RESUMEN
Cortical function relies on the balanced activation of excitatory and inhibitory neurons. However, little is known about the organization and dynamics of shaft excitatory synapses onto cortical inhibitory interneurons. Here, we use the excitatory postsynaptic marker PSD-95, fluorescently labeled at endogenous levels, as a proxy for excitatory synapses onto layer 2/3 pyramidal neurons and parvalbumin-positive (PV+) interneurons in the barrel cortex of adult mice. Longitudinal in vivo imaging under baseline conditions reveals that, although synaptic weights in both neuronal types are log-normally distributed, synapses onto PV+ neurons are less heterogeneous and more stable. Markov model analyses suggest that the synaptic weight distribution is set intrinsically by ongoing cell-type-specific dynamics, and substantial changes are due to accumulated gradual changes. Synaptic weight dynamics are multiplicative, i.e., changes scale with weights, although PV+ synapses also exhibit an additive component. These results reveal that cell-type-specific processes govern cortical synaptic strengths and dynamics.
Asunto(s)
Homólogo 4 de la Proteína Discs Large/fisiología , Potenciales Postsinápticos Excitadores/fisiología , Interneuronas/fisiología , Inhibición Neural , Parvalbúminas/metabolismo , Células Piramidales/fisiología , Sinapsis/fisiología , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Plasticidad NeuronalRESUMEN
Hypobaric hypoxia (HH) is a typical characteristic of high altitude environment and causes a spectrum of pathophysiological effects, including headaches, gliovascular dysfunction and cognitive retardation. Here, we sought to understand the mechanisms underlying cognitive deficits under HH exposure. Our results showed that hypobaric hypoxia exposure impaired cognitive function and suppressed dendritic spine density accompanied with increased neck length in both basal and apical hippocampal CA1 region neurons in mice. The expression of PSD95, a vital synaptic scaffolding molecule, is down-regulated by hypobaric hypoxia exposure and post-transcriptionally regulated by cold-inducible RNA-binding protein (Cirbp) through 3'-UTR region binding. PSD95 expressing alleviates hypoxia-induced dendritic spine morphology changes of hippocampal neurons and memory deterioration. Moreover, overexpressed Cirbp in hippocampus rescues HH-induced abnormal expression of PSD95 and attenuates hypoxia-induced dendritic spine injury and cognitive retardation. Thus, our findings reveal a novel mechanism that Cirbp-PSD-95 axis appears to play an essential role in HH-induced cognitive dysfunction in mice.
Asunto(s)
Mal de Altura/fisiopatología , Región CA1 Hipocampal/patología , Trastornos del Conocimiento/prevención & control , Espinas Dendríticas/ultraestructura , Homólogo 4 de la Proteína Discs Large/fisiología , Proteínas de Unión al ARN/fisiología , Regiones no Traducidas 3' , Animales , Reacción de Prevención , Secuencia de Bases , Células Cultivadas , Trastornos del Conocimiento/etiología , Homólogo 4 de la Proteína Discs Large/biosíntesis , Homólogo 4 de la Proteína Discs Large/genética , Regulación de la Expresión Génica , Genes Reporteros , Vectores Genéticos/administración & dosificación , Trastornos de la Memoria/etiología , Trastornos de la Memoria/prevención & control , Ratones , Ratones Endogámicos C57BL , Prueba del Laberinto Acuático de Morris , Neuronas/fisiología , Neuronas/ultraestructura , Prueba de Campo Abierto , Proteínas de Unión al ARN/biosíntesis , Proteínas de Unión al ARN/genética , Distribución Aleatoria , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Long-term depression (LTD) of synaptic strength can take multiple forms and contribute to circuit remodeling, memory encoding or erasure. The generic term LTD encompasses various induction pathways, including activation of NMDA, mGlu or P2X receptors. However, the associated specific molecular mechanisms and effects on synaptic physiology are still unclear. We here compare how NMDAR- or P2XR-dependent LTD affect synaptic nanoscale organization and function in rodents. While both LTDs are associated with a loss and reorganization of synaptic AMPARs, only NMDAR-dependent LTD induction triggers a profound reorganization of PSD-95. This modification, which requires the autophagy machinery to remove the T19-phosphorylated form of PSD-95 from synapses, leads to an increase in AMPAR surface mobility. We demonstrate that these post-synaptic changes that occur specifically during NMDAR-dependent LTD result in an increased short-term plasticity improving neuronal responsiveness of depressed synapses. Our results establish that P2XR- and NMDAR-mediated LTD are associated to functionally distinct forms of LTD.
