RESUMEN
Plasticity of synaptic transmission underlies learning and memory. It is accompanied by changes in the density and size of synapses, collectively called structural plasticity. Therefore, understanding the mechanism of structural plasticity is critical for understanding the mechanism of synaptic plasticity. In this chapter, we describe the procedures and equipment required to image structural plasticity of a single dendritic spine, which hosts excitatory synapses in the central nervous system, and underlying molecular interactions/biochemical reactions using two-photon fluorescence lifetime microscopy (2P-FLIM) in combination with Förster resonance energy transfer (FRET)-based biosensors.
Asunto(s)
Espinas Dendríticas , Transferencia Resonante de Energía de Fluorescencia , Microscopía de Fluorescencia por Excitación Multifotónica , Plasticidad Neuronal , Espinas Dendríticas/metabolismo , Espinas Dendríticas/ultraestructura , Espinas Dendríticas/fisiología , Plasticidad Neuronal/fisiología , Animales , Transferencia Resonante de Energía de Fluorescencia/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Sinapsis/metabolismo , Sinapsis/fisiología , Ratones , Técnicas Biosensibles/métodosRESUMEN
Proteome analyses of the postsynaptic density (PSD), a proteinaceous specialization beneath the postsynaptic membrane of excitatory synapses, have identified several thousands of proteins. While proteins with predictable functions have been well studied, functionally uncharacterized proteins are mostly overlooked. In this study, we conducted a comprehensive meta-analysis of 35 PSD proteome datasets, encompassing a total of 5,869 proteins. Employing a ranking methodology, we identified 97 proteins that remain inadequately characterized. From this selection, we focused our detailed analysis on the highest-ranked protein, FAM81A. FAM81A interacts with PSD proteins, including PSD-95, SynGAP, and NMDA receptors, and promotes liquid-liquid phase separation of those proteins in cultured cells or in vitro. Down-regulation of FAM81A in cultured neurons causes a decrease in the size of PSD-95 puncta and the frequency of neuronal firing. Our findings suggest that FAM81A plays a crucial role in facilitating the interaction and assembly of proteins within the PSD, and its presence is important for maintaining normal synaptic function. Additionally, our methodology underscores the necessity for further characterization of numerous synaptic proteins that still lack comprehensive understanding.
Asunto(s)
Separación de Fases , Proteoma , Proteoma/metabolismo , Homólogo 4 de la Proteína Discs Large/metabolismo , Sinapsis/metabolismo , Membranas SinápticasRESUMEN
Synaptic plasticity plays a crucial role in memory formation by regulating the communication between neurons. Although actin polymerization has been linked to synaptic plasticity and dendritic spine stability, the causal link between actin polymerization and memory encoding has not been identified yet. It is not clear whether actin polymerization and structural changes in dendritic spines are a driver or a consequence of learning and memory. Using an extrinsically disordered form of the protein kinase LIMK1, which rapidly and precisely acts on ADF/cofilin, a direct modifier of actin, we induced long-term enlargement of dendritic spines and enhancement of synaptic transmission in the hippocampus on command. The activation of extrinsically disordered LIMK1 in vivo improved memory encoding and slowed cognitive decline in aged mice exhibiting reduced cofilin phosphorylation. The engineered memory by an extrinsically disordered LIMK1 supports a direct causal link between actin-mediated synaptic transmission and memory.
Asunto(s)
Actinas , Hipocampo , Ratones , Animales , Actinas/metabolismo , Hipocampo/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Fosforilación/fisiología , Plasticidad Neuronal/fisiologíaRESUMEN
Interactions between an enzyme kinase, an ion channel and cytoskeletal proteins maintain the structure of synapses involved in memory formation.
