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1.
J Neurophysiol ; 118(1): 532-543, 2017 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-28356471

RESUMEN

N-methyl-d-aspartate receptors (NMDARs) govern synaptic plasticity, development, and neuronal response to insult. Prolonged activation of NMDARs such as during an insult may activate secondary currents or modulate Mg2+ sensitivity, but the conditions under which these occur are not fully defined. We reexamined the effect of prolonged NMDAR activation in juvenile mouse hippocampal slices. NMDA (10 µM) elicited current with the expected negative-slope conductance in the presence of 1.2 mM Mg2+ However, several minutes of continued NMDA exposure elicited additional inward current at -70 mV. A higher concentration of NMDA (100 µM) elicited the current more rapidly. The additional current was not dependent on Ca2+, network activity, or metabotropic NMDAR function and did not persist on agonist removal. Voltage ramps revealed no alteration of either reversal potential or NMDA-elicited conductance between -30 mV and +50 mV. The result was a more linear NMDA current-voltage relationship. The current linearization was also induced in interneurons and in mature dentate granule neurons but not immature dentate granule cells, dissociated cultured hippocampal neurons, or nucleated patches excised from CA1 pyramidal neurons. Comparative simulations of NMDA application to a CA1 pyramidal neuron and to a cultured neuron revealed that linearization can be explained by space-clamp errors arising from gradual recruitment of distal dendritic NMDARs. We conclude that persistent secondary currents do not strongly contribute to NMDAR responses in juvenile mouse hippocampus and careful discernment is needed to exclude contributions of clamp artifacts to apparent secondary currents.NEW & NOTEWORTHY We report that upon sustained activation of NMDARs in juvenile mouse hippocampal neurons there is apparent loss of Mg2+ block at negative membrane potentials. However, the phenomenon is explained by loss of dendritic voltage clamp, leading to a linear current-voltage relationship. Our results give a specific example of how spatial voltage errors in voltage-clamp recordings can readily be misinterpreted as biological modulation.


Asunto(s)
Magnesio/metabolismo , Potenciales de la Membrana/fisiología , N-Metilaspartato/metabolismo , Neuronas/metabolismo , Técnicas de Placa-Clamp , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Artefactos , Calcio/metabolismo , Cationes Bivalentes/metabolismo , Células Cultivadas , Simulación por Computador , Agonistas de Aminoácidos Excitadores/farmacología , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones Endogámicos C57BL , Modelos Neurológicos , N-Metilaspartato/farmacología , Neuronas/efectos de los fármacos , Ratas , Receptores de N-Metil-D-Aspartato/agonistas , Técnicas de Cultivo de Tejidos
2.
J Biol Chem ; 289(9): 6249-57, 2014 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-24385443

RESUMEN

Reversible attachment and removal of palmitate or other long-chain fatty acids on proteins has been hypothesized, like phosphorylation, to control diverse biological processes. Indeed, palmitate turnover regulates Ras trafficking and signaling. Beyond this example, however, the functions of palmitate turnover on specific proteins remain poorly understood. Here, we show that a mechanism regulating G protein-coupled receptor signaling in neuronal cells requires palmitate turnover. We used hexadecyl fluorophosphonate or palmostatin B to inhibit enzymes in the serine hydrolase family that depalmitoylate proteins, and we studied R7 regulator of G protein signaling (RGS)-binding protein (R7BP), a palmitoylated allosteric modulator of R7 RGS proteins that accelerate deactivation of Gi/o class G proteins. Depalmitoylation inhibition caused R7BP to redistribute from the plasma membrane to endomembrane compartments, dissociated R7BP-bound R7 RGS complexes from Gi/o-gated G protein-regulated inwardly rectifying K(+) (GIRK) channels and delayed GIRK channel closure. In contrast, targeting R7BP to the plasma membrane with a polybasic domain and an irreversibly attached lipid instead of palmitate rendered GIRK channel closure insensitive to depalmitoylation inhibitors. Palmitate turnover therefore is required for localizing R7BP to the plasma membrane and facilitating Gi/o deactivation by R7 RGS proteins on GIRK channels. Our findings broaden the scope of biological processes regulated by palmitate turnover on specific target proteins. Inhibiting R7BP depalmitoylation may provide a means of enhancing GIRK activity in neurological disorders.