Asunto(s)
Homólogo 4 de la Proteína Discs Large/fisiología , Depresión Sináptica a Largo Plazo/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Adenosina Trifosfato/administración & dosificación , Animales , Autofagia/fisiología , Células Cultivadas , Homólogo 4 de la Proteína Discs Large/deficiencia , Femenino , Hipocampo/citología , Hipocampo/fisiología , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Potenciales Postsinápticos Miniatura/fisiología , Modelos Neurológicos , N-Metilaspartato/administración & dosificación , Plasticidad Neuronal/fisiología , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Ratas , Ratas Sprague-Dawley , Receptores AMPA/fisiología , Receptores Purinérgicos P2X/fisiologíaRESUMEN
Cognitive processes that require spatial information rely on synaptic plasticity in the dorsal CA1 area (dCA1) of the hippocampus. Since the function of the hippocampus is impaired in aged individuals, it remains unknown how aged animals make spatial choices. Here, we used IntelliCage to study behavioral processes that support spatial choices of aged female mice living in a group. As a proxy of training-induced synaptic plasticity, we analyzed the morphology of dendritic spines and the expression of a synaptic scaffold protein, PSD-95. We observed that spatial choice training in young adult mice induced correlated shrinkage of dendritic spines and downregulation of PSD-95 in dCA1. Moreover, long-term depletion of PSD-95 by shRNA in dCA1 limited correct choices to a reward corner, while reward preference was intact. In contrast, old mice used behavioral strategies characterized by an increased tendency for perseverative visits and social interactions. This strategy resulted in a robust preference for the reward corner during the spatial choice task. Moreover, training decreased the correlation between PSD-95 expression and the size of dendritic spines. Furthermore, PSD-95 depletion did not impair place choice or reward preference in old mice. Thus, our data indicate that while young mice require PSD-95-dependent synaptic plasticity in dCA1 to make correct spatial choices, old animals observe cage mates and stick to a preferred corner to seek the reward. This strategy is resistant to the depletion of PSD-95 in the CA1 area. Overall, our study demonstrates that aged mice combine alternative behavioral and molecular strategies to approach and consume rewards in a complex environment.SIGNIFICANCE STATEMENT It remains poorly understood how aging affects behavioral and molecular processes that support cognitive functions. It is, however, essential to understand these processes to develop therapeutic interventions that support successful cognitive aging. Our data indicate that while young mice require PSD-95-dependent synaptic plasticity in dCA1 to make correct spatial choices (i.e., choices that require spatial information), old animals observe cage mates and stick to a preferred corner to seek the reward. This strategy is resistant to the depletion of PSD-95 in the CA1 area. Overall, our study demonstrates that aged mice combine alternative behavioral and molecular strategies to approach and consume rewards in a complex environment. Second, the contribution of PSD-95-dependent synaptic functions in spatial choice changes with age.
Asunto(s)
Región CA1 Hipocampal/fisiología , Conducta de Elección/fisiología , Homólogo 4 de la Proteína Discs Large/fisiología , Percepción Espacial/fisiología , Envejecimiento/fisiología , Envejecimiento/psicología , Animales , Espinas Dendríticas/fisiología , Homólogo 4 de la Proteína Discs Large/genética , Ambiente , Femenino , Regulación de la Expresión Génica/genética , Ratones , Ratones Endogámicos C57BL , Plasticidad Neuronal/fisiología , Recompensa , Interacción SocialRESUMEN
α-Synuclein (α-syn)-induced neurotoxicity has been generally accepted as a key step in the pathogenesis of Parkinson's disease (PD). Microtubule-associated protein tau, which is considered second only to α-syn, has been repeatedly linked with PD in association studies. However, the underlying interaction between these two PD-related proteins in vivo remains unclear. To investigate how the expression of tau affects α-syn-induced neurodegeneration in vivo, we generated triple transgenic mice that overexpressed α-syn A53T mutation in the midbrain dopaminergic neurons (mDANs) with different expression levels of tau. Here, we found that tau had no significant effect on the A53T α-syn-mediated mDANs degeneration. However, tau knockout could modestly promote the formation of α-syn aggregates, accelerate the severe and progressive degeneration of parvalbumin-positive (PV+) neurons in substantia nigra pars reticulata (SNR), accompanied with anxiety-like behavior in aged PD-related α-syn A53T mice. The mechanisms may be associated with A53T α-syn-mediated specifically successive impairment of N-methyl-d-aspartate receptor subunit 2B (NR2B), postsynaptic density-95 (PSD-95) and microtubule-associated protein 1A (MAP1A) in PV+ neurons. Our study indicates that MAP1A may play a beneficial role in preserving the survival of PV+ neurons, and that inhibition of the impairment of NR2B/PSD-95/MAP1A pathway, may be a novel and preferential option to ameliorate α-syn-induced neurodegeneration.
