RESUMEN
NMDA receptors (NMDARs) are ionotropic glutamate receptors that play a key role in excitatory neurotransmission. The number and subtype of surface NMDARs are regulated at several levels, including their externalization, internalization, and lateral diffusion between the synaptic and extrasynaptic regions. Here, we used novel anti-GFP (green fluorescent protein) nanobodies conjugated to either the smallest commercially available quantum dot 525 (QD525) or the several nanometer larger (and thus brighter) QD605 (referred to as nanoGFP-QD525 and nanoGFP-QD605, respectively). Targeting the yellow fluorescent protein-tagged GluN1 subunit in rat hippocampal neurons, we compared these two probes to a previously established larger probe, a rabbit anti-GFP IgG together with a secondary IgG conjugated to QD605 (referred to as antiGFP-QD605). The nanoGFP-based probes allowed faster lateral diffusion of the NMDARs, with several-fold increased median values of the diffusion coefficient (D). Using thresholded tdTomato-Homer1c signals to mark synaptic regions, we found that the nanoprobe-based D values sharply increased at distances over 100 nm from the synaptic edge, while D values for antiGFP-QD605 probe remained unchanged up to a 400 nm distance. Using the nanoGFP-QD605 probe in hippocampal neurons expressing the GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits, we detected subunit-dependent differences in the synaptic localization of NMDARs, D value, synaptic residence time, and synaptic-extrasynaptic exchange rate. Finally, we confirmed the applicability of the nanoGFP-QD605 probe to study differences in the distribution of synaptic NMDARs by comparing to data obtained with nanoGFPs conjugated to organic fluorophores, using universal point accumulation imaging in nanoscale topography and direct stochastic optical reconstruction microscopy.SIGNIFICANCE STATEMENT Our study systematically compared the localization and mobility of surface NMDARs containing GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits expressed in rodent hippocampal neurons, using anti-green fluorescent protein (GFP) nanobodies conjugated to the quantum dot 605 (nanoGFP-QD605), as well as nanoGFP probes conjugated with small organic fluorophores. Our comprehensive analysis showed that the method used to delineate the synaptic region plays an important role in the study of synaptic and extrasynaptic pools of NMDARs. In addition, we showed that the nanoGFP-QD605 probe has optimal parameters for studying the mobility of NMDARs because of its high localization accuracy comparable to direct stochastic optical reconstruction microscopy and longer scan time compared with universal point accumulation imaging in nanoscale topography. The developed approaches are readily applicable to the study of any GFP-labeled membrane receptors expressed in mammalian neurons.
Asunto(s)
Receptores de N-Metil-D-Aspartato , Anticuerpos de Dominio Único , Ratas , Animales , Conejos , Receptores de N-Metil-D-Aspartato/metabolismo , Anticuerpos de Dominio Único/metabolismo , Sinapsis/fisiología , Hipocampo/metabolismo , Neuronas/metabolismo , Inmunoglobulina G/metabolismo , MamíferosRESUMEN
Alzheimer's disease (AD) is a progressive neurodegenerative disorder and currently there is no efficient treatment. The classic drug-design strategy based on the "one-molecule-one-target" paradigm was found to be ineffective in the case of multifactorial diseases like AD. A novel multi-target-directed ligand strategy based on the assumption that a single compound consisting of two or more distinct pharmacophores is able to hit multiple targets has been proposed as promising. Herein, we investigated 7-methoxytacrine - memantine heterodimers developed with respect to the multi-target-directed ligand theory. The spectroscopic, microscopic and cell culture methods were used for systematic investigation of the interference of the heterodimers with ß-secretase (BACE1) activity, Aß peptide amyloid fibrillization (amyloid theory) and interaction with M1 subtype of muscarinic (mAChRs), nicotinic (nAChRs) acetylcholine receptors (cholinergic theory) and N-methyl-d-aspartate receptors (NMDA) (glutamatergic theory). The drug-like properties of selected compounds have been evaluated from the point of view of blood-brain barrier penetration and cell proliferation. We have confirmed the multipotent effect of novel series of compounds. They inhibited effectively Aß peptide amyloid fibrillization and affected the BACE1 activity. Moreover, they have AChE inhibitory potency but they could not potentiate cholinergic transmission via direct interaction with cholinergic receptors. All compounds were reported to act as an antagonist of both M1 muscarinic and muscle-type nicotinic receptors. We have found that 7-methoxytacrine - memantine heterodimers are able to hit multiple targets associated with Alzheimer's disease and thus, have a potential clinical impact for slowing or blocking the neurodegenerative process related to this disease.