Asunto(s)
Proteínas Portadoras/metabolismo , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Lipoilación/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Proteínas RGS/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Proteínas Portadoras/genética , Línea Celular Tumoral , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lipoilación/efectos de los fármacos , Ratones , Propiolactona/análogos & derivados , Propiolactona/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas RGS/genética , Receptores Acoplados a Proteínas G/genética
3.
Proc Natl Acad Sci U S A ; 109(49): 19977-82, 2012 Dec 04.
Artículo en Inglés | MEDLINE | ID: mdl-23169654

RESUMEN

G-protein-activated inward-rectifying K(+) (GIRK) channels hyperpolarize neurons to inhibit synaptic transmission throughout the nervous system. By accelerating G-protein deactivation kinetics, the regulator of G-protein signaling (RGS) protein family modulates the timing of GIRK activity. Despite many investigations, whether RGS proteins modulate GIRK activity in neurons by mechanisms involving kinetic coupling, collision coupling, or macromolecular complex formation has remained unknown. Here we show that GIRK modulation occurs by channel assembly with R7-RGS/Gß5 complexes under allosteric control of R7 RGS-binding protein (R7BP). Elimination of R7BP occludes the Gß5 subunit that interacts with GIRK channels. R7BP-bound R7-RGS/Gß5 complexes and Gßγ dimers interact noncompetitively with the intracellular domain of GIRK channels to facilitate rapid activation and deactivation of GIRK currents. By disrupting this allosterically regulated assembly mechanism, R7BP ablation augments GIRK activity. This enhanced GIRK activity increases the drug effects of agonists acting at G-protein-coupled receptors that signal via GIRK channels, as indicated by greater antinociceptive effects of GABA(B) or µ-opioid receptor agonists. These findings show that GIRK current modulation in vivo requires channel assembly with allosterically regulated RGS protein complexes, which provide a target for modulating GIRK activity in neurological disorders in which these channels have crucial roles, including pain, epilepsy, Parkinson's disease and Down syndrome.


Asunto(s)
Regulación Alostérica/fisiología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Proteínas RGS/metabolismo , Análisis de Varianza , Animales , Transferencia de Energía por Resonancia de Bioluminiscencia , Cartilla de ADN/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Células HEK293 , Hipocampo/citología , Hipocampo/fisiología , Humanos , Immunoblotting , Inmunoprecipitación , Ratones , Ratones Noqueados , Microscopía Fluorescente , Mutagénesis , Proteínas RGS/genética
4.
Bioorg Med Chem ; 22(3): 1040-8, 2014 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-24411196

RESUMEN

A three-step synthetic pathway has been employed to synthesize a small library of 2-(4-arylpiperidin-1-yl)-1-(1H-indol-3-yl)ethanone and 2-(4-arylpiperidin-1-yl)-1-(1H-indol-3-yl)ethane-1,2-dione derivatives that have been screened in [(3)H]ifenprodil competition binding assay. Some compounds exhibited significant binding affinity at nanomolar concentration, the most active being ligand 35 (IC50=5.5nM). Docking experiments suggested the main interactions between 35 and GluN2B-containing NMDA receptors. Notably, the compound 35 reduced NMDA-mediated excitatory post-synaptic currents recorded in mouse hippocampal slices indicating antagonistic effects (50nM). Moreover, the compound 35 has shown antioxidant effects in a preliminary screening, thus suggesting that it might be considered prototype for future drug development of novel 'dual target' neuroprotective agents.


Asunto(s)
Anticonvulsivantes/farmacología , Indoles/química , Receptores de N-Metil-D-Aspartato/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Anticonvulsivantes/química , Antioxidantes/química , Antioxidantes/farmacología , Unión Competitiva , Técnicas de Química Sintética , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Ligandos , Ratones , Ratones Endogámicos DBA , Ratones Endogámicos , Simulación del Acoplamiento Molecular , Estructura Molecular , Piperidinas/metabolismo , Bibliotecas de Moléculas Pequeñas/síntesis química
5.
Mol Pharmacol ; 83(2): 354-66, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23144238

