RESUMEN
Peripheral inflammation induces sensitization of nociceptive spinal cord neurons. Both spinal tumor necrosis factor (TNF) and neuronal membrane insertion of Ca2+ permeable AMPA receptor (AMPAr) contribute to spinal sensitization and resultant pain behavior, molecular mechanisms connecting these two events have not been studied in detail. Intrathecal (i.t.) injection of TNF-blockers attenuated paw carrageenan-induced mechanical and thermal hypersensitivity. Levels of GluA1 and GluA4 from dorsal spinal membrane fractions increased in carrageenan-injected rats compared to controls. In the same tissue, GluA2 levels were not altered. Inflammation-induced increases in membrane GluA1 were prevented by i.t. pre-treatment with antagonists to TNF, PI3K, PKA and NMDA. Interestingly, administration of TNF or PI3K inhibitors followed by carrageenan caused a marked reduction in plasma membrane GluA2 levels, despite the fact that membrane GluA2 levels were stable following inhibitor administration in the absence of carrageenan. TNF pre-incubation induced increased numbers of Co2+ labeled dorsal horn neurons, indicating more neurons with Ca2+ permeable AMPAr. In parallel to Western blot results, this increase was blocked by antagonism of PI3K and PKA. In addition, spinal slices from GluA1 transgenic mice, which had a single alanine replacement at GluA1 ser 845 or ser 831 that prevented phosphorylation, were resistant to TNF-induced increases in Co2+ labeling. However, behavioral responses following intraplantar carrageenan and formalin in the mutant mice were no different from littermate controls, suggesting a more complex regulation of nociception. Co-localization of GluA1, GluA2 and GluA4 with synaptophysin on identified spinoparabrachial neurons and their relative ratios were used to assess inflammation-induced trafficking of AMPAr to synapses. Inflammation induced an increase in synaptic GluA1, but not GluA2. Although total GluA4 also increased with inflammation, co-localization of GluA4 with synaptophysin, fell short of significance. Taken together these data suggest that peripheral inflammation induces a PI3K and PKA dependent TNFR1 activated pathway that culminates with trafficking of calcium permeable AMPAr into synapses of nociceptive dorsal horn projection neurons.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Células del Asta Posterior/metabolismo , Radiculopatía/patología , Receptores AMPA/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Calcio/metabolismo , Carragenina/toxicidad , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Etanercept/uso terapéutico , Femenino , Masculino , Ratones , Células del Asta Posterior/patología , Células del Asta Posterior/ultraestructura , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Radiculopatía/inducido químicamente , Radiculopatía/tratamiento farmacológico , Ratas Sprague-Dawley , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Sinaptofisina/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Genetic factors significantly influence susceptibility for substance abuse and mood disorders. Rodent studies have begun to elucidate a role of Cav1.3 L-type Ca2+ channels in neuropsychiatric-related behaviors, such as addictive and depressive-like behaviors. Human studies have also linked the CACNA1D gene, which codes for the Cav1.3 protein, with bipolar disorder. However, the neurocircuitry and the molecular mechanisms underlying the role of Cav1.3 in neuropsychiatric phenotypes are not well established. In the present study, we directly manipulated Cav1.3 channels in Cav1.2 dihydropyridine insensitive mutant mice and found that ventral tegmental area (VTA) Cav1.3 channels mediate cocaine-related and depressive-like behavior through a common nucleus accumbens (NAc) shell calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (CP-AMPAR) mechanism that requires GluA1 phosphorylation at S831. Selective activation of VTA Cav1.3 with (±)-BayK-8644 (BayK) enhanced cocaine conditioned place preference and cocaine psychomotor activity while inducing depressive-like behavior, an effect not observed in S831A phospho-mutant mice. Infusion of the CP-AMPAR-specific blocker Naspm into the NAc shell reversed the cocaine and depressive-like phenotypes. In addition, activation of VTA Cav1.3 channels resulted in social behavioral deficits. In contrast to the cocaine- and depression-related phenotypes, GluA1/A2 AMPARs in the NAc core mediated social deficits, independent of S831-GluA1 phosphorylation. Using a candidate gene analysis approach, we also identified single-nucleotide polymorphisms in the CACNA1D gene associated with cocaine dependence in human subjects. Together, our findings reveal novel, overlapping mechanisms through which VTA Cav1.3 mediates cocaine-related, depressive-like and social phenotypes, suggesting that Cav1.3 may serve as a target for the treatment of neuropsychiatric symptoms.