Asunto(s)
Proteínas del Citoesqueleto , Espinas DendríticasRESUMEN
Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a signaling protein required for long-term memory. When activated by Ca2+/CaM, it sustains activity even after the Ca2+ dissipates. In addition to the well-known autophosphorylation-mediated mechanism, interaction with specific binding partners also persistently activates CaMKII. A long-standing model invokes two distinct S and T sites. If an interactor binds at the T-site, then it will preclude autoinhibition and allow substrates to be phosphorylated at the S site. Here, we specifically test this model with X-ray crystallography, molecular dynamics simulations, and biochemistry. Our data are inconsistent with this model. Co-crystal structures of four different activators or substrates show that they all bind to a single continuous site across the kinase domain. We propose a mechanistic model where persistent CaMKII activity is facilitated by high-affinity binding partners that kinetically compete with autoinhibition by the regulatory segment to allow substrate phosphorylation.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Procesamiento Proteico-Postraduccional , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Dominio Catalítico , FosforilaciónRESUMEN
Dendritic spines constitute the major compartments of excitatory post-synapses. They undergo activity-dependent enlargement, which is thought to increase the synaptic efficacy underlying learning and memory. The activity-dependent spine enlargement requires activation of signaling pathways leading to promotion of actin polymerization within the spines. However, the molecular machinery that suffices for that structural plasticity remains unclear. Here, we demonstrate that shootin1a links polymerizing actin filaments in spines with the cell-adhesion molecules N-cadherin and L1-CAM, thereby mechanically coupling the filaments to the extracellular environment. Synaptic activation enhances shootin1a-mediated actin-adhesion coupling in spines. Promotion of actin polymerization is insufficient for the plasticity; the enhanced actin-adhesion coupling is required for polymerizing actin filaments to push against the membrane for spine enlargement. By integrating cell signaling, cell adhesion, and force generation into the current model of actin-based machinery, we propose molecular machinery that is sufficient to trigger the activity-dependent spine structural plasticity.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Actinas/metabolismo , Animales , Espinas Dendríticas/metabolismo , Humanos , Ratones , Plasticidad NeuronalRESUMEN
Synaptic connections in neuronal circuits change in response to neuronal activity patterns. This can induce a persistent change in the efficacy of synaptic transmission, a phenomenon known as synaptic plasticity. One form of plasticity, long-term potentiation (LTP) has been extensively studied as the cellular basis of memory. In LTP, the potentiated synaptic transmission persists along with structural changes in the synapses. Many studies have sought to identify the "memory molecule" or the "molecular engram". Ca2+/calmodulin-dependent protein kinase II (CaMKII) is probably the most well-studied candidate for the memory molecule. However, consensus has not yet been reached on a very basic aspect: how CaMKII is regulated during LTP. Here, I propose a new model of CaMKII regulation: reciprocal activation within a kinase effector complex (RAKEC) that is made between CaMKII and its effector protein, which is mediated by a persistent interaction between CaMKII and a pseudosubstrate sequence on T-lymphoma invasion and metastasis protein 1 (Tiam1), resulting in reciprocal activation of these two molecules. Through the RAKEC mechanism, CaMKII can maintain memory as biochemical activity in a synapse-specific manner. In this review, the detailed mechanism of the RAKEC and its expansion for the maintenance of LTP is described.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calcio/metabolismo , Memoria/fisiología , Plasticidad Neuronal/fisiología , Citoesqueleto de Actina/metabolismo , AnimalesRESUMEN
A stimulation inducing long-term potentiation (LTP) of synaptic transmission induces a persistent expansion of dendritic spines, a phenomenon known as structural LTP (sLTP). We previously proposed that the formation of a reciprocally activating kinase-effector complex (RAKEC) between CaMKII and Tiam1, an activator of the small G-protein Rac1, locks CaMKII into an active conformation, which in turn maintains the phosphorylation status of Tiam1. This makes Rac1 persistently active, specifically in the stimulated spine. To understand the significance of the CaMKII-Tiam1 RAKEC in vivo, we generated a Tiam1 mutant knock-in mouse line in which critical residues for CaMKII binding were mutated into alanines. We confirmed the central role of this interaction on sLTP by observing that KI mice showed reduced Rac1 activity, had smaller spines and a diminished sLTP as compared to their wild-type littermates. Moreover, behavioral tests showed that the novel object recognition memory of these animals was impaired. We thus propose that the CaMKII-Tiam1 interaction regulates spine morphology in vivo and is required for memory storage.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Espinas Dendríticas/metabolismo , Aprendizaje/fisiología , Potenciación a Largo Plazo/fisiología , Memoria/fisiología , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T/metabolismo , Animales , Hipocampo/metabolismo , Ratones Transgénicos , Neuronas/metabolismo , Fosforilación , Reconocimiento en Psicología/fisiología , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T/genéticaAsunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Animales , Encéfalo , Memoria , Ratones , NeuronasRESUMEN
Long-term synaptic plasticity requires a mechanism that converts short Ca2+ pulses into persistent biochemical signaling to maintain changes in the synaptic structure and function. Here, we present a novel mechanism of a positive feedback loop, formed by a reciprocally activating kinase-effector complex (RAKEC) in dendritic spines, enabling the persistence and confinement of a molecular memory. We found that stimulation of a single spine causes the rapid formation of a RAKEC consisting of CaMKII and Tiam1, a Rac-GEF. This interaction is mediated by a pseudo-autoinhibitory domain on Tiam1, which is homologous to the CaMKII autoinhibitory domain itself. Therefore, Tiam1 binding results in constitutive CaMKII activation, which in turn persistently phosphorylates Tiam1. Phosphorylated Tiam1 promotes stable actin-polymerization through Rac1, thereby maintaining the structure of the spine during LTP. The RAKEC can store biochemical information in small subcellular compartments, thus potentially serving as a general mechanism for prolonged and compartmentalized signaling.