RESUMEN

NMDA receptor (NMDAR) antagonists are dissociative anesthetics, drugs of abuse, and are of therapeutic interest in neurodegeneration and neuropsychiatric disease. Many well-known NMDAR antagonists are positively charged, voltage-dependent channel blockers. We recently showed that the hydrophobic anion dipicrylamine (DPA) negatively regulates GABA(A) receptor function by a mechanism indistinguishable from that of sulfated neurosteroids. Because sulfated neurosteroids also modulate NMDARs, here we examined the effects of DPA on NMDAR function. In rat hippocampal neurons DPA inhibited currents gated by 300 µM NMDA with an IC(50) of 2.3 µM. Neither onset nor offset of antagonism exhibited dependence on channel activation but exhibited a noncompetitive profile. DPA antagonism was independent of NMDAR subunit composition and was similar at extrasynaptic and total receptor populations. Surprisingly, similar to cationic channel blockers but unlike sulfated neurosteroids, DPA antagonism was voltage dependent. Onset and offset of DPA antagonism were nearly 10-fold faster than DPA-induced increases in membrane capacitance, suggesting that membrane interactions do not directly explain antagonism. Furthermore, voltage dependence did not derive from association of DPA with a site on NMDARs directly accessible to the outer membrane leaflet, assessed by DPA translocation experiments. Consistent with the expected lack of channel block, DPA antagonism did not interact with permeant ions. Therefore, we speculate that voltage dependence may arise from interactions of DPA with the inherent voltage dependence of channel gating. Overall, we conclude that DPA noncompetitively inhibits NMDA-induced current by a novel voltage-dependent mechanism and represents a new class of anionic NMDAR antagonists.


Asunto(s)
Picratos/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Animales , Aniones/farmacología , Línea Celular , Femenino , Células HEK293 , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , N-Metilaspartato/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neurotransmisores/farmacología , Oocitos/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Xenopus laevis/metabolismo
6.
Eur J Pharmacol ; 938: 175389, 2023 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-36435235

RESUMEN

Microglia represent the resident immune system in the brain. They mediate neuroinflammatory processes and have been described as important regulators of homeostasis in the central nervous system (CNS). Among several players and mechanisms contributing to microglial function in inflammation, ATP and glutamate have been shown to be involved in microgliosis. In this study, we focused on receptor subtypes that respond to these neurotransmitters, purinergic ionotropic P2X7 receptor and metabotropic glutamate mGlu5 receptor. We found that both receptors are functionally expressed in a murine microglia cell line, BV2 cells, and we performed patch-clamp experiments to measure purinergic ionotropic P2X7 receptor ion flux in control condition and after metabotropic glutamate mGlu5 receptor activation. The selective purinergic ionotropic P2X7 receptor agonist, 2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate (BzATP, 100 µM), elicited a robust current that was prevented by the selective purinergic ionotropic P2X7 receptor antagonist A438079 (10 µM). When BV2 cells were acutely stimulated with the selective metabotropic glutamate mGlu5 agonist, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG, 200 µM), purinergic ionotropic P2X7 receptor current was increased. This positive modulation was prevented by the selective metabotropic glutamate mGlu5 receptor antagonist 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP, 1 µM). Moreover, nitric oxide synthesis elicited by purinergic ionotropic P2X7 receptor activation was enhanced by metabotropic glutamate mGlu5 receptor co-stimulation. Taken together, our results suggest an important crosstalk between ATP and glutamate in inflammation. Pro-inflammatory effects mediated by purinergic ionotropic P2X7 receptor might be exacerbated by simultaneous exposure of microglia to ATP and glutamate, suggesting new pharmacological targets to modulate neuroinflammation.


Asunto(s)
Microglía , Receptor del Glutamato Metabotropico 5 , Receptores Purinérgicos P2X7 , Animales , Ratones , Adenosina Trifosfato/farmacología , Células Cultivadas , Agonistas de Aminoácidos Excitadores , Ácido Glutámico/metabolismo , Inflamación/metabolismo , Receptor del Glutamato Metabotropico 5/metabolismo , Receptores Purinérgicos P2X7/metabolismo
7.
J Neurophysiol ; 107(9): 2532-40, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22279196

RESUMEN

Past research has shown that calcium influx through NMDA receptors (NMDARs) depresses GABA(A) currents. We examined upstream triggers of this suppression, including involvement of target synaptic GABA(A) receptors and the NMDARs triggering suppression. In hippocampal neurons, conditioning with 20 µM NMDA for 20 s caused 50% suppression of GABA responses. The suppression was delayed by ≈ 60 s following NMDA application and persisted for at least 5 min following conditioning. Pharmacology experiments suggested a shift in both the sensitivity to GABA and a loss of functional receptors. NMDA conditioning strongly suppressed inhibitory postsynaptic currents and speeded decay kinetics. Synaptic NMDAR conditioning was necessary to suppress GABA current in pyramidal neurons; extrasynaptic NMDAR activation did not suppress, even when matched to synaptic activation. We found no evidence that specific synaptic NMDAR subunits mediate depression of GABA responses. Although physical colocalization of glutamate and GABA(A) receptors is mostly likely in extrasynaptic regions, our evidence suggests that NMDAR-induced suppression of GABA responsiveness prominently affects precise, moment-to-moment signaling from synaptic receptors to synaptic receptors.