Asunto(s)
Afecto/fisiología , Canales de Calcio Tipo L/metabolismo , Cocaína/farmacología , Inhibidores de Captación de Dopamina/farmacología , Núcleo Accumbens/metabolismo , Área Tegmental Ventral/metabolismo , Afecto/efectos de los fármacos , Animales , Canales de Calcio Tipo L/genética , Trastornos Relacionados con Cocaína/metabolismo , Condicionamiento Psicológico/efectos de los fármacos , Condicionamiento Psicológico/fisiología , Depresión/metabolismo , Modelos Animales de Enfermedad , Estudios de Asociación Genética , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Núcleo Accumbens/efectos de los fármacos , Densidad Postsináptica/efectos de los fármacos , Densidad Postsináptica/metabolismo , Receptores AMPA/metabolismo , Conducta Social , Área Tegmental Ventral/efectos de los fármacosRESUMEN
When chronically silenced, cortical and hippocampal neurons homeostatically upregulate excitatory synaptic function. However, the subcellular position of such changes on the dendritic tree is not clear. We exploited the cable-filtering properties of dendrites to derive a parameter, the dendritic filtering index (DFI), to map the spatial distribution of synaptic currents. Our analysis indicates that young rat cortical neurons globally scale AMPA receptor-mediated currents, while mature hippocampal neurons do not, revealing distinct homeostatic strategies between brain regions and developmental stages. The DFI presents a useful tool for mapping the dendritic origin of synaptic currents and the location of synaptic plasticity changes.
Asunto(s)
Dendritas/fisiología , Homeostasis/fisiología , Plasticidad Neuronal/fisiología , Animales , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/fisiología , Dendritas/efectos de los fármacos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Homeostasis/efectos de los fármacos , Inmunohistoquímica , Ratones Endogámicos C57BL , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Potenciales Postsinápticos Miniatura/fisiología , Plasticidad Neuronal/efectos de los fármacos , Técnicas de Placa-Clamp , Ratas Sprague-Dawley , Receptores AMPA , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacologíaRESUMEN
Synapse formation in the developing brain depends on the coordinated activity of synaptogenic proteins, some of which have been implicated in a number of neurodevelopmental disorders. Here, we show that the sushi repeat-containing protein X-linked 2 (SRPX2) gene encodes a protein that promotes synaptogenesis in the cerebral cortex. In humans, SRPX2 is an epilepsy- and language-associated gene that is a target of the foxhead box protein P2 (FoxP2) transcription factor. We also show that FoxP2 modulates synapse formation through regulating SRPX2 levels and that SRPX2 reduction impairs development of ultrasonic vocalization in mice. Our results suggest FoxP2 modulates the development of neural circuits through regulating synaptogenesis and that SRPX2 is a synaptogenic factor that plays a role in the pathogenesis of language disorders.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Trastornos del Lenguaje/genética , Lenguaje , Proteínas del Tejido Nervioso/fisiología , Sinapsis/fisiología , Vocalización Animal , Animales , Corteza Cerebral/citología , Epilepsia/genética , Factores de Transcripción Forkhead/genética , Humanos , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Proteínas de Neoplasias , Proteínas del Tejido Nervioso/genética , Neuronas/fisiología , TransfecciónRESUMEN
The GluA4-containing Ca(2+)-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (Ca-AMPARs) were previously shown to mediate excitotoxicity through mechanisms involving the activator protein-1 (AP-1), a c-Jun N-terminal kinase (JNK) substrate. To further investigate JNK involvement in excitotoxic pathways coupled to Ca-AMPARs we used HEK293 cells expressing GluA4-containing Ca-AMPARs (HEK-GluA4). Cell death induced by overstimulation of Ca-AMPARs was mediated, at least in part, by JNK. Importantly, JNK activation downstream of these receptors was dependent on the extracellular Ca(2+) concentration. In our quest for a molecular link between Ca-AMPARs and the JNK pathway we found that the JNK interacting protein-1 (JIP-1) interacts with the GluA4 subunit of AMPARs through the N-terminal domain. In vivo, the excitotoxin kainate promoted the association between GluA4 and JIP-1 in the rat hippocampus. Taken together, our results show that the JNK pathway is activated by Ca-AMPARs upon excitotoxic stimulation and suggest that JIP-1 may contribute to the propagation of the excitotoxic signal.