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Amantadina/farmacología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Tacrina/análogos & derivados , Enfermedad de Alzheimer/metabolismo , Amantadina/análogos & derivados , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Células CHO , Colinesterasas/metabolismo , Cricetulus , Dimerización , Inhibidores Enzimáticos/química , Células HEK293 , Humanos , Terapia Molecular Dirigida , Receptor Muscarínico M1/antagonistas & inhibidores , Receptor Muscarínico M1/metabolismo , Receptores Colinérgicos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Tacrina/química , Tacrina/farmacología , XenopusRESUMEN
NMDA receptors (NMDARs) comprise a subclass of neurotransmitter receptors whose surface expression is regulated at multiple levels, including processing in the endoplasmic reticulum (ER), intracellular trafficking via the Golgi apparatus, internalization, recycling, and degradation. With respect to early processing, NMDARs are regulated by the availability of GluN subunits within the ER, the presence of ER retention and export signals, and posttranslational modifications, including phosphorylation and palmitoylation. However, the role of N-glycosylation, one of the most common posttranslational modifications, in regulating NMDAR processing has not been studied in detail. Using biochemistry, confocal and electron microscopy, and electrophysiology in conjunction with a lentivirus-based molecular replacement strategy, we found that NMDARs are released from the ER only when two asparagine residues in the GluN1 subunit (Asn-203 and Asn-368) are N-glycosylated. Although the GluN2A and GluN2B subunits are also N-glycosylated, their N-glycosylation sites do not appear to be essential for surface delivery of NMDARs. Furthermore, we found that removing N-glycans from native NMDARs altered the receptor affinity for glutamate. Our results suggest a novel mechanism by which neurons ensure that postsynaptic membranes contain sufficient numbers of functional NMDARs.
Asunto(s)
N-Metilaspartato/metabolismo , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transmisión Sináptica , Animales , Células COS , Chlorocebus aethiops , Retículo Endoplásmico/metabolismo , Glicosilación , Aparato de Golgi/metabolismo , Células HEK293 , Humanos , N-Metilaspartato/química , Neuronas/química , Polisacáridos/metabolismo , Ratas , Receptores de N-Metil-D-Aspartato/química , Sinapsis/metabolismoRESUMEN
In mammals, excitatory synapses contain two major types of ionotropic glutamate receptors: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and N-methyl-d-aspartate receptors (NMDARs). Both receptor types are comprised of several subunits that are post-translationally modified by N-glycosylation. However, the precise N-glycans that are attached to these receptor types are largely unknown. Here, we used biochemistry to confirm that native NMDARs are extensively N-glycosylated; moreover, we found that the NMDAR GluN2B subunit differs from GluN1 subunits with respect to endoglycosidase H sensitivity. Next, we used a complete panel of lectins to determine the glycan composition of NMDARs in both cerebellar tissue and cultured cerebellar granule cells. Our experiments identified 23 lectins that pulled down both the GluN1 and GluN2B NMDAR subunits. We then performed an electrophysiological analysis using representative lectins and found that pre-incubating cerebellar granule cells with the AAL, WGA, or ConA alters the receptor's biophysical properties; this lectin-mediated effect was eliminated when the cells were deglycosylated with peptide-N-glycosidase F. Similar lectin-mediated effects were observed using HEK293 cells that express recombinant GluN1/GluN2B receptors. Finally, using mutant recombinant GluN subunits expressed in HEK293 cells, we found that 11 out of 12 predicted N-glycosylation sites in GluN1 and 7 out of 7 N-glycosylation sites in GluN2B are occupied by N-glycans. These data provide new insight into the role that N-glycosylation plays in regulating the function of NMDA receptors in the central nervous system. All animal experiments were performed in accordance with relevant institutional ethics guidelines and regulations with respect to protecting animal welfare. We examined the N-glycan composition of NMDA receptors (NMDARs) using deglycosylating enzymes, lectin-based biochemistry, and electrophysiology. Our results revealed that cerebellar NMDARs associate with 23 different lectins that have unique specificities for glycan structures. Furthermore, we found that 11 out of 12 predicted N-glycosylation sites in GluN1 and 7 out of 7 N-glycosylation sites in GluN2B are occupied by N-glycans. These data shed light on the glycan composition of NMDARs, revealing potential targets for the development of novel therapeutic approaches.
