabolismo , Receptores Nicotínicos/metabolismo , Corteza Visual/metabolismo , Acetilcolina/farmacología , Aconitina/análogos & derivados , Aconitina/farmacología , Animales , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/fisiología , Estimulación Eléctrica , Electrofisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Lidocaína/análogos & derivados , Lidocaína/farmacología , Mediciones Luminiscentes , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Conducción Nerviosa/efectos de los fármacos , Conducción Nerviosa/fisiología , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/metabolismo , Vías Nerviosas/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neurosecreción/fisiología , Antagonistas Nicotínicos/farmacología , Ratas , Ratas Wistar , Receptores Muscarínicos/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Corteza Visual/efectos de los fármacos , Corteza Visual/fisiología
17.
J Neurochem ; 107(4): 952-63, 2008 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-18990115

RESUMEN

Gymnodimines (GYMs) are phycotoxins exhibiting unusual structural features including a spirocyclic imine ring system and a trisubstituted tetrahydrofuran embedded within a 16-membered macrocycle. The toxic potential and the mechanism of action of GYM-A, highly purified from contaminated clams, have been assessed. GYM-A in isolated mouse phrenic hemidiaphragm preparations produced a concentration- and time-dependent block of twitch responses evoked by nerve stimulation, without affecting directly elicited muscle twitches, suggesting that it may block the muscle nicotinic acetylcholine (ACh) receptor (nAChR). This was confirmed by the blockade of miniature endplate potentials and the recording of subthreshold endplate potentials in GYM-A paralyzed frog and mouse isolated neuromuscular preparations. Patch-clamp recordings in Xenopus skeletal myocytes revealed that nicotinic currents evoked by constant iontophoretical ACh pulses were blocked by GYM-A in a reversible manner. GYM-A also blocked, in a voltage-independent manner, homomeric human alpha7 nAChR expressed in Xenopus oocytes. Competition-binding assays confirmed that GYM-A is a powerful ligand interacting with muscle-type nAChR, heteropentameric alpha3beta2, alpha4beta2, and chimeric alpha7-5HT(3) neuronal nAChRs. Our data show for the first time that GYM-A broadly targets nAChRs with high affinity explaining the basis of its neurotoxicity, and also pave the way for designing specific tests for accurate GYM-A detection in shellfish samples.


Asunto(s)
Compuestos Heterocíclicos con 3 Anillos/farmacología , Hidrocarburos Cíclicos/farmacología , Iminas/farmacología , Células Musculares/efectos de los fármacos , Unión Neuromuscular/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacología , Animales , Conducta Animal/efectos de los fármacos , Bivalvos/química , Bungarotoxinas/metabolismo , Células Cultivadas , Cromatografía Líquida de Alta Presión/métodos , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Estimulación Eléctrica/métodos , Expresión Génica/efectos de los fármacos , Compuestos Heterocíclicos con 3 Anillos/análisis , Compuestos Heterocíclicos con 3 Anillos/química , Compuestos Heterocíclicos con 3 Anillos/clasificación , Humanos , Hidrocarburos Cíclicos/análisis , Hidrocarburos Cíclicos/química , Hidrocarburos Cíclicos/clasificación , Iminas/análisis , Iminas/química , Iminas/clasificación , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Células Musculares/metabolismo , Unión Neuromuscular/fisiología , Unión Neuromuscular/efectos de la radiación , Oocitos , Técnicas de Placa-Clamp , Unión Proteica/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa 7
18.
J Soc Biol ; 202(2): 143-60, 2008.
Artículo en Francés | MEDLINE | ID: mdl-18547512

RESUMEN

Synaptic plasticity is the cellular mechanism underlying the phenomena of learning and memory. Much of the research on synaptic plasticity is based on the postulate of Hebb (1949) who proposed that, when a neuron repeatedly takes part in the activation of another neuron, the efficacy of the connections between these neurons is increased. Plasticity has been extensively studied, and often demonstrated through the processes of LTP (Long Term Potentiation) and LTD (Long Term Depression), which represent an increase and a decrease of the efficacy of long-term synaptic transmission. This review summarizes current knowledge concerning the cellular mechanisms of LTP and LTD, whether at the level of excitatory synapses, which have been the most studied, or at the level of inhibitory synapses. However, if we consider neuronal networks rather than the individual synapses, the consequences of synaptic plasticity need to be considered on a large scale to determine if the activity of networks are changed or not. Homeostatic plasticity takes into account the mechanisms which control the efficacy of synaptic transmission for all the synaptic inputs of a neuron. Consequently, this new concept deals with the coordinated activity of excitatory and inhibitory networks afferent to a neuron which maintain a controlled level of excitability during the acquisition of new information related to the potentiation or to the depression of synaptic efficacy. We propose that the protocols of stimulation used to induce plasticity at the synaptic level set up a "homeostatic potentiation" or a "homeostatic depression" of excitation and inhibition at the level of the neuronal networks. The coordination between excitatory and inhibitory circuits allows the neuronal networks to preserve a level of stable activity, thus avoiding episodes of hyper- or hypo-activity during the learning and memory phases.