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Calcio/metabolismo , Activación Enzimática/fisiología , MAP Quinasa Quinasa 4/metabolismo , Receptores AMPA/metabolismo , Transducción de Señal/fisiología , Animales , Western Blotting , Electroforesis en Gel de Poliacrilamida , Activación Enzimática/efectos de los fármacos , Agonistas de Aminoácidos Excitadores/farmacología , Células HEK293 , Humanos , Inmunoprecipitación , Ácido Kaínico/farmacología , Masculino , Ratas , Ratas Wistar , Receptores AMPA/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , TransfecciónRESUMEN
Synaptic development is an activity-dependent process utilizing coordinated network activity to drive synaptogenesis and subsequent refinement of immature connections. Hippocampal CA3 pyramidal neurons (PYRs) exhibit intense burst firing (BF) early in development, concomitant with the period of mossy fibre (MF) development. However, whether developing MF-PYR synapses utilize PYR BF to promote MF synapse maturation remains unknown. Recently, we demonstrated that transient tonic depolarization of postsynaptic PYRs induces a persistent postsynaptic form of long-term depression (depolarization-induced long-term depression, DiLTD) at immature MF-PYR synapses. DiLTD induction is NMDAR independent but does require postsynaptic Ca(2+) influx through L-type voltage gated Ca(2+) channels (L-VGCCs), and is expressed as a reduction in AMPAR function through the loss of GluR2-lacking AMPARs present at immature MF-PYR synapses. Here we examined whether more physiologically relevant phasic L-VGCC activation by PYR action potential (AP) BF activity patterns can trigger DiLTD. Using combined electrophysiological and Ca(2+) imaging approaches we demonstrate that PYR BF effectively drives L-VGCC activation and that brief periods of repetitive PYR BF, produced by direct current injection or intrinsic network activity induces NMDAR-independent LTD by promoting Ca(2+) influx through the activated L-VGCCs. This BF induced LTD, just like DiLTD, is specific for developing MF-PYR synapses, is PICK1 dependent, and is expressed postsynaptically. Our results demonstrate that DiLTD can be induced by phasic L-VGCC activation driven by PYR BF, suggesting the engagement of natural PYR network activity patterns for MF synapse maturation.
Asunto(s)
Potenciales de Acción/fisiología , Relojes Biológicos/fisiología , Depresión Sináptica a Largo Plazo/fisiología , Fibras Musgosas del Hipocampo/fisiología , Células Piramidales/fisiología , Sinapsis/fisiología , Potenciales Sinápticos/fisiología , Animales , Células Cultivadas , Ratones , Ratones Endogámicos C57BLRESUMEN
Neuronal activity regulated pentraxin (Narp) is a secreted, synaptic protein that has been implicated in modulating synaptic transmission. However, it is unclear how Narp secretion is regulated. Since we noted prominent Narp immunostaining in vasopressin neurons of the hypothalamus and in the posterior pituitary, we assessed whether it, like vasopressin, is released into the systemic circulation in an activity-dependent fashion. Consistent with this hypothesis, electron microscopic studies of the posterior pituitary demonstrated that Narp is located in secretory vesicles containing vasopressin. Using affinity chromatography, we detected Narp in plasma and found that these levels are markedly decreased by hypophysectomy. In addition, we confirmed that injection of a viral Narp construct into the hypothalamus restores plasma Narp levels in Narp knockout mice. In checking for activity-dependent secretion of Narp from the posterior pituitary, we found that several stimuli known to trigger vasopressin release, i.e. hypovolemia, dehydration and endotoxin, elevate plasma Narp levels. Taken together, these findings provide compelling evidence that Narp is secreted from vasopressin neurons in an activity-dependent fashion.