Asunto(s)
Fenómenos Electrofisiológicos/fisiología , Neuronas/metabolismo , Polisacáridos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismo , Animales , Ácido Glutámico/metabolismo , Células HEK293 , Humanos , Ratas , Transducción de Señal/fisiologíaRESUMEN
NMDA receptors (NMDARs) are glutamate-gated ion channels that mediate excitatory neurotransmission in the CNS. Although these receptors are in direct contact with plasma membrane, lipid-NMDAR interactions are little understood. In the present study, we aimed at characterizing the effect of cholesterol on the ionotropic glutamate receptors. Whole-cell current responses induced by fast application of NMDA in cultured rat cerebellar granule cells (CGCs) were almost abolished (reduced to 3%) and the relative degree of receptor desensitization was increased (by seven-fold) after acute cholesterol depletion by methyl-ß-cyclodextrin. Both of these effects were fully reversible by cholesterol repletion. By contrast, the responses mediated by AMPA/kainate receptors were not affected by cholesterol depletion. Similar results were obtained in CGCs after chronic inhibition of cholesterol biosynthesis by simvastatin and acute enzymatic cholesterol degradation to 4-cholesten-3-one by cholesterol oxidase. Fluorescence anisotropy measurements showed that membrane fluidity increased after methyl-ß-cyclodextrin pretreatment. However, no change in fluidity was observed after cholesterol enzymatic degradation, suggesting that the effect of cholesterol on NMDARs is not mediated by changes in membrane fluidity. Our data show that diminution of NMDAR responses by cholesterol depletion is the result of a reduction of the open probability, whereas the increase in receptor desensitization is the result of an increase in the rate constant of entry into the desensitized state. Surface NMDAR population, agonist affinity, single-channel conductance and open time were not altered in cholesterol-depleted CGCs. The results of our experiments show that cholesterol is a strong endogenous modulator of NMDARs.
Asunto(s)
Cerebelo/citología , Cerebelo/fisiología , Colesterol Oxidasa/farmacología , Colesterol/fisiología , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/fisiología , Simvastatina/farmacología , Animales , Anticolesterolemiantes/farmacología , Células Cultivadas , Cerebelo/efectos de los fármacos , Colesterol/deficiencia , Fenómenos Electrofisiológicos/fisiología , Femenino , Masculino , Fluidez de la Membrana/efectos de los fármacos , Fluidez de la Membrana/fisiología , Lípidos de la Membrana/fisiología , Conducción Nerviosa/fisiología , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Transmisión Sináptica/fisiología , beta-Ciclodextrinas/farmacologíaRESUMEN
N-methyl-d-aspartate (NMDA) receptors are glutamate ionotropic receptors that play critical roles in synaptic transmission, plasticity, and excitotoxicity. The functional NMDA receptors, heterotetramers composed mainly of two NR1 and two NR2 subunits, likely pass endoplasmic reticulum quality control before they are released from the endoplasmic reticulum and trafficked to the cell surface. However, the mechanism underlying this process is not clear. Using truncated and mutated NMDA receptor subunits expressed in heterologous cells, we found that the M3 domains of both NR1 and NR2 subunits contain key amino acid residues that contribute to the regulation of the number of surface functional NMDA receptors. These key residues are critical neither for the interaction between the NR1 and NR2 subunits nor for the formation of the functional receptors, but rather they regulate the early trafficking of the receptors. We also found that the identified key amino acid residues within both NR1 and NR2 M3 domains contribute to the regulation of the surface expression of unassembled NR1 and NR2 subunits. Thus, our data identify the unique role of the membrane domains in the regulation of the number of surface NMDA receptors.
Asunto(s)
Receptores de N-Metil-D-Aspartato/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Transporte de Proteínas , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
N-methyl-D-aspartate (NMDA) receptors are glutamate ion channels that are critically involved in excitatory synaptic transmission and plasticity. The functional NMDA receptor is a heterotetramer composed mainly of GluN1 and GluN2 subunits. It is generally thought that only correctly assembled NMDA receptors can pass the quality control checkpoint in the endoplasmic reticulum (ER) and are transported to the cell surface membranes. The molecular mechanisms underlying these processes remain poorly understood. Using chimeric and mutated GluN1 subunits expressed in heterologous cells, we identified a single amino acid residue within the fourth membrane domain (M4) of GluN1 subunit, L830, that regulates the surface number of NMDA receptors. Our experiments show that this residue is not critical for the interaction between GluN1 and GluN2 subunits or for the formation of functional receptors, but rather that it regulates the forward trafficking of the NMDA receptors. The surface expression of both GluN2A- and GluN2B-containing receptors is regulated by the L830 residue in a similar manner. We also found that the L830 residue is not involved in the trafficking of individually expressed GluN1 subunits. Our data reveal a critical role of the single amino acid residue within the GluN1 M4 domain in the surface delivery of functional NMDA receptors.