Asunto(s)
Mutación , Degeneración Nerviosa , Enfermedad de Parkinson/etiología , Parvalbúminas/análisis , Sustancia Negra/patología , alfa-Sinucleína/genética , Proteínas tau/fisiología , Animales , Homólogo 4 de la Proteína Discs Large/fisiología , Proteínas de Homeodominio/fisiología , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/fisiología , Enfermedad de Parkinson/patología , Fragmentos de Péptidos/fisiología , Agregado de Proteínas , Receptores de N-Metil-D-Aspartato/fisiología , Factores de Transcripción/fisiología , alfa-Sinucleína/fisiología , Proteínas tau/química , Proteínas tau/genéticaRESUMEN
Determining the number of synapses that are present in different brain regions is crucial to understand brain connectivity as a whole. Membrane-associated guanylate kinases (MAGUKs) are a family of scaffolding proteins that are expressed in excitatory glutamatergic synapses. We used genetic labeling of two of these proteins (PSD95 and SAP102), and Spinning Disc confocal Microscopy (SDM), to estimate the number of fluorescent puncta in the CA1 area of the hippocampus. We also used FIB-SEM, a three-dimensional electron microscopy technique, to calculate the actual numbers of synapses in the same area. We then estimated the ratio between the three-dimensional densities obtained with FIB-SEM (synapses/µm3) and the bi-dimensional densities obtained with SDM (puncta/100 µm2). Given that it is impractical to use FIB-SEM brain-wide, we used previously available SDM data from other brain regions and we applied this ratio as a conversion factor to estimate the minimum density of synapses in those regions. We found the highest densities of synapses in the isocortex, olfactory areas, hippocampal formation and cortical subplate. Low densities were found in the pallidum, hypothalamus, brainstem and cerebellum. Finally, the striatum and thalamus showed a wide range of synapse densities.
Asunto(s)
Encéfalo/fisiología , Homólogo 4 de la Proteína Discs Large/fisiología , Guanilato-Quinasas/fisiología , Hipocampo/fisiología , Proteínas de la Membrana/fisiología , Sinapsis/fisiología , Animales , Encéfalo/ultraestructura , Hipocampo/ultraestructura , Masculino , Ratones , Ratones Noqueados , Microscopía Electrónica , Sinapsis/ultraestructuraRESUMEN
BACKGROUND: Experimental evidence shows postnatal exposure to anesthesia negatively affects brain development. The PDZ2 domain, mediating protein-protein interactions of the postsynaptic density-95 protein, serves as a molecular target for several inhaled anesthetics. The authors hypothesized that early postnatal disruption of postsynaptic density-95 PDZ2 domain interactions has persistent effects on dendritic spines and cognitive function. METHODS: One-week-old mice were exposed to 1.5% isoflurane for 4 h or injected with 8 mg/kg active postsynaptic density-95 wild-type PDZ2 peptide along with their respective controls. A subset of these mice also received 4 mg/kg of the nitric oxide donor molsidomine. Hippocampal spine density, long-term potentiation, novel object recognition memory, and fear learning and memory were evaluated in mice. RESULTS: Exposure of 7-day-old mice to isoflurane or postsynaptic density-95 wild-type PDZ2 peptide relative to controls causes: (1) a long-term decrease in mushroom spines at 7 weeks (mean ± SD [spines per micrometer]): control (0.8 ± 0.2) versus isoflurane (0.4 ± 0.2), P < 0.0001, and PDZ2MUT (0.7 ± 0.2) versus PDZ2WT (0.4 ± 0.2), P < 0.001; (2) deficits in object recognition at 6 weeks (mean ± SD [recognition index]): naïve (70 ± 8) versus isoflurane (55 ± 14), P = 0.010, and control (65 ± 13) versus isoflurane (55 ± 14), P = 0.045, and PDZ2MUT (64 ±11) versus PDZ2WT (53 ± 18), P = 0.045; and (3) deficits in fear learning at 7 weeks and memory at 8 weeks (mean ± SD [% freezing duration]): Learning, control (69 ± 12) versus isoflurane (52 ± 13), P < 0.0001, and PDZ2MUT (65 ± 14) versus PDZ2WT (55 ± 14) P = 0.011, and Memory, control (80 ± 17) versus isoflurane (56 ± 23), P < 0.0001 and PDZ2MUT (73 ± 18) versus PDZ2WT (44 ± 19) P < 0.0001. Impairment in long-term potentiation has fully recovered here at 7 weeks (mean ± SD [% baseline]): control (140 ± 3) versus isoflurane (137 ± 8), P = 0.560, and PDZ2MUT (136 ± 17) versus PDZ2WT (128 ± 11), P = 0.512. The isoflurane induced decrease in mushroom spines was preventable by introduction of a nitric oxide donor. CONCLUSIONS: Early disruption of PDZ2 domain-mediated protein-protein interactions mimics isoflurane in decreasing mushroom spine density and causing learning and memory deficits in mice. Prevention of the decrease in mushroom spine density with a nitric oxide donor supports a role for neuronal nitric oxide synthase pathway in mediating this cellular change associated with cognitive impairment.