Asunto(s)
Proteína C-Reactiva/metabolismo , Actividad Motora/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuronas/fisiología , Vasopresinas/fisiología , Adenoviridae/genética , Animales , Cromatografía de Afinidad , ADN Complementario/biosíntesis , ADN Complementario/genética , Deshidratación/fisiopatología , Vectores Genéticos , Humanos , Hipovolemia/fisiopatología , Inmunohistoquímica , Lipopolisacáridos/toxicidad , Ratones , Ratones Noqueados , Microscopía Electrónica , Microscopía Inmunoelectrónica , Proteínas del Tejido Nervioso/sangre , Hipófisis/metabolismo , Ratas , Ratas Sprague-Dawley , Restricción Física , Estrés Psicológico/metabolismo , Estrés Psicológico/fisiopatologíaRESUMEN
Accumulating evidence from both genetic and clinico-pharmacological studies suggests that D-serine, an endogenous coagonist to the NMDA subtype glutamate receptor, may be implicated in schizophrenia (SZ). Although an association of genes for D-serine degradation, such as D-amino acid oxidase and G72, has been reported, a role for D-serine in SZ has been unclear. In this study, we identify and characterize protein interacting with C-kinase (PICK1) as a protein interactor of the D-serine synthesizing enzyme, serine racemase (SR). The binding of endogenous PICK1 and SR requires the PDZ domain of PICK1. The gene coding for PICK1 is located at chromosome 22q13, a region frequently linked to SZ. In a case-control association study using well-characterized Japanese subjects, we observe an association of the PICK1 gene with SZ, which is more prominent in disorganized SZ. Our findings implicating PICK1 as a susceptibility gene for SZ are consistent with a role for D-serine in the disease.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Nucleares/metabolismo , Racemasas y Epimerasas/metabolismo , Esquizofrenia/enzimología , Esquizofrenia/genética , Serina/metabolismo , Adulto , Animales , Astrocitos/metabolismo , Proteínas Portadoras/genética , Estudios de Casos y Controles , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , Ratones , Persona de Mediana Edad , Proteínas Nucleares/genética , Polimorfismo de Nucleótido Simple , Esquizofrenia/clasificación , Serina/biosíntesisRESUMEN
Caspases are proteases involved in various physiological and pathological processes in the nervous system, including development and pathogenesis. GRASP-1 is a recently identified neuronal substrate of caspase-3-subfamily caspases. It is a Ras-guanine nucleotide exchange factor (RasGEF) that interacts with the glutamate receptor interacting protein (GRIP). This alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor/GRIP protein complex has been proposed to be involved in AMPA receptor synaptic targeting. The caspase-3 cleavage of GRASP-1 separates the N-terminal RasGEF catalytic domain from the C-terminal GRIP-interacting region, potentially disrupting regulation of the RasGEF activity by GRIP. To examine the regulation and regional distribution of the caspase-3 cleavage of GRASP-1 in vivo, we generated a cleavage site-specific antibody, termed CGP, against the cleaved N-terminal fragment of GRASP-1. Using this antibody, we have examined the caspase cleavage of GRASP-1 during postnatal development and following ischemia in mice. We found that caspase cleavage of GRASP-1 occurs in specific brain regions in a time-dependent manner during development and ischemia. This data provides an important account of the brain areas that might require caspase-3 activity in postnatal development and ischemic damage, which has not been documented. It also demonstrates that the CGP antibody is a powerful tool for studying neuronal activity of the caspase-3-subfamily caspases in vivo.