Asunto(s)
Actinas/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Espinas Dendríticas/metabolismo , Potenciación a Largo Plazo/fisiología , Neuronas/metabolismo , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Espinas Dendríticas/ultraestructura , Retroalimentación Fisiológica , Factores de Intercambio de Guanina Nucleótido/metabolismo , Hipocampo/citología , Potenciación a Largo Plazo/efectos de los fármacos , Microscopía Confocal , Neuronas/ultraestructura , Fosforilación , Polimerizacion , Pironas/farmacología , Quinolinas/farmacología , Ratas , Proteína de Unión al GTP rac1/antagonistas & inhibidoresRESUMEN
Glutathione-labile protecting groups for phosphodiester moieties in oligonucleotides were designed, synthesized, and incorporated into oligonucleotides. The protecting groups on the phosphodiester moieties were cleaved in a buffer containing 10 mM glutathione, which was used as a model of intracellular fluid. Cellular uptake of oligonucleotides bearing glutathione-labile protecting groups was strongly affected by the location and number of the protecting groups.
Asunto(s)
Diseño de Fármacos , Glutatión/química , Oligonucleótidos/farmacocinética , Células HeLa , Humanos , Estructura Molecular , Oligonucleótidos/síntesis química , Oligonucleótidos/químicaRESUMEN
Extracellular signal-regulated kinase 5 (ERK5) regulates diverse physiological responses such as proliferation, differentiation, and gene expression. Previously, we demonstrated that ERK5 is essential for neurite outgrowth and catecholamine biosynthesis in PC12 cells and sympathetic neurons. However, it remains unclear how ERK5 regulates the activity of ion channels, which are important for membrane excitability. Thus, we examined the effect of ERK5 on the ion channel activity in the PC12 cells that overexpress both ERK5 and the constitutively active MEK5 mutant. The gene and protein expression levels of voltage-dependent Ca2+ and K⺠channels were determined by RT-qPCR or Western blotting. The A-type K⺠current was recorded using the whole-cell patch clamp method. In these ERK5-activated cells, the gene expression levels of voltage-dependent L- and P/Q-type Ca2+ channels did not alter, but the N-type Ca2+ channel was slightly reduced. In contrast, those of Kv4.2 and Kv4.3, which are components of the A-type current, were significantly enhanced. Unexpectedly, the protein levels of Kv4.2 were not elevated by ERK5 activation, but the phosphorylation levels were increased by ERK5 activation. By electrophysiological analysis, the inactivation time constant of the A-type current was prolonged by ERK5 activation, without changes in the peak current. Taken together, ERK5 inhibits an inactivation of the A-type current by phosphorylation of Kv4.2, which may contribute to the neuronal differentiation process.
Asunto(s)
Proteína Quinasa 7 Activada por Mitógenos/genética , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Canales de Potasio Shal/genética , Canales de Potasio Shal/metabolismo , Animales , Catecolaminas/biosíntesis , Diferenciación Celular , Regulación de la Expresión Génica , Potenciales de la Membrana , Neuronas/citología , Neuronas/metabolismo , Células PC12 , Técnicas de Placa-Clamp , Fosforilación , Ratas , Transducción de SeñalRESUMEN
Cell-permeable oligodeoxyribonucleotides (ODNs) bearing reduction-activated protecting groups were synthesized as oligonucleotide pro-drugs. Although these oligonucleotides were amenable to solid-phase DNA synthesis and purification, the protecting group on their phosphodiester moiety could be readily cleaved by nitroreductase and NADH. Moreover, these compounds exhibited good nuclease resistance against 3'-exonuclease and endonuclease and good stability in human serum. Fluorescein-labeled ODNs modified with reduction-activated protecting groups showed better cellular uptake compared with that of naked ODNs.