Asunto(s)
Anestésicos por Inhalación/toxicidad , Cognición/efectos de los fármacos , Espinas Dendríticas/efectos de los fármacos , Homólogo 4 de la Proteína Discs Large/antagonistas & inhibidores , Isoflurano/toxicidad , Animales , Animales Recién Nacidos , Cognición/fisiología , Espinas Dendríticas/patología , Espinas Dendríticas/fisiología , Homólogo 4 de la Proteína Discs Large/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos , Péptidos/farmacología , Densidad Postsináptica/efectos de los fármacos , Densidad Postsináptica/patología , Densidad Postsináptica/fisiologíaRESUMEN
CRIPT, the cysteine-rich PDZ-binding protein, binds to the third PDZ domain of PSD-95 (postsynaptic density protein 95) family proteins and directly binds microtubules, linking PSD-95 family proteins to the neuronal cytoskeleton. Here, we show that overexpression of a full-length CRIPT leads to a modest decrease, and knockdown of CRIPT leads to an increase in dendritic branching in cultured rat hippocampal neurons. Overexpression of truncated CRIPT lacking the PDZ domain-binding motif, which does not bind to PSD-95, significantly decreases dendritic arborization. Conversely, overexpression of a full-length CRIPT significantly increases the number of immature and mature dendritic spines, and this effect is not observed when CRIPT∆PDZ is overexpressed. Competitive inhibition of CRIPT binding to the third PDZ domain of PSD-95 with PDZ3-binding peptides resulted in differential effects on dendritic arborization based on the origin of respective peptide sequence. These results highlight multifunctional roles of CRIPT during development and underscore the significance of the interaction between CRIPT and the third PDZ domain of PSD-95.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Homólogo 4 de la Proteína Discs Large/fisiología , Hipocampo/citología , Plasticidad Neuronal/fisiología , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencias de Aminoácidos , Animales , Unión Competitiva , Células Cultivadas , Espinas Dendríticas/fisiología , Espinas Dendríticas/ultraestructura , Técnicas de Silenciamiento del Gen , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Unión Proteica , Mapeo de Interacción de Proteínas , Interferencia de ARN , ARN Interferente Pequeño/genética , RatasRESUMEN
OBJECTIVE: To explore the effects of sevoflurane on the latency and error times of the passive avoidance and levels of PSD-95 and AMPA receptors in the hippocampus. We evaluated the effects of sevoflurane on short-term memory in adult mice and explored the possible mechanism. METHODS: 144 Kunming mice (2-3 months, 30-35 g) were randomly divided into two groups A (n = 64) and B (n = 80) and received the dark-avoidance (DA) and step-down avoidance (SA) tests, respectively. The groups DA and SA were further divided into control (inhaled 40% O2 2 h) and sevoflurane (3.3% sevoflurane and 40% O2 2 h) subgroups. Before inhalation intervention, all mice were trained to be familiar with the Morris water maze (MWM). According to the test points of behavioral indicators, 8 mice were randomly selected from each subgroup at point 12 h (T1), 24 h (T2), 48 h (T3), and 72 h (T4) after inhalation intervention. The step-through latency and error times were measured in 5 min. After the behavioral test, the mice were killed and the tissues of the hippocampus were taken for hematoxylin and eosin (H&E) staining. The expression level of PSD-95 and AMPA receptors in the hippocampus was detected by immunohistochemistry and Western Blot. The changes of synaptic transmission were measured via electrophysiology analysis of hippocampal slices. RESULTS: The mice in the control subgroups found the platform in a shorter pathway than those in the sevoflurane subgroups during an MWM test. The step-through latency of T1 and T2 in the sevoflurane subgroup was shorter than baseline time, and the error times were increased in 5 min and higher than baseline time when compared with the control subgroup (P < 0.05) in the A and B groups. Compared with the control subgroup, the expression level of PSD-95 and AMPA receptors in the hippocampus was decreased at T1 and T2 in the sevoflurane subgroup (P < 0.05). The nerve cells were partially swelling. Electrophysiology analysis showed that the levels of PSD-95 and AMPA receptor expression were associated with synaptic transmission. CONCLUSION: Sevoflurane impaired short-term memory in adult mice by inhibiting the expression of PSD-95 and AMPA receptors in the hippocampus, which led to the decrease in synaptic transmission.