Asunto(s)
Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Caspasas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Receptores AMPA/metabolismo , Factores de Intercambio de Guanina Nucleótido ras/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Animales Recién Nacidos , Especificidad de Anticuerpos/inmunología , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatología , Caspasa 3 , Dominio Catalítico/fisiología , Muerte Celular/fisiología , Células Cultivadas , Lateralidad Funcional/fisiología , Inmunohistoquímica , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/fisiopatología , Proteínas de la Membrana , Ratones , Neostriado/metabolismo , Neostriado/fisiopatología , Neuronas/citología , Estructura Terciaria de Proteína/fisiologíaRESUMEN
Phosphorylation of AMPA receptor subunits is believed to regulate channel function and synaptic plasticity. Extensive biochemical and molecular studies have identified sites of PKA, PKC and CamKII phosphorylation in the C-termini of the GluR1 and 4 subunits. Recent studies have shown GluR1 phosphorylation to be bidirectionally altered during long-term potentiation (LTP) and long-term depression (LTD) in the hippocampus. The majority of AMPA receptors in the brain are believed to contain the GluR2 subunit that also contains potential sites for protein phosphorylation. Here we characterize PKC phosphorylation on the GluR2 subunit using biochemical and molecular techniques. Site-directed mutagenesis confirmed that this phosphorylation occurs on Serine 863 and Serine 880 of the GluR2 subunit C-terminus. Site identification allowed the generation of phosphorylation site-specific antibodies to facilitate the examination of GluR2 modification in primary neuronal culture. These studies confirmed that GluR2 is modified in response to the activation of PKC and suggests that phosphorylation of the ubiquitous GluR2 subunit may be important in the regulation of excitatory synaptic transmission.
Asunto(s)
Proteína Quinasa C/metabolismo , Receptores AMPA/metabolismo , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Línea Celular , Sistema Libre de Células/enzimología , Sistema Libre de Células/metabolismo , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/enzimología , Corteza Cerebral/metabolismo , Mapeo Epitopo/métodos , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Proteína Quinasa C/aislamiento & purificación , Conejos , Ratas , Receptores AMPA/genética , Receptores AMPA/inmunología , Serina/metabolismo , TransfecciónRESUMEN
The interaction of PDZ domain-containing proteins with the C termini of alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) receptors has been suggested to be important in the regulation of receptor targeting to excitatory synapses. Recent studies have shown that the rapid internalization of AMPA receptors at synapses may mediate, at least in part, the expression of long-term depression (LTD). We have previously shown that phosphorylation of Ser-880 on the AMPA receptor GluR2 subunit differentially regulated the interaction of GluR2 with the PDZ domain-containing proteins GRIP1 and PICK1. Here, we show that induction of LTD in hippocampal slices increases phosphorylation of Ser-880 within the GluR2 C-terminal PDZ ligand, suggesting that the modulation of GluR2 interaction with GRIP1 and PICK1 may regulate AMPA receptor internalization during LTD. Moreover, postsynaptic intracellular perfusion of GluR2 C-terminal peptides that disrupt GluR2 interaction with PICK1 inhibit the expression of hippocampal LTD. These results suggest that the interaction of GluR2 with PICK1 may play a regulatory role in the expression of LTD in the hippocampus.
Asunto(s)
Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/fisiología , Plasticidad Neuronal/fisiología , Receptores AMPA/fisiología , 2-Amino-5-fosfonovalerato/farmacología , Animales , Carbazoles/farmacología , Dendritas/efectos de los fármacos , Dendritas/fisiología , Inhibidores Enzimáticos/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Indoles/farmacología , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/fisiología , Masculino , Plasticidad Neuronal/efectos de los fármacos , Ácido Ocadaico/farmacología , Ésteres del Forbol/farmacología , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Subunidades de Proteína , Ratas , Ratas Sprague-Dawley , Receptores AMPA/efectos de los fármacos , Serina/metabolismoRESUMEN
Excitatory post-synaptic currents in the CNS are primarily mediated by alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors in response to glutamate. Internalization of cell-surface receptors has been shown to be one mechanism by which to control receptor function. To test for agonist control of AMPA receptor plasma membrane expression we used biochemical assays to study AMPA receptor internalization and insertion processes. In heterologous cells, we observed a slow constitutive internalization and a rapid agonist-induced internalization of AMPA receptors. To our surprise, however, agonist treatment had no effect on the steady-state levels of AMPA receptors on the cell surface. To examine whether this could be explained by an agonist-induced increase in the insertion rate of AMPA receptors into the plasma membrane we developed an assay to independently measure receptor insertion. Remarkably, agonist treatment of cells also dramatically increased AMPA receptor plasma membrane insertion rates. In addition, using an assay to measure recycling of internalized pools we found that internalized receptors are rapidly recycled to the cell surface. These results suggest that agonist-induced receptor internalization is coupled to increases in receptor recycling. This increase in receptor flux through intracellular pools may allow for rapid changes in receptor surface expression by independent regulatory control of internalization and insertion.