Asunto(s)
N-Metilaspartato/farmacología , Inhibición Neural/fisiología , Receptor Cross-Talk/fisiología , Receptores de GABA-A/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Potenciales Sinápticos/fisiología , Animales , Animales Recién Nacidos , Células Cultivadas , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Inhibición Neural/efectos de los fármacos , Técnicas de Cultivo de Órganos , Ratas , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de Neurotransmisores/agonistas , Receptores de Neurotransmisores/fisiología , Potenciales Sinápticos/efectos de los fármacos
8.
J Neurosci ; 30(8): 2871-9, 2010 Feb 24.
Artículo en Inglés | MEDLINE | ID: mdl-20181584

RESUMEN

Voltage-sensitive dyes are important tools for assessing network and single-cell excitability, but an untested premise in most cases is that the dyes do not interfere with the parameters (membrane potential, excitability) that they are designed to measure. We found that popular members of several different families of voltage-sensitive dyes modulate GABA(A) receptor with maximum efficacy and potency similar to clinically used GABA(A) receptor modulators. Di-4-ANEPPS and DiBAC4(3) potentiated GABA function with micromolar and high nanomolar potency, respectively, and yielded strong maximum effects similar to barbiturates and neurosteroids. Newer blue oxonols had biphasic effects on GABA(A) receptor function at nanomolar and micromolar concentrations, with maximum potentiation comparable to that of saturating benzodiazepine effects. ANNINE-6 and ANNINE-6plus had no detectable effect on GABA(A) receptor function. Even dyes with no activity on GABA(A) receptors at baseline induced photodynamic enhancement of GABA(A) receptors. The basal effects of dyes were sufficient to prolong IPSCs and to dampen network activity in multielectrode array recordings. Therefore, the dual effects of voltage-sensitive dyes on GABAergic inhibition require caution in dye use for studies of excitability and network activity.


Asunto(s)
Colorantes Fluorescentes/farmacología , GABAérgicos/farmacología , Hipocampo/metabolismo , Receptores de GABA-A/metabolismo , Coloración y Etiquetado/métodos , Imagen de Colorante Sensible al Voltaje/métodos , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Colorantes Fluorescentes/química , GABAérgicos/química , Agonistas del GABA/farmacología , Antagonistas del GABA/farmacología , Hipocampo/citología , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/fisiología , Microscopía Confocal , Microscopía Fluorescente , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oocitos , Técnicas de Placa-Clamp , Ratas , Receptores de GABA-A/efectos de los fármacos , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Xenopus laevis
9.
Neuropharmacology ; 183: 108358, 2021 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-33115614

RESUMEN

Neuroactive steroids are an ascendant class of treatment for neuropsychiatric illness. Effects on ligand-gated neurotransmitter receptors appear to be a major mechanism of action. Here we describe a neuroactive steroid with a unique constellation of receptor actions. MQ-221 is a sulfated, 3ß-hydroxy neurosteroid analogue that inhibits NMDAR function but also potentiates GABAAR function, thereby exhibiting unusual but potentially clinically desirable effects. Although the compound also exhibited features of other sulfated steroids, namely activation-dependent inhibition of GABAAR function, net potentiation dominated under physiological conditions. Potentiation of GABAAR function was distinct from the mechanism governing potentiation by anesthetic neurosteroids. Inhibition of NMDAR function showed weaker channel activation dependence than pregnanolone sulfate (3α5ßPS). MQ-221 was unique among four stereoisomers explored in the pattern of effects at GABAA and NMDARs. Taken together, MQ-221 may represent a new class of compound with unique psychoactive effects and beneficial prospects for treating neuropsychiatric disorders.