Asunto(s)
Memoria a Corto Plazo/efectos de los fármacos , Sevoflurano/efectos adversos , Animales , Encéfalo/metabolismo , China , Homólogo 4 de la Proteína Discs Large/metabolismo , Homólogo 4 de la Proteína Discs Large/fisiología , Femenino , Hipocampo/metabolismo , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Memoria a Corto Plazo/fisiología , Ratones , Neuronas/metabolismo , Receptores AMPA/metabolismo , Receptores AMPA/fisiología , Sevoflurano/farmacologíaRESUMEN
AMPA receptors and interacting proteins are importantly involved in mediating stress-dependent plasticity. Previously we reported that GluA1-containing AMPA receptors and their interaction with PDZ-proteins are required for the experience-dependent expression of behavioral despair in the forced swim test. However, it is unclear if the expression of GluA1-containing AMPA receptors is affected by this type of behavior. Here we investigated in wild type mice, whether hippocampal gene or protein levels of GluA1 or associated PDZ proteins is altered following forced swim stress. We show that expression of Dlg4 (the gene coding for PSD-95) was strongly reduced after two days of forced swimming. In contrast, levels of Dlg1, Gria1, and Gria2 (coding for SAP97, GluA1, and GluA2 respectively) were not affected after one or two days of forced swimming. The changes in gene expression largely did not translate to the protein level. These findings indicate a limited acute effect of forced swim stress on the expression of the investigated targets and suggest that the acute involvement of GluA1-containing AMPA receptors tor forced swim behavior is a result of non-genomic mechanisms.
Asunto(s)
Homólogo 1 de la Proteína Discs Large/metabolismo , Homólogo 4 de la Proteína Discs Large/metabolismo , Hipocampo/metabolismo , Receptores AMPA/metabolismo , Animales , Western Blotting , Homólogo 1 de la Proteína Discs Large/análisis , Homólogo 1 de la Proteína Discs Large/fisiología , Homólogo 4 de la Proteína Discs Large/análisis , Homólogo 4 de la Proteína Discs Large/fisiología , Femenino , Regulación de la Expresión Génica , Hipocampo/química , Hipocampo/fisiología , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores AMPA/análisis , Receptores AMPA/fisiología , Estrés Fisiológico/fisiología , NataciónRESUMEN
Nitric oxide (NO) is a neurotransmitter synthesized in the brain by neuronal nitric oxide synthase (nNOS). Using immunohistochemistry and confocal imaging in the inferior colliculus (IC, auditory midbrain) of the guinea pig (Cavia porcellus, male and female), we show that nNOS occurs in two distinct cellular distributions. We confirm that, in the cortices of the IC, a subset of neurons show cytoplasmic labeling for nNOS, whereas in the central nucleus (ICc), such neurons are not present. However, we demonstrate that all neurons in the ICc do in fact express nNOS in the form of discrete puncta found at the cell membrane. Our multi-labeling studies reveal that nNOS puncta form multiprotein complexes with NMDA receptors, soluble guanylyl cyclase (sGC), and PSD95. These complexes are found apposed to glutamatergic terminals, which is indicative of synaptic function. Interestingly, these glutamatergic terminals express both vesicular glutamate transporters 1 and 2 denoting a specific source of brainstem inputs. With in vivo electrophysiological recordings of multiunit activity in the ICc, we found that local application of NMDA enhances sound-driven activity in a concentration-dependent and reversible fashion. This response is abolished by blockade of nNOS or sGC, indicating that the NMDA effect is mediated solely via the NO and cGMP signaling pathway. This discovery of a ubiquitous, but highly localized, expression of nNOS throughout the ICc and demonstration of the dramatic influence of the NMDA activated NO pathway on sound-driven neuronal activity imply a key role for NO signaling in auditory processing.SIGNIFICANCE STATEMENT We show that neuronal nitric oxide synthase (nNOS), the enzyme that synthesizes nitric oxide (NO), occurs as puncta in apparently all neurons in the central nucleus of the inferior colliculus (ICc) in the auditory midbrain. Punctate nNOS appears at glutamatergic synapses in a complex with glutamate NMDA receptors (NMDA-Rs), soluble guanylyl cyclase (sGC, the NO receptor), and PSD95 (a protein that anchors receptors and enzymes at the postsynaptic density). We show that NMDA-R modulation of sound-driven activity in the ICc is solely mediated by activation of nNOS and sGC. The presence of nNOS throughout this sensory nucleus argues for a major role of NO in hearing. Furthermore, this punctate form of nNOS expression may exist and have gone unnoticed in other brain regions.