Asunto(s)
Membrana Celular/metabolismo , Agonistas de Aminoácidos Excitadores/farmacología , Potenciación a Largo Plazo/fisiología , Plasticidad Neuronal/fisiología , Receptores AMPA/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacología , Línea Celular , Humanos , Riñón/citología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Receptores AMPA/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , TransfecciónRESUMEN
Spine function requires precise control of the actin cytoskeleton. Kalirin-7, a GDP/GTP exchange factor for Rac1, interacts with PDZ proteins such as PSD-95, colocalizing with PSD-95 at synapses of cultured hippocampal neurons. PSD-95 and Kalirin-7 interact in vivo and in heterologous expression systems. In primary cortical neurons, transfected Kalirin-7 is targeted to spines and increases the number and size of spine-like structures. A Kalirin-7 mutant unable to interact with PDZ proteins remains in the cell soma, inducing local formation of aberrant filopodial neurites. Kalirin-7 with an inactivated GEF domain reduces the number of spines below control levels. These results provide evidence that PDZ proteins target Kalirin-7 to the PSD, where it regulates dendritic morphogenesis through Rac1 signaling to the actin cytoskeleton.
Asunto(s)
Proteínas Portadoras , Dendritas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Neuronas/metabolismo , Actinas/metabolismo , Secuencias de Aminoácidos/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión/fisiología , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Dendritas/ultraestructura , Homólogo 4 de la Proteína Discs Large , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Hipocampo/citología , Hipocampo/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Morfogénesis/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/ultraestructura , Estructura Terciaria de Proteína/fisiología , Ratas , Ratas Sprague-Dawley , Sinapsis/metabolismo , Transfección , Técnicas del Sistema de Dos Híbridos , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Phosphorylation is one of the principal regulatory mechanisms in the nervous system. Several different procedures used to characterize the phosphorylation state of neuronal proteins are described in this unit, including analysis of phosphorylation in situ, phosphoamino acid analysis, and phosphopeptide map analysis. In addition, there is a protocol describing in vitro phosphorylation of fusion proteins. These methods are often combined to provide a comprehensive evaluation of the phosphorylation state of a particular protein.
Asunto(s)
Mapeo Peptídico/métodos , Fosfoproteínas/metabolismo , Animales , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/fisiología , Células Cultivadas , Humanos , Fosfopéptidos/genética , Fosfopéptidos/metabolismo , Fosfoproteínas/genética , FosforilaciónRESUMEN
The synaptic localization, clustering, and immobilization of neurotransmitter receptors and ion channels play important roles in synapse formation and synaptic transmission. Although several proteins have been identified that interact with AMPA receptors and that may regulate their synaptic targeting, little is known about the interaction of AMPA receptors with the cytoskeleton. In studies examining the interaction of the AMPA receptor GluR1 subunit with neuronal proteins, we determined that GluR1 interacts with the 4.1G and 4.1N proteins, homologs of the erythrocyte membrane cytoskeletal protein 4.1. Using the yeast two-hybrid system and a heterologous cell system, we demonstrated that both 4.1G and 4.1N bind to a membrane proximal region of the GluR1 C terminus, and that a region within the C-terminal domain of 4.1G or 4.1N is sufficient to mediate the interaction. We also found that 4.1N can associate with GluR1 in vivo and colocalizes with AMPA receptors at excitatory synapses. Disruption of the interaction of GluR1 with 4.1N or disruption of actin filaments decreased the surface expression of GluR1 in heterologous cells. Moreover, disruption of actin filaments in cultured cortical neurons dramatically reduced the level of surface AMPA receptors. These results suggest that protein 4.1N may link AMPA receptors to the actin cytoskeleton.