Asunto(s)
Neuroesteroides/farmacología , Receptores de GABA-A/fisiología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Animales , Hipocampo/fisiología , Pregnanolona/farmacología , Ratas Sprague-Dawley
10.
Pharmacol Res ; 61(2): 157-61, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19782136

RESUMEN

Alzheimer's disease is associated to a cerebral amyloid angiopathy with dysregulation of cerebral blood flow (CBF). In vitro studies have shown that short-term application of beta-amyloid (Abeta) peptides to isolated vessels affects vascular tone within 1h, but no studies have examined the effect of long-term incubation with Abeta. Here we evaluate the effect of Abeta((1-40)) and Abeta((25-35)) in rat basilar artery for up to 24h. Basilar artery segments were incubated with 25microeta((1-40)) or Abeta((25-35)), for 6 or 24h. After treatment, arteries were mounted in a wire myograph, in physiological salt solution gassed with O(2)/CO(2), in the absence of Abeta, and challenged with vasoconstrictors and vasodilators. Vasomotor responses were not significantly changed by 6h treatment with Abeta peptides whereas 24h treatment with either Abeta((25-35)) or Abeta((1-40)) increased vasoconstriction to 5-hydroxytryptamine (5-HT) and reduced endothelium-dependent vasodilatation to acetylcholine (ACh). Analysis of endothelial cells did not show apoptotic changes associated to endothelial dysfunction, as assessed by TUNEL immunostaining and examination of nuclear morphology, but basal phosphorylation of endothelial nitric oxide synthase (at serine 1177) appeared reduced. These data suggest that long incubation with Abeta peptides induces an alteration of endothelial function in isolated basilar artery, involving eNOS activity without changing cell morphology. This endothelial dysfunction may play a role in the pathogenesis of CBF dysregulation occurring in cerebral amyloid angiopathy and Alzheimer's disease.


Asunto(s)
Péptidos beta-Amiloides/metabolismo , Arteria Basilar/metabolismo , Endotelio Vascular/metabolismo , Fragmentos de Péptidos/metabolismo , Vasodilatación , Acetilcolina/farmacología , Animales , Arteria Basilar/efectos de los fármacos , Arteria Basilar/fisiopatología , Angiopatía Amiloide Cerebral/metabolismo , Angiopatía Amiloide Cerebral/fisiopatología , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Masculino , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación , Ratas , Ratas Sprague-Dawley , Serotonina/farmacología , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Vasoconstricción , Vasoconstrictores/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología
12.
Neuropharmacology ; 144: 91-103, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30332607

RESUMEN

Positive modulators of NMDA receptors are important candidates for therapeutic development to treat psychiatric disorders including autism and schizophrenia. Sulfated neurosteroids have been studied as positive allosteric modulators of NMDA receptors for years, but we understand little about the cellular fate of these compounds, an important consideration for drug development. Here we focus on a visualizable sulfated neurosteroid analogue, KK-169. As expected of a pregnenolone sulfate analogue, the compound strongly potentiates NMDA receptor function, is an antagonist of GABAA receptors, exhibits occlusion with pregnenolone sulfate potentiation, and requires receptor domains important for pregnenolone sulfate potentiation. KK-169 exhibits somewhat higher potency than the natural parent, pregnenolone sulfate. The analogue contains a side-chain alkyne group, which we exploited for retrospective click labeling of neurons. Although the anionic sulfate group is expected to hinder cell entry, we detected significant accumulation of KK-169 in neurons with even brief incubations. Adding a photolabile diazirine group revealed that the expected plasma membrane localization of KK-169 is likely lost during fixation. Overall, our studies reveal new facets of the structure-activity relationship of neurosteroids at NMDA receptors, and their intracellular distribution suggests that sulfated neurosteroids could have unappreciated targets in addition to plasma membrane receptors.


Asunto(s)
Membrana Celular/efectos de los fármacos , Citoplasma/efectos de los fármacos , Agonistas de Aminoácidos Excitadores/farmacología , Receptores de N-Metil-D-Aspartato/agonistas , Regulación Alostérica , Animales , Membrana Celular/metabolismo , Células Cultivadas , Química Clic , Citoplasma/metabolismo , Agonistas de Aminoácidos Excitadores/química , Ácido Glutámico/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Ratones , Oocitos , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Relación Estructura-Actividad , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Xenopus laevis
13.
Cell Death Dis ; 10(1): 24, 2019 01 10.
Artículo en Inglés | MEDLINE | ID: mdl-30631041

