RESUMEN
Biochemical signaling pathways in living cells are often highly organized into spatially segregated volumes, membranes, scaffolds, subcellular compartments, and organelles comprising small numbers of interacting molecules. At this level of granularity stochastic behavior dominates, well-mixed continuum approximations based on concentrations break down and a particle-based approach is more accurate and more efficient. We describe and validate a new version of the open-source MCell simulation program (MCell4), which supports generalized 3D Monte Carlo modeling of diffusion and chemical reaction of discrete molecules and macromolecular complexes in solution, on surfaces representing membranes, and combinations thereof. The main improvements in MCell4 compared to the previous versions, MCell3 and MCell3-R, include a Python interface and native BioNetGen reaction language (BNGL) support. MCell4's Python interface opens up completely new possibilities for interfacing with external simulators to allow creation of sophisticated event-driven multiscale/multiphysics simulations. The native BNGL support, implemented through a new open-source library libBNG (also introduced in this paper), provides the capability to run a given BNGL model spatially resolved in MCell4 and, with appropriate simplifying assumptions, also in the BioNetGen simulation environment, greatly accelerating and simplifying model validation and comparison.
Asunto(s)
Método de Montecarlo , Programas Informáticos , Difusión , Simulación por Computador , Modelos Biológicos , Lenguajes de Programación , Biología Computacional/métodos , Transducción de Señal/fisiologíaRESUMEN
Huntington's disease (HD) is a genetically inherited neurodegenerative disorder caused by expansion of a polyglutamine (polyQ) repeat in the exon-1 of huntingtin protein (HTT). The expanded polyQ enhances the amyloidogenic propensity of HTT exon 1 (HTTex1), which forms a heterogeneous mixture of assemblies with a broad neurotoxicity spectrum. While predominantly intracellular, monomeric and aggregated mutant HTT species are also present in the cerebrospinal fluids of HD patients, however, their biological properties are not well understood. To explore the role of extracellular mutant HTT in aggregation and toxicity, we investigated the uptake and amplification of recombinant HTTex1 assemblies in cell culture models. We find that small HTTex1 fibrils preferentially enter human neurons and trigger the amplification of neurotoxic assemblies; astrocytes or epithelial cells are not permissive. The amplification of HTTex1 in neurons depletes endogenous HTT protein with non-pathogenic polyQ repeat, activates apoptotic caspase-3 pathway and induces nuclear fragmentation. Using a panel of novel monoclonal antibodies and genetic mutation, we identified epitopes within the N-terminal 17 amino acids and proline-rich domain of HTTex1 to be critical in neural uptake and amplification. Synaptosome preparations from the brain homogenates of HD mice also contain mutant HTT species, which enter neurons and behave similar to small recombinant HTTex1 fibrils. These studies suggest that amyloidogenic extracellular mutant HTTex1 assemblies may preferentially enter neurons, propagate and promote neurodegeneration.
Asunto(s)
Astrocitos/metabolismo , Células Epiteliales/metabolismo , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/metabolismo , Neuronas/metabolismo , Agregación Patológica de Proteínas/metabolismo , Proteínas Amiloidogénicas/genética , Proteínas Amiloidogénicas/metabolismo , Animales , Apoptosis , Caspasa 3 , Exones , Técnicas de Sustitución del Gen , Humanos , Proteína Huntingtina/genética , Ratones , Ratones Transgénicos , Mutación , Péptidos/genética , Agregación Patológica de Proteínas/genética , SinaptosomasRESUMEN
Ca2+/calmodulin-dependent protein kinase II (CaMKII) accounts for up to 2 percent of all brain protein and is essential to memory function. CaMKII activity is known to regulate dynamic shifts in the size and signaling strength of neuronal connections, a process known as synaptic plasticity. Increasingly, computational models are used to explore synaptic plasticity and the mechanisms regulating CaMKII activity. Conventional modeling approaches may exclude biophysical detail due to the impractical number of state combinations that arise when explicitly monitoring the conformational changes, ligand binding, and phosphorylation events that occur on each of the CaMKII holoenzyme's subunits. To manage the combinatorial explosion without necessitating bias or loss in biological accuracy, we use a specialized syntax in the software MCell to create a rule-based model of a twelve-subunit CaMKII holoenzyme. Here we validate the rule-based model against previous experimental measures of CaMKII activity and investigate molecular mechanisms of CaMKII regulation. Specifically, we explore how Ca2+/CaM-binding may both stabilize CaMKII subunit activation and regulate maintenance of CaMKII autophosphorylation. Noting that Ca2+/CaM and protein phosphatases bind CaMKII at nearby or overlapping sites, we compare model scenarios in which Ca2+/CaM and protein phosphatase do or do not structurally exclude each other's binding to CaMKII. Our results suggest a functional mechanism for the so-called "CaM trapping" phenomenon, wherein Ca2+/CaM may structurally exclude phosphatase binding and thereby prolong CaMKII autophosphorylation. We conclude that structural protection of autophosphorylated CaMKII by Ca2+/CaM may be an important mechanism for regulation of synaptic plasticity.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calmodulina/metabolismo , Animales , Sitios de Unión , Fenómenos Biofísicos , Calcio/metabolismo , Biología Computacional , Estabilidad de Enzimas , Hipocampo/metabolismo , Humanos , Modelos Moleculares , Modelos Neurológicos , Plasticidad Neuronal , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Unión Proteica , Estructura Cuaternaria de Proteína , Subunidades de ProteínaRESUMEN