Asunto(s)
Membrana Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Neuropéptidos/metabolismo , Receptores AMPA/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Animales , Sitios de Unión/genética , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Células COS , Células Cultivadas , Proteínas del Citoesqueleto/genética , Humanos , Immunoblotting , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutagénesis Sitio-Dirigida , Neuropéptidos/genética , Pruebas de Precipitina , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Ratas , Ratas Sprague-Dawley , Receptores AMPA/genética , Sinapsis/metabolismo , Tiazoles/farmacología , Tiazolidinas , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
PSD-95, DLG, ZO-1 (PDZ) domain-mediated protein interactions have been shown to play important roles in the regulation of glutamate receptor function at excitatory synapses. Recent studies demonstrating the rapid regulation of AMPA receptor function during synaptic plasticity have suggested that AMPA receptor interaction with PDZ domain-containing proteins may be dynamically modulated. Here we show that PKC phosphorylation of the AMPA receptor GluR2 subunit differentially modulates its interaction with the PDZ domain-containing proteins GRIP1 and PICK1. The serine residue [serine-880 (Ser880)] in the GluR2 C-terminal sequence (IESVKI) critical for PDZ domain binding is a substrate of PKC and is phosphorylated in vivo. In vitro binding and coimmunoprecipitation studies show that phosphorylation of serine-880 within the GluR2 PDZ ligand significantly decreases GluR2 binding to GRIP1 but not to PICK1. Immunostaining of cultured hippocampal neurons demonstrates that the Ser880-phosphorylated GluR2 subunits are enriched and colocalized with PICK1 in the dendrites, with very little staining observed at excitatory synapses. Interestingly, PKC activation in neurons increases the Ser880 phosphorylation of GluR2 subunits and recruits PICK1 to excitatory synapses. Moreover, PKC stimulation in neurons results in rapid internalization of surface GluR2 subunits. These results suggest that GluR2 phosphorylation of serine-880 may be important in the regulation of the AMPA receptor internalization during synaptic plasticity.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Receptores AMPA/metabolismo , Animales , Proteínas Portadoras/metabolismo , Células Cultivadas , Proteínas del Citoesqueleto , Humanos , Riñón/citología , Riñón/metabolismo , Ligandos , Mutagénesis Sitio-Dirigida , Plasticidad Neuronal/genética , Neuronas/citología , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Coactivador 2 del Receptor Nuclear , Fosforilación/efectos de los fármacos , Proteína Quinasa C/metabolismo , Estructura Terciaria de Proteína , Ratas , Receptores AMPA/genética , Serina/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Factores de Transcripción/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Compartmentalization of glutamate receptors with the signaling enzymes that regulate their activity supports synaptic transmission. Two classes of binding proteins organize these complexes: the MAGUK proteins that cluster glutamate receptors and AKAPs that anchor kinases and phosphatases. In this report, we demonstrate that glutamate receptors and PKA are recruited into a macromolecular signaling complex through direct interaction between the MAGUK proteins, PSD-95 and SAP97, and AKAP79/150. The SH3 and GK regions of the MAGUKs mediate binding to the AKAP. Cell-based studies indicate that phosphorylation of AMPA receptors is enhanced by a SAP97-AKAP79 complex that directs PKA to GluR1 via a PDZ domain interaction. As AMPA receptor phosphorylation is implicated in regulating synaptic plasticity, these data suggest that a MAGUK-AKAP complex may be centrally involved.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Nucleósido-Fosfato Quinasa/metabolismo , Receptores de Glutamato/metabolismo , Transducción de Señal/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Unión Competitiva/fisiología , Western Blotting , Homólogo 4 de la Proteína Discs Large , Guanilato-Quinasas , Hipocampo/citología , Hipocampo/metabolismo , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Microscopía Confocal , Proteínas del Tejido Nervioso/fisiología , Neuronas/metabolismo , Pruebas de Precipitina , Unión Proteica/fisiología , Ratas , Receptores AMPA/metabolismo , Receptores AMPA/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