Asunto(s)
Transporte de Proteínas/fisiología , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Aminoácidos/genética , Animales , Células COS , Chlorocebus aethiops , Retículo Endoplásmico/metabolismo , Células HEK293 , Humanos , Mutagénesis/fisiología , Estructura Cuaternaria de Proteína/fisiología , Estructura Terciaria de Proteína/fisiología , Receptores de Superficie Celular/genética , Receptores de N-Metil-D-Aspartato/genéticaRESUMEN
N-methyl-D-aspartaterecepro receptor (NMDARs) are a subclass of glutamate receptors, which play an essential role in excitatory neurotransmission, but their excessive overactivation by glutamate leads to excitotoxicity. NMDARs are hence a valid pharmacological target for the treatment of neurodegenerative disorders; however, novel drugs targeting NMDARs are often associated with specific psychotic side effects and abuse potential. Motivated by currently available treatment against neurodegenerative diseases involving the inhibitors of acetylcholinesterase (AChE) and NMDARs, administered also in combination, we developed a dually-acting compound 7-phenoxytacrine (7-PhO-THA) and evaluated its neuropsychopharmacological and drug-like properties for potential therapeutic use. Indeed, we have confirmed the dual potency of 7-PhO-THA, i.e. potent and balanced inhibition of both AChE and NMDARs. We discovered that it selectively inhibits the GluN1/GluN2B subtype of NMDARs via an ifenprodil-binding site, in addition to its voltage-dependent inhibitory effect at both GluN1/GluN2A and GluN1/GluN2B subtypes of NMDARs. Furthermore, whereas NMDA-induced lesion of the dorsal hippocampus confirmed potent anti-excitotoxic and neuroprotective efficacy, behavioral observations showed also a cholinergic component manifesting mainly in decreased hyperlocomotion. From the point of view of behavioral side effects, 7-PhO-THA managed to avoid these, notably those analogous to symptoms of schizophrenia. Thus, CNS availability and the overall behavioral profile are promising for subsequent investigation of therapeutic use.
Asunto(s)
Fármacos Neuroprotectores/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Tacrina/farmacología , Animales , Células HEK293 , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Masculino , Fármacos Neuroprotectores/química , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Tacrina/químicaRESUMEN
N-methyl-d-aspartate receptors (NMDARs) play an essential role in regulating glutamatergic neurotransmission. Recently, pathogenic missense mutations were identified in genes encoding NMDAR subunits; however, their effect on NMDAR activity is often poorly understood. Here, we examined whether three previously identified pathogenic mutations (M641I, A645S, and Y647S) in the M3 domain of the GluN1 subunit affect the receptor's surface delivery, agonist sensitivity, Mg2+ block, and/or inhibition by the FDA-approved NMDAR blocker memantine. When expressed in HEK293 cells, we found reduced surface expression of GluN1-M641I/GluN2A, GluN1-Y647S/GluN2A, and GluN1-Y647S/GluN2B receptors; other mutation-bearing NMDAR combinations, including GluN1/GluN3A receptors, were expressed at normal surface levels. When expressed in rat hippocampal neurons, we consistently found reduced surface expression of the GluN1-M641I and GluN1-Y647S subunits when compared with wild-type GluN1 subunit. At the functional level, we found that GluN1-M641I/GluN2 and GluN1-A645S/GluN2 receptors expressed in HEK293 cells have wild-type EC50 values for both glutamate and glycine; in contrast, GluN1-Y647S/GluN2 receptors do not produce glutamate-induced currents. In the presence of a physiological concentration of Mg2+, we found that GluN1-M641I/GluN2 receptors have a lower memantine IC50 and slower offset kinetics, whereas GluN1-A645S/GluN2 receptors have a higher memantine IC50 and faster offset kinetics when compared to wild-type receptors. Finally, we found that memantine was the most neuroprotective in hippocampal neurons expressing GluN1-M641I subunits, followed by neurons expressing wild-type GluN1 and then GluN1-A645S subunits in an NMDA-induced excitotoxicity assay. These results indicate that specific pathogenic mutations in the M3 domain of the GluN1 subunit differentially affect the trafficking and functional properties of NMDARs.