Asunto(s)
Oligonucleótidos/síntesis química , Oligonucleótidos/metabolismo , Secuencia de Bases , Técnicas de Química Sintética , Estabilidad de Medicamentos , Humanos , NAD/metabolismo , Nitrorreductasas/metabolismo , Oligonucleótidos/química , Oligonucleótidos/genética , Oxidación-Reducción , PermeabilidadRESUMEN
Since the discovery of long-term potentiation (LTP) about a half-century ago, Ca2+ /CaM-dependent protein kinase II (CaMKII) has been one of the most extensively studied components of the molecular machinery that regulate plasticity. This unique dodecameric kinase complex plays pivotal roles in LTP by phosphorylating substrates through elaborate regulatory mechanisms, and is known to be both necessary and sufficient for LTP. In addition to acting as a kinase, CaMKII has been postulated to have structural roles because of its extraordinary abundance and diverse interacting partners. It now is becoming clear that these two functions of CaMKII cooperate closely for the induction of both functional and structural synaptic plasticity of dendritic spines. Because of its extraordinary abundance within neuronal cells, calmodulin kinase CaMKII has been believed to act as a structural protein as well as an enzyme during synaptic plasticity. In this review, we summarized studies in CaMKII field and provide an insight into how enzymatic and structural functions of CaMKII cooperate with each other for long-term potentiation (LTP) in neurons. This article is part of a mini review series: "Synaptic Function and Dysfunction in Brain Diseases".
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Hipocampo/enzimología , Potenciación a Largo Plazo/fisiología , Animales , Espinas Dendríticas/enzimología , Humanos , Microtúbulos/enzimología , Plasticidad Neuronal/fisiologíaRESUMEN
Hypofunction of the N-methyl-D-aspartate type glutamate receptor (NMDAR) has been implicated in the pathogenesis of schizophrenia. Here, we investigated the significance of a common human genetic variation of the NMDAR NR3B subunit that inserts 4 bases within the coding region (insCGTT) in the pathogenesis of schizophrenia. The cDNA carrying this polymorphism generates a truncated protein, which is electrophysiologically non-functional in heterologous expression systems. Among 586 schizophrenia patients and 754 healthy controls, insCGTT was significantly overrepresented in patients compared to controls (odds ratio = 1.37, p = 0.035). Among 121 schizophrenia patients and 372 healthy controls, genetic analyses of normal individuals revealed that those carrying insCGTT have a predisposition to schizotypal personality traits (F1,356 = 4.69, p = 0.031). Furthermore, pre-pulse inhibition, a neurobiological trait disturbed in patients with schizophrenia, was significantly impaired in patients carrying insCGTT compared with those with the major allele (F1,116 = 5.72, p = 0.018, F1,238 = 4.46, p = 0.036, respectively). These results indicate that a naturally occurring null variant in NR3B could be a risk factor of schizophrenia.
Asunto(s)
Receptores de N-Metil-D-Aspartato/genética , Esquizofrenia/genética , Adulto , Estudios de Casos y Controles , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Esquizofrenia/patologíaRESUMEN
Synapses store information by long-lasting modifications of their structure and molecular composition, but the precise chronology of these changes has not been studied at single-synapse resolution in real time. Here we describe the spatiotemporal reorganization of postsynaptic substructures during long-term potentiation (LTP) at individual dendritic spines. Proteins translocated to the spine in four distinct patterns through three sequential phases. In the initial phase, the actin cytoskeleton was rapidly remodeled while active cofilin was massively transported to the spine. In the stabilization phase, cofilin formed a stable complex with F-actin, was persistently retained at the spine, and consolidated spine expansion. In contrast, the postsynaptic density (PSD) was independently remodeled, as PSD scaffolding proteins did not change their amount and localization until a late protein synthesis-dependent third phase. Our findings show how and when spine substructures are remodeled during LTP and explain why synaptic plasticity rules change over time.