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Corteza Auditiva/fisiología , Mesencéfalo/fisiología , Óxido Nítrico Sintasa de Tipo I/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Transducción de Señal/fisiología , Animales , Percepción Auditiva/fisiología , GMP Cíclico/fisiología , Homólogo 4 de la Proteína Discs Large/fisiología , Femenino , Cobayas , Colículos Inferiores/citología , Colículos Inferiores/fisiología , Masculino , Óxido Nítrico/fisiología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Guanilil Ciclasa Soluble/metabolismo , Sinapsis/fisiología , Proteínas de Transporte Vesicular de Glutamato/metabolismoRESUMEN
The disc-large (DLG)-membrane-associated guanylate kinase (MAGUK) family of proteins forms a central signaling hub of the glutamate receptor complex. Among this family, some proteins regulate developmental maturation of glutamatergic synapses, a process vulnerable to aberrations, which may lead to neurodevelopmental disorders. As is typical for paralogs, the DLG-MAGUK proteins postsynaptic density (PSD)-95 and PSD-93 share similar functional domains and were previously thought to regulate glutamatergic synapses similarly. Here, we show that they play opposing roles in glutamatergic synapse maturation. Specifically, PSD-95 promoted, whereas PSD-93 inhibited maturation of immature α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptor (AMPAR)-silent synapses in mouse cortex during development. Furthermore, through experience-dependent regulation of its protein levels, PSD-93 directly inhibited PSD-95's promoting effect on silent synapse maturation in the visual cortex. The concerted function of these two paralogs governed the critical period of juvenile ocular dominance plasticity (jODP), and fine-tuned visual perception during development. In contrast to the silent synapse-based mechanism of adjusting visual perception, visual acuity improved by different mechanisms. Thus, by controlling the pace of silent synapse maturation, the opposing but properly balanced actions of PSD-93 and PSD-95 are essential for fine-tuning cortical networks for receptive field integration during developmental critical periods, and imply aberrations in either direction of this process as potential causes for neurodevelopmental disorders.
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Homólogo 4 de la Proteína Discs Large/fisiología , Guanilato-Quinasas/fisiología , Proteínas de la Membrana/fisiología , Sinapsis/metabolismo , Animales , Homólogo 4 de la Proteína Discs Large/metabolismo , Fármacos actuantes sobre Aminoácidos Excitadores , Femenino , Ácido Glutámico/metabolismo , Guanilato-Quinasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/fisiología , Receptores AMPA/metabolismo , Receptores de Glutamato/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de Señal , Transmisión Sináptica/fisiología , Corteza Visual/metabolismoRESUMEN
Synapses are semi-membraneless, protein-dense, sub-micron chemical reaction compartments responsible for signal processing in each and every neuron. Proper formation and dynamic responses to stimulations of synapses, both during development and in adult, are fundamental to functions of mammalian brains, although the molecular basis governing formation and modulation of compartmentalized synaptic assemblies is unclear. Here, we used a biochemical reconstitution approach to show that, both in solution and on supported membrane bilayers, multivalent interaction networks formed by major excitatory postsynaptic density (PSD) scaffold proteins led to formation of PSD-like assemblies via phase separation. The reconstituted PSD-like assemblies can cluster receptors, selectively concentrate enzymes, promote actin bundle formation, and expel inhibitory postsynaptic proteins. Additionally, the condensed phase PSD assemblies have features that are distinct from those in homogeneous solutions and fit for synaptic functions. Thus, we have built a molecular platform for understanding how neuronal synapses are formed and dynamically regulated.