Asunto(s)
Agonistas de Aminoácidos Excitadores/administración & dosificación , Antagonistas de Aminoácidos Excitadores/administración & dosificación , Mutación/genética , Proteínas del Tejido Nervioso/genética , Subunidades de Proteína/genética , Receptores de N-Metil-D-Aspartato/genética , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Células HEK293 , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Humanos , Masculino , Mutación/efectos de los fármacos , Proteínas del Tejido Nervioso/agonistas , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Subunidades de Proteína/agonistas , Subunidades de Proteína/antagonistas & inhibidores , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Propiedades de Superficie/efectos de los fármacosRESUMEN
Although numerous pathogenic mutations have been identified in various subunits of N-methyl-D-aspartate receptors (NMDARs), ionotropic glutamate receptors that are central to glutamatergic neurotransmission, the functional effects of these mutations are often unknown. Here, we combined in silico modelling with microscopy, biochemistry, and electrophysiology in cultured HEK293 cells and hippocampal neurons to examine how the pathogenic missense mutation S688Y in the GluN1 NMDAR subunit affects receptor function and trafficking. We found that the S688Y mutation significantly increases the EC50 of both glycine and D-serine in GluN1/GluN2A and GluN1/GluN2B receptors, and significantly slows desensitisation of GluN1/GluN3A receptors. Moreover, the S688Y mutation reduces the surface expression of GluN3A-containing NMDARs in cultured hippocampal neurons, but does not affect the trafficking of GluN2-containing receptors. Finally, we found that the S688Y mutation reduces Ca2+ influx through NMDARs and reduces NMDA-induced excitotoxicity in cultured hippocampal neurons. These findings provide key insights into the molecular mechanisms that underlie the regulation of NMDAR subtypes containing pathogenic mutations.
Asunto(s)
Glicina/farmacología , Hipocampo/metabolismo , Mutación , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Glicinérgicos/farmacología , Células HEK293 , Hipocampo/citología , Hipocampo/efectos de los fármacos , Humanos , Ligandos , Modelos Moleculares , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Neuronas/efectos de los fármacos , Dominios Proteicos , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/genéticaRESUMEN
The role of negatively charged amino acids in the F-loop of the beta 4 subunit in channel activation and desensitization was studied using the patch-clamp technique. The selected amino acids were changed to their neutral analogs via point mutations. Whole-cell currents were recorded in COS cells transiently transfected with the alpha 3 beta 4 nicotinic acetylcholine receptor. The application of acetylcholine (ACh), nicotine (Nic), cytisine (Cyt), carbamylcholine (CCh) and epibatidine (Epi) to cells clamped at -40 mV produced inward currents which displayed biphasic desensitization. The EC50 of Epi and Nic were increased by a factor of 3-6 due to mutations D191N or D192N. Only Epi remained an agonist in the double-mutated receptors with EC50 increased 17-fold. The interaction of the receptors with the competitive antagonist (+)tubocurarine (TC) was weakened almost 3-fold in the double-mutated receptors. The mutations increased the proportion of the slower desensitization component and increased the response plateau, resulting in decreased receptor desensitization. The double mutation substantially accelerated the return from long-term desensitization induced by Epi.
Asunto(s)
Aminoácidos/química , Aminoácidos/metabolismo , Neuronas/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Secuencia de Aminoácidos , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Células COS , Chlorocebus aethiops , Cinética , Datos de Secuencia Molecular , Mutación/genética , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Estructura Secundaria de Proteína , Piridinas/farmacología , Ratas , Alineación de Secuencia , Relación Estructura-Actividad , Tubocurarina/farmacologíaRESUMEN
N-methyl-d-aspartate receptors (NMDARs) play an essential role in excitatory neurotransmission within the mammalian central nervous system (CNS). NMDARs are heteromultimers containing GluN1, GluN2, and/or GluN3 subunits, thus giving rise to a wide variety of subunit combinations, each with unique functional and pharmacological properties. Importantly, GluN1/GluN3A and GluN1/GluN3B receptors form glycine-gated receptors. Here, we combined electrophysiology with rapid solution exchange in order to determine whether the presence of specific N-glycans and/or interactions with specific lectins regulates the functional properties of GluN1/GluN3A and GluN1/GluN3B receptors expressed in human embryonic kidney 293 (HEK293) cells. We found that removing putative N-glycosylation sites alters the functional properties of GluN1/GluN3B receptors, but has no effect on GluN1/GluN3A receptors. Moreover, we found that the functional properties of both GluN1/GluN3A and GluN1/GluN3B receptors are modulated by a variety of lectins, including Concanavalin A (ConA), Wheat Germ Agglutinin (WGA), and Aleuria Aurantia Lectin (AAL), and this effect is likely mediated by a reduction in GluN1 subunit-mediated desensitization. We also found that AAL has the most profound effect on GluN1/GluN3 receptors, and this effect is mediated partly by a single N-glycosylation site on the GluN3 subunit (specifically, N565 on GluN3A and N465 on GluN3B). Finally, we found that lectins mediate their effect only when applied to non-activated receptors and have no effect when applied in the continuous presence of glycine. These findings provide further evidence to distinguish GluN1/GluN3 receptors from the canonical GluN1/GluN2 receptors and offer insight into how GluN1/GluN3 receptors may be regulated in the mammalian CNS.