Asunto(s)
Espinas Dendríticas/fisiología , Hipocampo/citología , Potenciación a Largo Plazo/fisiología , Neuronas/ultraestructura , Sinapsis/fisiología , Animales , Animales Recién Nacidos , Células Cultivadas , Espinas Dendríticas/ultraestructura , Ácido Glutámico/farmacología , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Modelos Biológicos , Proteínas del Tejido Nervioso/metabolismo , Técnicas de Cultivo de Órganos , Densidad Postsináptica/metabolismo , Densidad Postsináptica/ultraestructura , Ratas , Receptores de Neurotransmisores/metabolismo , Sinapsis/genética , Sinapsis/ultraestructura , Transducción GenéticaRESUMEN
Most excitatory synapses reside on small protrusions located on the dendritic shaft of neurons called dendritic spines. Neuronal activity regulates the number and structure of spines in both developing and mature brains. Such morphological changes are mediated by the modification of the actin cytoskeleton, the major structural component of spines. Because the number and size of spines is tightly correlated with the strength of synaptic transmission, the activity-dependent structural remodeling of a spine plays an important role in the modulation of synaptic transmission. The regulation of spine morphogenesis utilizes multiple intracellular signaling pathways that alter the dynamics of actin remodeling. Here, we will review recent studies examining the signaling pathways underlying activity-dependent actin remodeling at excitatory postsynaptic neurons.
Asunto(s)
Actinas/metabolismo , Calcio/metabolismo , Espinas Dendríticas/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rho/metabolismo , Animales , Señalización del Calcio , HumanosRESUMEN
Medulloblastoma is a highly prevalent pediatric central nervous system malignancy originating in the cerebellum, with a strong propensity for metastatic migration to the leptomeninges, which greatly increases mortality. While numerous investigations are focused on the molecular mechanisms of medulloblastoma histogenesis, the signaling pathways regulating migration are still poorly understood. Medulloblastoma likely arises from aberrant proliferative signaling in cerebellar granule precursor cells during development, and estrogen is a morphogen that promotes medulloblastoma cell migration. It has been previously shown that the calcium/calmodulin activated kinase kinase (CaMKK) pathway promotes cerebellar granule precursor migration and differentiation during normal cerebellar development via CaMKIV. Here we investigate the regulatory role of the CaMKK pathway in migration of the human medulloblastoma DAOY and cerebellar granule cells. Using pharmacological inhibitors and dominant negative approaches, we demonstrate that the CaMKK/CaMKI cascade regulates basal medulloblastoma cell migration via Rac1, in part by activation of the RacGEF, ßPIX. Additionally, pharmacological inhibition of CaMKK blocks both the estrogen induced Rac1 activation and medulloblastoma migration. The CaMKK signaling module described here is one of the first reported calcium regulated pathways that modulates medulloblastoma migration. Since tumor dissemination requires cell migration to ectopic sites, this CaMKK pathway may be a putative therapeutic target to limit medulloblastoma metastasis.
Asunto(s)
Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Movimiento Celular/efectos de los fármacos , Estrógenos/farmacología , Transducción de Señal/fisiología , Proteína de Unión al GTP rac1/metabolismo , Aminoquinolinas/farmacología , Animales , Animales Recién Nacidos , Bencimidazoles/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cerebelo/citología , Quelantes/farmacología , Interacciones Farmacológicas , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Inhibidores Enzimáticos/farmacología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Meduloblastoma/patología , Naftalimidas/farmacología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Técnicas de Cultivo de Órganos , Pirimidinas/farmacología , Pironas/farmacología , Quinolinas/farmacología , Ratas , Factores de Intercambio de Guanina Nucleótido Rho , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Transfección/métodos , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Proteína de Unión al GTP rac1/genéticaRESUMEN
Formation of the human brain during embryonic and postnatal development is an extraordinarily complex process resulting at maturity in billions of neurons with trillions of specialized connections called synapses. These synapses, composed of a varicosity or bouton from a presynaptic neuron that communicates with a dendritic spine of the postsynaptic neuron, comprise the neural network that is essential for complex behavioral phenomena and cognition. Inappropriate synapse formation or structure is thought to underlie several developmental neuropathologies. Even in the mature CNS, alterations in synapse structure and function continues to be a very dynamic process that is foundational to learning and memory as well as other adaptive abilities of the brain. This synaptic plasticity in mature neurons, which is often triggered by certain patterns of neural activity, is again multifaceted and involves post-translational modifications (e.g. phosphorylation) and subcellular relocalization or trafficking (endocytosis/exocytosis) of existing synaptic proteins, initiation of protein synthesis from existing mRNAs localized in dendrites or spines, and triggering of new gene transcription in the nucleus. These various cellular processes support varying temporal components of synaptic plasticity that begin within 1-2 min but can persist for hours to days. This review will give a critical assessment of activity-dependent molecular modulations of synapses reported over the past couple years. Owing to space limitations, it will focus on mammalian excitatory (i.e. glutamatergic) synapses and will not consider several activity-independent signaling pathways (e.g. ephrinB receptor) that also modulate spine and synapse formation.