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Neurogénesis , Plasticidad Neuronal , Densidad Postsináptica , Sinapsis/fisiología , Animales , Encéfalo/fisiología , Homólogo 4 de la Proteína Discs Large/fisiología , Hipocampo/fisiología , Luz , Ratones , Microscopía Confocal , Neuronas/fisiología , Dispersión de Radiación , Transducción de Señal , Transmisión SinápticaRESUMEN
Voltage-gated K+ (Kv ) channels are major determinants of membrane potential in vascular smooth muscle cells (VSMCs) and regulate the diameter of small cerebral arteries and arterioles. However, the intracellular structures that govern the expression and function of vascular Kv channels are poorly understood. Scaffolding proteins including postsynaptic density 95 (PSD95) recently were identified in rat cerebral VSMCs. Primarily characterized in neurons, the PSD95 scaffold has more than 50 known binding partners, and it can mediate macromolecular signaling between cell-surface receptors and ion channels. In cerebral arteries, Shaker-type Kv 1 channels appear to associate with the PSD95 molecular scaffold, and PSD95 is required for the normal expression and vasodilator influence of members of this K+ channel gene family. Furthermore, recent findings suggest that the ß1-subtype adrenergic receptor is expressed in cerebral VSMCs and forms a functional vasodilator complex with Kv 1 channels on the PSD95 scaffold. Activation of ß1-subtype adrenergic receptors in VSMCs enables protein kinase A-dependent phosphorylation and opening of Kv 1 channels in the PSD95 complex; the subsequent K+ efflux mediates membrane hyperpolarization and vasodilation of small cerebral arteries. Early evidence from other studies suggests that other families of Kv channels and scaffolding proteins are expressed in VSMCs. Future investigations into these macromolecular complexes that modulate the expression and function of Kv channels may reveal unknown signaling cascades that regulate VSMC excitability and provide novel targets for ion channel-based medications to optimize vascular tone.
Asunto(s)
Circulación Cerebrovascular , Homólogo 4 de la Proteína Discs Large/fisiología , Canales de Potasio con Entrada de Voltaje/metabolismo , Receptores Adrenérgicos beta/fisiología , Animales , Homólogo 4 de la Proteína Discs Large/metabolismo , Humanos , Músculo Liso Vascular/química , Músculo Liso Vascular/citología , Ratas , Receptores Adrenérgicos beta/metabolismoRESUMEN
Ubiquitination-directed proteasomal degradation of synaptic proteins, presumably mediated by lysine 48 (K48) of ubiquitin, is a key mechanism in synapse and neural circuit remodeling. However, more than half of polyubiquitin (polyUb) species in the mammalian brain are estimated to be non-K48; among them, the most abundant is Lys 63 (K63)-linked polyUb chains that do not tag substrates for degradation but rather modify their properties and activity. Virtually nothing is known about the role of these nonproteolytic polyUb chains at the synapse. Here we report that K63-polyUb chains play a significant role in postsynaptic protein scaffolding and synaptic strength and plasticity. We found that the postsynaptic scaffold PSD-95 (postsynaptic density protein 95) undergoes K63 polyubiquitination, which markedly modifies PSD-95's scaffolding potentials, enables its synaptic targeting, and promotes synapse maturation and efficacy. TNF receptor-associated factor 6 (TRAF6) is identified as a direct E3 ligase for PSD-95, which, together with the E2 complex Ubc13/Uev1a, assembles K63-chains on PSD-95. In contrast, CYLD (cylindromatosis tumor-suppressor protein), a K63-specific deubiquitinase enriched in postsynaptic densities, cleaves K63-chains from PSD-95. We found that neuronal activity exerts potent control of global and synaptic K63-polyUb levels and, through NMDA receptors, drives rapid, CYLD-mediated PSD-95 deubiquitination, mobilizing and depleting PSD-95 from synapses. Silencing CYLD in hippocampal neurons abolishes NMDA-induced chemical long-term depression. Our results unveil a previously unsuspected role for nonproteolytic polyUb chains in the synapse and illustrate a mechanism by which a PSD-associated K63-linkage-specific ubiquitin machinery acts on a major postsynaptic scaffold to regulate synapse organization, function, and plasticity.