The PDZ domain-containing proteins, such as PSD-95 and GRIP, have been suggested to be involved in the targeting of glutamate receptors, a process that plays a critical role in the efficiency of synaptic transmission and plasticity. To address the molecular mechanisms underlying AMPA receptor synaptic localization, we have identified several GRIP-associated proteins (GRASPs) that bind to distinct PDZ domains within GRIP. GRASP-1 is a neuronal rasGEF associated with GRIP and AMPA receptors in vivo. Overexpression of GRASP-1 in cultured neurons specifically reduced the synaptic targeting of AMPA receptors. In addition, the subcellular distribution of both AMPA receptors and GRASP-1 was rapidly regulated by the activation of NMDA receptors. These results suggest that GRASP-1 may regulate neuronal ras signaling and contribute to the regulation of AMPA receptor distribution by NMDA receptor activity.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Isoformas de Proteínas/metabolismo , Receptores AMPA/metabolismo , Factores de Intercambio de Guanina Nucleótido ras/metabolismo , Secuencia de Aminoácidos/genética , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Caspasa 3 , Caspasas/metabolismo , Clonación Molecular , Péptidos y Proteínas de Señalización Intracelular , Masculino , Proteínas de la Membrana , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/fisiología , Especificidad por Sustrato , Sinapsis/metabolismo , Distribución Tisular , Factores de Intercambio de Guanina Nucleótido ras/genéticaRESUMEN
Bidirectional changes in the efficacy of neuronal synaptic transmission, such as hippocampal long-term potentiation (LTP) and long-term depression (LTD), are thought to be mechanisms for information storage in the brain. LTP and LTD may be mediated by the modulation of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazloe proprionic acid) receptor phosphorylation. Here we show that LTP and LTD reversibly modify the phosphorylation of the AMPA receptor GluR1 subunit. However, contrary to the hypothesis that LTP and LTD are the functional inverse of each other, we find that they are associated with phosphorylation and dephosphorylation, respectively, of distinct GluR1 phosphorylation sites. Moreover, the site modulated depends on the stimulation history of the synapse. LTD induction in naive synapses dephosphorylates the major cyclic-AMP-dependent protein kinase (PKA) site, whereas in potentiated synapses the major calcium/calmodulin-dependent protein kinase II (CaMKII) site is dephosphorylated. Conversely, LTP induction in naive synapses and depressed synapses increases phosphorylation of the CaMKII site and the PKA site, respectively. LTP is differentially sensitive to CaMKII and PKA inhibitors depending on the history of the synapse. These results indicate that AMPA receptor phosphorylation is critical for synaptic plasticity, and that identical stimulation conditions recruit different signal-transduction pathways depending on synaptic history.
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Plasticidad Neuronal/fisiología , Receptores AMPA/metabolismo , Sinapsis/fisiología , Animales , Sitios de Unión , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Electrofisiología , Inhibidores Enzimáticos/farmacología , Hipocampo/metabolismo , Hipocampo/fisiología , Técnicas In Vitro , Potenciación a Largo Plazo/fisiología , Masculino , Ratones , Modelos Neurológicos , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Ratas , Ratas Long-Evans , Serina/metabolismo , Transducción de SeñalRESUMEN
The activation of cAMP-dependent protein kinase regulates the physiological activity of AMPA-type glutamate receptors. In this study, phosphorylation of the AMPA receptor subunit GluR1 at Ser(845) was increased in neostriatal slices by activation of D1-type dopamine receptors and by inhibitors of protein phosphatase 1/protein phosphatase 2A. In contrast, Ser(831), a residue which, when phosphorylated by protein kinase C or calcium/calmodulin-dependent kinase II, increases AMPA receptor channel conductance, was unaffected by either D1 or D2 receptor agonists in neostriatal slices. The phosphorylation of Ser(845), but not Ser(831), was strongly increased in neostriatum in vivo in response to the psychostimulants cocaine and methamphetamine. The effects of dopamine and psychostimulants on the phosphorylation of GluR1 were attenuated in dopamine and cAMP-regulated phosphoprotein M(r) 32 kDa (DARPP-32) knock-out mice. These results identify DARPP-32 and AMPA-type glutamate receptors as likely essential cellular effectors for psychostimulant actions.