Asunto(s)
Homeostasis/fisiología , Modelos Biológicos , Plasticidad Neuronal/fisiología , Animales , Humanos , Aprendizaje/fisiología , Potenciación a Largo Plazo/fisiología , Depresión Sináptica a Largo Plazo/fisiología , Memoria/fisiología , Red Nerviosa/fisiología , Sinapsis/fisiología
19.
Eur J Neurosci ; 26(2): 289-301, 2007 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-17650107

RESUMEN

In the cortex, N-methyl-D-aspartate receptors (NMDARs) play a critical role in the control of synaptic plasticity processes. We have previously shown in rat visual cortex that the application of a high-frequency stimulation (HFS) protocol used to induce long-term potentiation in layer 2/3 leads to a parallel potentiation of excitatory and inhibitory inputs received by cortical layer 5 pyramidal neurones without changing the excitation/inhibition balance of the pyramidal neurone, indicating a homeostatic control of this parameter. We show here that the blockade of NMDARs of the neuronal network prevents the potentiation of excitatory and inhibitory inputs, and this result leaves open to question the role of the NMDAR isoform involved in the induction of long-term potentiation, which is actually being strongly debated. In postnatal day (P)18-23 rat cortical slices, the blockade of synaptic NR2B-containing NMDARs prevents the induction of the potentiation induced by the HFS protocol, whereas the blockade of NR2A-containing NMDARs reduced the potentiation itself. In P29-P32 cortical slices, the specific activation of NR2A-containing receptors fully ensures the potentiation of excitatory and inhibitory inputs. These results constitute the first report of a functional shift in subunit composition of NMDARs during the critical period (P12-P36), which explains the relative contribution of both NR2B- and NR2A-containing NMDARs in synaptic plasticity processes. These effects of the HFS protocol are mediated by the activation of synaptic NMDARs but our results also indicate that the homeostatic control of the excitation/inhibition balance is independent of NMDAR activation and is due to specialized recurrent interactions between excitatory and inhibitory networks.


Asunto(s)
Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/fisiología , Células Piramidales/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , 2-Amino-5-fosfonovalerato/farmacología , Animales , Corteza Cerebral/citología , Estimulación Eléctrica , Electrofisiología , Plasticidad Neuronal/fisiología , Técnicas de Placa-Clamp , Células Piramidales/efectos de los fármacos , Ratas , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Sinapsis/fisiología , Corteza Visual/citología , Corteza Visual/crecimiento & desarrollo , Corteza Visual/fisiología
20.
J Biol Chem ; 281(25): 17164-17172, 2006 Jun 23.
Artículo en Inglés | MEDLINE | ID: mdl-16621796

RESUMEN

Vacuolar proton ATPase accumulates protons inside various intracellular organelles such as synaptic vesicles; its membrane domain V0 could also be involved in membrane fusion. These different functions could require vacuolar proton ATPases possessing different V0 subunit a isoforms. In vertebrates, four genes encode isoforms a1-a4, and a1 variants are also generated by alternative splicing. We identified a novel a1 splice variant a1-IV and showed that the two a1 variants containing exon C are specifically expressed in neurons. Single neurons coexpress a2, a1-I, and a1-IV, and these subunit a isoforms are targeted to different membrane compartments. Recombinant a2 was accumulated in the trans-Golgi network, and a1-I was concentrated in axonal varicosities, whereas a1-IV was sorted to both distal dendrites and axons. Our results indicate that alternative splicing of exon N controls differential sorting of a1 variants to nerve terminals or distal dendrites, whereas exon C regulates their neuronal expression.


Asunto(s)
Empalme Alternativo , Neuronas/metabolismo , ATPasas de Translocación de Protón Vacuolares/biosíntesis , Animales , Exones , Regulación de la Expresión Génica , Hipocampo/citología , Hipocampo/embriología , Microscopía Fluorescente , Técnicas de Placa-Clamp , Isoformas de Proteínas , Protones , Ratas , Distribución Tisular , Transfección , ATPasas de Translocación de Protón Vacuolares/genética
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