Asunto(s)
Homólogo 4 de la Proteína Discs Large/fisiología , Hipocampo/fisiología , Neuronas/fisiología , Poliubiquitina/metabolismo , Densidad Postsináptica , Complejo de la Endopetidasa Proteasomal/metabolismo , Sinapsis/fisiología , Animales , Hipocampo/citología , Lisina , Ratones , Ratones Noqueados , Neuronas/citología , UbiquitinaciónRESUMEN
BACKGROUND Emotional state can be affected by different training loads. The aim of this study was to explore the changes of rat emotional state, as well as the mRNA and protein expressions of N-methyl-D-aspartate receptors (NMDARs), postsynaptic density 95 (PSD-95), and kinesin family member 17 (KIF-17) in the hippocampus, by long-term moderate-intensity and high-intensity training models in rats. MATERIAL AND METHODS The exercise model of SD rats was set up by treadmill running of moderate and high intensities for 4 weeks. The rats in the moderate-intensity training group were given endurance training with increasing intensity, while rats in the high-intensity training group were given high-speed training, and those in the normal control group were also established. The body weights of rats were measured before and after exercise to determine weight reduction. Real-time PCR and Western blotting were used to detect the mRNA and protein expressions of NMDARs, PSD-95, and KIF-17 in hippocampus of rats under different training loads. RESULTS Compared with the control group, the rats in the moderate-intensity training group had better body condition and emotional state, while the rats in the high-intensity training group had poor body condition and emotional state. The mRNA and protein expression of PSD-95, KIF-17, and NMDARs in the moderate-intensity training group were significantly elevated (P<0.05) while those in the high-intensity training group were suppressed (P<0.05). CONCLUSIONS Different training loads have remarkable influences on the cognition, emotion, and mental status of rats, and can affect the mRNA and protein expressions of NMDARs, PSD-95, and KIF-17 in rats. Appropriate training loads alleviate hypoxia damage to the hippocampus, and also effectively improve hippocampus function.
Asunto(s)
Emociones/fisiología , Condicionamiento Físico Animal/fisiología , Animales , Western Blotting , Peso Corporal , Homólogo 4 de la Proteína Discs Large/metabolismo , Homólogo 4 de la Proteína Discs Large/fisiología , Regulación de la Expresión Génica/genética , Hipocampo/metabolismo , Hipocampo/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cinesinas/metabolismo , Cinesinas/fisiología , Masculino , Densidad Postsináptica , ARN Mensajero , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologíaRESUMEN
Phosphorylation regulates surface and synaptic expression of NMDA receptors (NMDARs). Both the tyrosine kinase Fyn and the tyrosine phosphatase striatal-enriched protein tyrosine phosphatase (STEP) are known to target the NMDA receptor subunit GluN2B on tyrosine 1472, which is a critical residue that mediates NMDAR endocytosis. STEP reduces the surface expression of NMDARs by promoting dephosphorylation of GluN2B Y1472, whereas the synaptic scaffolding protein postsynaptic density protein 95 (PSD-95) stabilizes the surface expression of NMDARs. However, nothing is known about a potential functional interaction between STEP and PSD-95. We now report that STEP61 binds to PSD-95 but not to other PSD-95 family members. We find that PSD-95 expression destabilizes STEP61 via ubiquitination and degradation by the proteasome. Using subcellular fractionation, we detect low amounts of STEP61 in the PSD fraction. However, STEP61 expression in the PSD is increased upon knockdown of PSD-95 or in vivo as detected in PSD-95-KO mice, demonstrating that PSD-95 excludes STEP61 from the PSD. Importantly, only extrasynaptic NMDAR expression and currents were increased upon STEP knockdown, as is consistent with low STEP61 localization in the PSD. Our findings support a dual role for PSD-95 in stabilizing synaptic NMDARs by binding directly to GluN2B but also by promoting synaptic exclusion and degradation of the negative regulator STEP61.