Asunto(s)
Lectinas/farmacología , Subunidades de Proteína/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Células Cultivadas , Glicina/farmacología , Glicosilación/efectos de los fármacos , Humanos , Lectinas/antagonistas & inhibidores , Potenciales de la Membrana/fisiología , Polisacáridos/metabolismo , Unión ProteicaRESUMEN
N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that play an essential role in mediating excitatory neurotransmission in the mammalian central nervous system (CNS). Functional NMDARs are tetramers composed of GluN1, GluN2A-D, and/or GluN3A-B subunits, giving rise to a wide variety of NMDAR subtypes with unique functional properties. Here, we examined the surface delivery and functional properties of NMDARs containing mutations in the glycine-binding sites in GluN1 and GluN3A subunits expressed in mammalian cell lines and primary rat hippocampal neurons. We found that the structural features of the glycine-binding sites in both GluN1 and GluN3A subunits are correlated with receptor forward trafficking to the cell surface. In addition, we found that a potentially clinically relevant mutation in the glycine-binding site of the human GluN3A subunit significantly reduces surface delivery of NMDARs. Taken together, these findings provide novel insight into how NMDARs are regulated by their glycine-binding sites and may provide important information regarding the role of NMDARs in both physiological and pathophysiological processes in the mammalian CNS.
Asunto(s)
Glicina/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Células HEK293 , Hipocampo/citología , Humanos , Mutación/genética , Neuronas/metabolismo , Dominios Proteicos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Ratas , Relación Estructura-ActividadRESUMEN
BACKGROUND: Alzheimer's disease (AD) is the most common form of dementia in the elderly. It is characterized as a multi-factorial disorder with a prevalent genetic component. Due to the unknown etiology, current treatment based on acetylcholinesterase (AChE) inhibitors and N-methyl-D-aspartate receptors (NMDAR) antagonist is effective only temporary. It seems that curative treatment will necessarily be complex due to the multifactorial nature of the disease. In this context, the so-called "multi-targeting" approach has been established. OBJECTIVES: The aim of this study was to develop a multi-target-directed ligand (MTDL) combining the support for the cholinergic system by inhibition of AChE and at the same time ameliorating the burden caused by glutamate excitotoxicity mediated by the NMDAR receptors. METHODS: We have applied common approaches of organic chemistry to prepare a hybrid of 6-chlorotacrine and memantine. Then, we investigated its blocking ability towards AChE and NMDRS in vitro, as well as its neuroprotective efficacy in vivo in the model of NMDA-induced lessions. We also studied cytotoxic potential of the compound and predicted the ability to cross the blood-brain barrier. RESULTS: A novel molecule formed by combination of 6-chlorotacrine and memantine proved to be a promising multipotent hybrid capable of blocking the action of AChE as well as NMDARs. The presented hybrid surpassed the AChE inhibitory activity of the parent compound 6-Cl-THA twofold. According to results it has been revealed that our novel hybrid blocks NMDARs in the same manner as memantine, potently inhibits AChE and is predicted to cross the blood-brain barrier via passive diffusion. Finally, the MTDL design strategy was indicated by in vivo results which showed that the novel 6-Cl-THA-memantine hybrid displayed a quantitatively better neuroprotective effect than the parent compound memantine. CONCLUSION: We conclude that the combination of two pharmacophores with a synergistic mechanism of action into a single molecule offers great potential for the treatment of CNS disorders associated with cognitive decline and/or excitotoxicity mediated by NMDARs.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Inhibidores de la Colinesterasa/farmacología , Memantina/farmacología , Fármacos Neuroprotectores/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Tacrina/análogos & derivados , Acetilcolinesterasa/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Células CHO , Permeabilidad Capilar , Supervivencia Celular/efectos de los fármacos , Inhibidores de la Colinesterasa/síntesis química , Cricetulus , Ácido Glutámico/metabolismo , Células HEK293 , Humanos , Ligandos , Masculino , Memantina/síntesis química , Simulación del Acoplamiento Molecular , Fármacos Neuroprotectores/síntesis química , Ratas Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Tacrina/síntesis química , Tacrina/farmacología , Técnicas de Cultivo de TejidosRESUMEN