RESUMEN

Evidence is rapidly growing regarding a role of astroglial cells in the pathogenesis of Alzheimer's disease (AD), and the hippocampus is one of the important brain regions affected in AD. While primary astroglial cultures, both from wild-type mice and from rodent models of AD, have been useful for studying astrocyte-specific alterations, the limited cell number and short primary culture lifetime have limited the use of primary hippocampal astrocytes. To overcome these limitations, we have now established immortalized astroglial cell lines from the hippocampus of 3xTg-AD and wild-type control mice (3Tg-iAstro and WT-iAstro, respectively). Both 3Tg-iAstro and WT-iAstro maintain an astroglial phenotype and markers (glutamine synthetase, aldehyde dehydrogenase 1 family member L1 and aquaporin-4) but display proliferative potential until at least passage 25. Furthermore, these cell lines maintain the potassium inward rectifying (Kir) current and present transcriptional and proteomic profiles compatible with primary astrocytes. Importantly, differences between the 3Tg-iAstro and WT-iAstro cell lines in terms of calcium signaling and in terms of transcriptional changes can be re-conducted to the changes previously reported in primary astroglial cells. To illustrate the versatility of this model we performed shotgun mass spectrometry proteomic analysis and found that proteins related to RNA binding and ribosome are differentially expressed in 3Tg-iAstro vs WT-iAstro. In summary, we present here immortalized hippocampal astrocytes from WT and 3xTg-AD mice that might be a useful model to speed up research on the role of astrocytes in AD.


Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Astrocitos/metabolismo , Señalización del Calcio , Expresión Génica , Hipocampo/patología , Proteoma , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Transportador 2 de Aminoácidos Excitadores/metabolismo , Ácido Glutámico/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mapas de Interacción de Proteínas , Transmisión Sináptica , Transfección
14.
Mol Neurobiol ; 54(6): 4081-4093, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-27318677

RESUMEN

Astrocytes regulate neuronal activity and blood brain barrier through tiny plasma membrane branches or astrocytic processes (APs) making contact with synapses and brain vessels. Several transmitters released by astrocytes and exerting their action on several receptor classes expressed by astrocytes themselves influence their physiology. Here we found that APs are dynamically modulated by purines. In live imaging experiments carried out in rat hippocampal astrocytes, Gq-coupled P2Y1 receptor blockade with the selective antagonist MRS2179 (1 µM) or inhibition of its effector phospholipase C using U73122 (3 µM) produced APs retraction, while stimulation of the same receptor with the selective agonist 2MeSADP (100 µM) increased their number. Since astrocytes, among other transmitters, release ATP by several mechanisms including connexin hemichannels, we used the connexin hemichannel inhibitor carbenoxolone (100 µM) and APs retraction was observed. In our system we then measured expression or function of channels important for modulation of volume transmission and K+ buffering, aquaporin-4, and K+ inward rectifying (Kir) channels, respectively. Aquaporin-4 expression level did not change whereas, in whole-cell patch-clamp recordings performed to measure Kir current, we observed an increase in K+ current in all conditions where APs number was reduced. These data are supporting the idea of a dynamic modulation of astrocytic processes by purinergic signal, strengthening the role of purines in brain homeostasis.


Asunto(s)
Astrocitos/metabolismo , Membrana Celular/metabolismo , Hipocampo/metabolismo , Receptores Purinérgicos P2Y1/metabolismo , Animales , Acuaporina 4/metabolismo , Señalización del Calcio , Células Cultivadas , Conexinas/metabolismo , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Ratas Sprague-Dawley , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismo
15.
Neuroscience ; 340: 551-562, 2017 01 06.
Artículo en Inglés | MEDLINE | ID: mdl-27816700

RESUMEN

The neuropeptide PACAP modulates synaptic transmission in the hippocampus exerting multiple effects through different receptor subtypes: the underlying mechanisms have not yet been completely elucidated. The neurotransmitter acetylcholine (ACh) also exerts a well-documented modulation of hippocampal synaptic transmission and plasticity. Since PACAP was shown to stimulate ACh release in the hippocampus, we tested whether PACAP acting through ACh might indirectly modulate glutamate-mediated synaptic transmission at a pre- and/or at a post-synaptic level. Using patch clamp on rat hippocampal slices, we tested PACAP effects on stimulation-evoked AMPA receptor-mediated excitatory post-synaptic currents (EPSCsAMPA) in the CA3-CA1 synapse and on spontaneous miniature EPSCs (mEPSCs) in CA1 pyramidal neurons. A subnanomolar dose of PACAP (0.5nM) decreased EPSCsAMPA amplitude, enhanced EPSC paired-pulse facilitation (PPF) and reduced mEPSC frequency, indicating a pre-synaptic decrease of glutamate release probability: these effects were abolished by simultaneous blockade of muscarinic and nicotinic ACh receptors, indicating the involvement of endogenous ACh. The effect of subnanomolar PACAP was abolished by a PAC1 receptor antagonist but not by a VPAC receptor blocker. At a higher concentration (10nM), PACAP inhibited EPSCsAMPA: this effect persisted in the presence of ACh receptor antagonists and did not involve any change in PPF or in mEPSC frequency, thus was not mediated by ACh and was exerted post- synaptically on CA1 pyramidal neurons. We suggest that a high-affinity PAC1 receptor pre-synaptically modulates hippocampal glutamatergic transmission acting through ACh. Therefore, administration of PACAP at very low doses might be envisaged in cognitive diseases with reduced cholinergic transmission.