N-methyl-D-aspartate receptors (NMDARs) play critical roles in both excitatory neurotransmission and synaptic plasticity. NMDARs containing the nonconventional GluN3A subunit have different functional properties compared to receptors comprised of GluN1/GluN2 subunits. Previous studies showed that GluN1/GluN2 receptors are regulated by N-glycosylation; however, limited information is available regarding the role of N-glycosylation in GluN3A-containing NMDARs. Using a combination of microscopy, biochemistry, and electrophysiology in mammalian cell lines and rat hippocampal neurons, we found that two asparagine residues (N203 and N368) in the GluN1 subunit and three asparagine residues (N145, N264 and N275) in the GluN3A subunit are required for surface delivery of GluN3A-containing NMDARs. Furthermore, deglycosylation and lectin-based analysis revealed that GluN3A subunits contain extensively modified N-glycan structures, including hybrid/complex forms of N-glycans. We also found (either using a panel of inhibitors or by studying human fibroblasts derived from patients with a congenital disorder of glycosylation) that N-glycan remodeling is not required for the surface delivery of GluN3A-containing NMDARs. Finally, we found that the surface mobility of GluN3A-containing NMDARs in hippocampal neurons is increased following incubation with 1-deoxymannojirimycin (DMM, an inhibitor of the formation of the hybrid/complex forms of N-glycans) and decreased in the presence of specific lectins. These findings provide new insight regarding the mechanisms by which neurons can regulate NMDAR trafficking and function.
RESUMEN
N-methyl-d-aspartate receptors (NMDARs) are ionotropic glutamate receptors that mediate excitatory neurotransmission in the mammalian central nervous system (CNS), and their dysregulation results in the aetiology of many CNS syndromes. Several NMDAR modulators have been used successfully in clinical trials (including memantine) and NMDARs remain a promising pharmacological target for the treatment of CNS syndromes. 1,2,3,4-Tetrahydro-9-aminoacridine (tacrine; THA) was the first approved drug for Alzheimer's disease (AD) treatment. 7-methoxyderivative of THA (7-MEOTA) is less toxic and showed promising results in patients with tardive dyskinesia. We employed electrophysiological recordings in HEK293â¯cells and rat neurones to examine the mechanism of action of THA and 7-MEOTA at the NMDAR. We showed that both THA and 7-MEOTA are "foot-in-the-door" open-channel blockers of GluN1/GluN2 receptors and that 7-MEOTA is a more potent but slower blocker than THA. We found that the IC50 values for THA and 7-MEOTA exhibited the GluN1/GluN2Aâ¯<â¯GluN1/GluN2Bâ¯<â¯GluN1/GluN2Câ¯=â¯GluN1/GluN2D relationship and that 7-MEOTA effectively inhibits human GluN1/GluN2A-M817V receptors that carry a pathogenic mutation. We also showed that 7-MEOTA is a "foot-in-the-door" open-channel blocker of GluN1/GluN3 receptors, although these receptors were not inhibited by memantine. In addition, the inhibitory potency of 7-MEOTA at synaptic and extrasynaptic hippocampal NMDARs was similar, and 7-MEOTA exhibited better neuroprotective activity when compared with THA and memantine in rats with NMDA-induced lesions of the hippocampus. Finally, intraperitoneal administration of 7-MEOTA attenuated MK-801-induced hyperlocomotion and pre-pulse inhibition deficit in rats. We conclude that 7-MEOTA may be considered for the treatment of diseases associated with the dysfunction of NMDARs.
Asunto(s)
Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Tacrina/análogos & derivados , Animales , Células Cultivadas , Maleato de Dizocilpina/antagonistas & inhibidores , Maleato de Dizocilpina/farmacología , Relación Dosis-Respuesta a Droga , Hipocampo/efectos de los fármacos , Humanos , Locomoción/efectos de los fármacos , Masculino , Memantina/farmacología , Mutación , Neuronas/fisiología , Inhibición Prepulso/efectos de los fármacos , Ratas , Receptores de N-Metil-D-Aspartato/genética , Tacrina/farmacologíaRESUMEN
The mechanism of tacrine as a precognitive drug has been considered to be complex and not fully understood. It has been reported to involve a wide spectrum of targets involving cholinergic, gabaergic, nitrinergic and glutamatergic pathways. Here, we review the effect of tacrine and its derivatives on the NMDA receptors (NMDAR) with a focus on the mechanism of action and biological consequences related to the Alzheimer's disease treatment. Our findings indicate that effect of tacrine on glutamatergic neurons is both direct and indirect. Direct NMDAR antagonistic effect is often reported by in vitro studies; however, it is achieved by high tacrine concentrations which are not likely to occur under clinical conditions. The impact on memory and behavioral testing can be ascribed to indirect effects of tacrine caused by influencing the NMDAR-mediated currents via M1 receptor activation, which leads to inhibition of Ca2+-activated potassium channels. Such inhibition prevents membrane repolarization leading to prolonged NMDAR activation and subsequently to long term potentiation. Considering these findings, we can conclude that tacrine-derivatives with dual cholinesterase and NMDARs modulating activity may represent a promising approach in the drug development for diseases associated with cognitive dysfunction, such as the Alzheimer disease.
Asunto(s)
Inhibidores de la Colinesterasa/farmacología , Neuronas/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Tacrina/farmacología , Animales , Humanos , Potenciales de la Membrana/efectos de los fármacos , Modelos Moleculares , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Tacrina/químicaRESUMEN
N-methyl-D-aspartate receptors (NMDARs) mediate synaptic plasticity, and their dysfunction is implicated in multiple brain disorders. NMDARs can be allosterically modulated by numerous compounds, including endogenous neurosteroid pregnanolone sulfate. Here, we identify the molecular basis of the use-dependent and voltage-independent inhibitory effect of neurosteroids on NMDAR responses. The site of action is located at the extracellular vestibule of the receptor's ion channel pore and is accessible after receptor activation. Mutations in the extracellular vestibule in the SYTANLAAF motif disrupt the inhibitory effect of negatively charged steroids. In contrast, positively charged steroids inhibit mutated NMDAR responses in a voltage-dependent manner. These results, in combination with molecular modeling, characterize structure details of the open configuration of the NMDAR channel. Our results provide a unique opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with dysfunction of the glutamate system.
Asunto(s)
Mutación , Pregnanolona , Receptores de N-Metil-D-Aspartato , Vestíbulo del Laberinto , Secuencias de Aminoácidos , Humanos , Pregnanolona/química , Pregnanolona/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Vestíbulo del Laberinto/química , Vestíbulo del Laberinto/metabolismoRESUMEN
N-methyl-D-aspartate (NMDA) receptors mediate fast excitatory synaptic transmission in the mammalian central nervous system. The activation of NMDA receptors plays a key role in brain development, synaptic plasticity, and memory formation, and is a major contributor to many neuropsychiatric disorders. Here, we investigated the mechanisms that underlie the trafficking of GluN1/GluN2C receptors. Using an approach combining molecular biology, microscopy, and electrophysiology in mammalian cell lines and cultured cerebellar granule cells, we found that the surface delivery of GluN2C-containing receptors is reduced compared to GluN2A- and GluN2B-containing receptors. Furthermore, we identified three distinct regions within the N-terminus, M3 transmembrane domain, and C-terminus of GluN2C subunits that are required for proper intracellular processing and surface delivery of NMDA receptors. These results shed new light on the regulation of NMDA receptor trafficking, and these findings can be exploited to develop new strategies for treating some forms of neuropsychiatric disorders.
RESUMEN
Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. There are three distinct subtypes of ionotropic glutamate receptors (GluRs) that have been identified including 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid receptors (AMPARs), N-methyl-D-aspartate receptors (NMDARs) and kainate receptors. The most common GluRs in mature synapses are AMPARs that mediate the fast excitatory neurotransmission and NMDARs that mediate the slow excitatory neurotransmission. There have been large numbers of recent reports studying how a single neuron regulates synaptic numbers and types of AMPARs and NMDARs. Our current research is centered primarily on NMDARs and, therefore, we will focus in this review on recent knowledge of molecular mechanisms occurring (1) early in the biosynthetic pathway of NMDARs, (2) in the transport of NMDARs after their release from the endoplasmic reticulum (ER); and (3) at the plasma membrane including excitatory synapses. Because a growing body of evidence also indicates that abnormalities in NMDAR functioning are associated with a number of human psychiatric and neurological diseases, this review together with other chapters in this issue may help to enhance research and to gain further knowledge of normal synaptic physiology as well as of the etiology of many human brain diseases.