Asunto(s)
Acetilcolina/metabolismo , Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Transmisión Sináptica/fisiología , Animales , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/administración & dosificación , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Ratas Sprague-Dawley , Ratas Wistar , Receptores AMPA/antagonistas & inhibidores , Receptores AMPA/metabolismo , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismo , Receptores de Tipo II del Péptido Intestinal Vasoactivo/metabolismo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/metabolismo , Técnicas de Cultivo de Tejidos
16.
Front Pharmacol ; 8: 812, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29170640

RESUMEN

Metabotropic glutamate (mGlu) receptor 5 is involved in neuroinflammation and has been shown to mediate reduced inflammation and neurotoxicity and to modify microglia polarization. On the other hand, blockade of mGlu5 receptor results in inhibition of microglia activation. To dissect this controversy, we investigated whether microvesicles (MVs) released from microglia BV2 cells could contribute to the communication between microglia and neurons and whether this interaction was modulated by mGlu5 receptor. Activation of purinergic ionotropic P2X7 receptor with the stable ATP analog benzoyl-ATP (100 µM) caused rapid MVs shedding from BV2 cells. Ionic currents through P2X7 receptor increased in BV2 cells pretreated for 24 h with the mGlu5 receptor agonist CHPG (200 µM) as by patch-clamp recording. This increase was blunted when microglia cells were activated by exposure to lipopolysaccharide (LPS; 0.1 µg/ml for 6 h). Accordingly, a greater amount of MVs formed after CHPG treatment, an effect prevented by the mGlu5 receptor antagonist MTEP (100 µM), as measured by expression of flotillin, a membrane protein enriched in MVs. Transferred MVs were internalized by SH-SY5Y neurons where they did not modify neuronal death induced by a low concentration of rotenone (0.1 µM for 24 h), but significantly increased rotenone neurotoxicity when shed from CHPG-treated BV2 cells. miR146a was increased in CHPG-treated MVs, an effect concealed in MVs from LPS-activated BV2 cells that showed per se an increase in miRNA146a levels. The present data support a role for microglia-shed MVs in mGlu5-mediated modulation of neuronal death and identify miRNAs as potential critical mediators of this interaction.

18.
FASEB J ; 16(14): 2006-8, 2002 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-12397084

RESUMEN

An ectopic reentrance into the cell cycle with ensuing DNA replication is required for neuronal apoptosis induced by beta-amyloid. Here, we investigate the repertoire of DNA polymerases expressed in beta-amyloid-treated neurons, and their specific role in DNA synthesis and apoptosis. We show that exposure of cultured cortical neurons to beta-amyloid induces the expression of DNA polymerase-beta, proliferating cell nuclear antigen, and the p49 and p58 subunits of DNA primase. Induction requires the activity of cyclin-dependent kinases. The knockdown of the p49 primase subunit prevents beta-amyloid-induced neuronal DNA synthesis and apoptosis. Similar effects are observed by knocking down DNA polymerase-beta or by using dideoxycytidine, a preferential inhibitor of this enzyme. Thus, the reparative enzyme DNA polymerase-beta unexpectedly mediates a large component of de novo DNA synthesis and apoptotic death in neurons exposed to beta-amyloid. These data indicate that DNA polymerases become death signals when erratically expressed by differentiated neurons.


Asunto(s)
Péptidos beta-Amiloides/farmacología , Apoptosis , ADN Polimerasa Dirigida por ADN/biosíntesis , Neuronas/enzimología , Fragmentos de Péptidos/farmacología , Animales , Ciclo Celular , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/enzimología , Replicación del ADN , ADN Polimerasa Dirigida por ADN/genética , Regulación de la Expresión Génica , Modelos Biológicos , Neuronas/citología , Neuronas/efectos de los fármacos , ARN Mensajero/biosíntesis , Ratas
19.
Brain Res ; 1047(1): 30-7, 2005 Jun 14.
Artículo en Inglés | MEDLINE | ID: mdl-15882840

RESUMEN

Nicergoline, a drug used for the treatment of Alzheimer's disease and other types of dementia, was tested for its ability to protect neurons against beta-amyloid toxicity. Pure cultures of rat cortical neurons were challenged with a toxic fragment of beta-amyloid peptide (betaAP(25-35)) and toxicity was assessed after 24 h. Micromolar concentrations of nicergoline or its metabolite, MDL, attenuated betaAP(25-35)-induced neuronal death, whereas MMDL (another metabolite of nicergoline), the alpha1-adrenergic receptor antagonist, prazosin, or the serotonin 5HT-2 receptor antagonist, methysergide, were inactive. Nicergoline increased the basal levels of Bcl-2 and reduced the increase in Bax levels induced by beta-amyloid, indicating that the drug inhibits the execution of an apoptotic program in cortical neurons. In mixed cultures of rat cortical cells containing both neurons and astrocytes, nicergoline and MDL were more efficacious than in pure neuronal cultures in reducing beta-amyloid neurotoxicity. Experiments carried out in pure cultures of astrocytes showed that a component of neuroprotection was mediated by a mechanism of glial-neuronal interaction. The conditioned medium of cultured astrocytes treated with nicergoline or MDL for 72-96 h (collected 24 h after drug withdrawal) was neuroprotective when transferred to pure neuronal cultures challenged with beta-amyloid. In cultured astrocytes, nicergoline increased the intracellular levels of transforming-growth factor-beta and glial-derived neurotrophic factor, two trophic factors that are known to protect neurons against beta-amyloid toxicity. These results raise the possibility that nicergoline reduces neurodegeneration in the Alzheimer's brain.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/antagonistas & inhibidores , Corteza Cerebral/efectos de los fármacos , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/toxicidad , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Astrocitos/metabolismo , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Células Cultivadas , Corteza Cerebral/metabolismo , Corteza Cerebral/fisiopatología , Técnicas de Cocultivo , Trastornos del Conocimiento/tratamiento farmacológico , Trastornos del Conocimiento/metabolismo , Trastornos del Conocimiento/fisiopatología , Trastornos de la Memoria/tratamiento farmacológico , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/fisiopatología , Factores de Crecimiento Nervioso/metabolismo , Neuronas/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Nicergolina/farmacología , Nicergolina/uso terapéutico , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/toxicidad , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Sprague-Dawley , Proteína X Asociada a bcl-2
20.
Life Sci ; 74(5): 643-50, 2003 Dec 19.
Artículo en Inglés | MEDLINE | ID: mdl-14623034

RESUMEN

Phenformin is a biguanide that has been largely used in the past for its anti-diabetic activity. A large body of evidence suggests additional effects of phenformin including antitumoral activity in different animal models in vivo. Thus, the present study has been conducted in order to elucidate possible mechanisms involved in the antitumoral effects of phenformin. In various tumoral cell lines (SH-SY5Y neuroblastoma and LNCaP prostate adenocarcinoma cells), increasing concentrations of phenformin (50-500 microM) induced a concentration-dependent inhibition of cell proliferation. This effect was not dependent on the ability of the drug to reduce glucose levels and was accompanied by induction of apoptotic cell death as measured by cytofluorometric analysis. In addition, a short-time incubation of SH-SY5Y cells with phenformin induced enhanced and transient expression of the cell cycle inhibitor p21 suggesting that phenformin causes inhibition of cell cycle progression prior to induction of apoptosis. These results demonstrate an activity at the cellular level of phenformin that supports its antitumoral effect observed in vivo.


Asunto(s)
Antineoplásicos , Hipoglucemiantes/farmacología , Fenformina/farmacología , Adenocarcinoma/patología , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/patología , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo , Glucosa/farmacología , Humanos , Masculino , Neuroblastoma/patología , Proteína Oncogénica p21(ras)/efectos de los fármacos , Neoplasias de la Próstata/patología , Sales de Tetrazolio , Tiazoles
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