Voltage-gated Cav1.2 and Cav1.3 (L-type) Ca2+ channels regulate neuronal excitability, synaptic plasticity, and learning and memory. Densin-180 (densin) is an excitatory synaptic protein that promotes Ca2+-dependent facilitation of voltage-gated Cav1.3 Ca2+ channels in transfected cells. Mice lacking densin (densin KO) exhibit defects in synaptic plasticity, spatial memory, and increased anxiety-related behaviors-phenotypes that more closely match those in mice lacking Cav1.2 than Cav1.3. Therefore, we investigated the functional impact of densin on Cav1.2. We report that densin is an essential regulator of Cav1.2 in neurons, but has distinct modulatory effects compared with its regulation of Cav1.3. Densin binds to the N-terminal domain of Cav1.2, but not that of Cav1.3, and increases Cav1.2 currents in transfected cells and in neurons. In transfected cells, densin accelerates the forward trafficking of Cav1.2 channels without affecting their endocytosis. Consistent with a role for densin in increasing the number of postsynaptic Cav1.2 channels, overexpression of densin increases the clustering of Cav1.2 in dendrites of hippocampal neurons in culture. Compared with wild-type mice, the cell surface levels of Cav1.2 in the brain, as well as Cav1.2 current density and signaling to the nucleus, are reduced in neurons from densin KO mice. We conclude that densin is an essential regulator of neuronal Cav1 channels and ensures efficient Cav1.2 Ca2+ signaling at excitatory synapses.SIGNIFICANCE STATEMENT The number and localization of voltage-gated Cav Ca2+ channels are crucial determinants of neuronal excitability and synaptic transmission. We report that the protein densin-180 is highly enriched at excitatory synapses in the brain and enhances the cell surface trafficking and postsynaptic localization of Cav1.2 L-type Ca2+ channels in neurons. This interaction promotes coupling of Cav1.2 channels to activity-dependent gene transcription. Our results reveal a mechanism that may contribute to the roles of Cav1.2 in regulating cognition and mood.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Señalización del Calcio/fisiología , Potenciales Postsinápticos Excitadores/fisiología , Neuronas/fisiología , Sialoglicoproteínas/metabolismo , Sinapsis/fisiología , Animales , Corteza Cerebral/fisiología , Activación del Canal Iónico/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transporte de Proteínas/fisiología , Transducción de Señal/fisiologíaRESUMEN
The strength of each excitatory synapse in the central nervous system is regulated by its prior activity in a process called synaptic plasticity. The initiation of synaptic plasticity occurs when calcium ions enter the postsynaptic compartment and encounter a subcellular structure called the postsynaptic density (PSD). The PSD is attached to the postsynaptic membrane just underneath the concentrated plaque of neurotransmitter receptors. It is comprised of a core set of 30-60 proteins, approximately 20 of which are scaffold proteins. The rest include protein kinases and phosphatases, some of which respond to calcium ion; small GTPases and their regulators; chaperones; ubiquitins; and proteases. The assembly of the PSD involves competitive binding among a variety of specific protein binding sites to form a dynamic network. A biochemical challenge for the future is to understand how the dynamic regulation of the structure, composition, and activity of the PSD mediates synaptic plasticity and how mutations in PSD proteins lead to mental and neurodegenerative diseases.
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Ácido Glutámico/metabolismo , Red Nerviosa/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Plasticidad Neuronal , Densidad Postsináptica/metabolismo , Animales , Humanos , Potenciación a Largo Plazo , Trastornos Mentales/genética , Trastornos Mentales/metabolismo , Trastornos Mentales/patología , Mutación , Red Nerviosa/citología , Red Nerviosa/patología , Proteínas del Tejido Nervioso/genética , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Densidad Postsináptica/genética , Densidad Postsináptica/patologíaRESUMEN
SynGAP is a Ras and Rap GTPase-activating protein (GAP) found in high concentration in the postsynaptic density (PSD) fraction from mammalian forebrain where it binds to PDZ domains of PSD-95. Phosphorylation of pure recombinant synGAP by Ca2+/calmodulin-dependent protein kinase II (CaMKII) shifts the balance of synGAP's GAP activity toward inactivation of Rap1; whereas phosphorylation by cyclin-dependent kinase 5 (CDK5) has the opposite effect, shifting the balance toward inactivation of HRas. These shifts in balance contribute to regulation of the numbers of surface AMPA receptors, which rise during synaptic potentiation (CaMKII) and fall during synaptic scaling (CDK5). Polo-like kinase 2 (Plk2/SNK), like CDK5, contributes to synaptic scaling. These two kinases act in concert to reduce the number of surface AMPA receptors following elevated neuronal activity by tagging spine-associated RapGAP protein (SPAR) for degradation, thus raising the level of activated Rap. Here we show that Plk2 also phosphorylates and regulates synGAP. Phosphorylation of synGAP by Plk2 stimulates its GAP activity toward HRas by 65%, and toward Rap1 by 16%. Simultaneous phosphorylation of synGAP by Plk2 and CDK5 at distinct sites produces an additive increase in GAP activity toward HRas (â¼230%) and a smaller, non-additive increase in activity toward Rap1 (â¼15%). Dual phosphorylation also produces an increase in GAP activity toward Rap2 (â¼40-50%), an effect not produced by either kinase alone. As we previously observed for CDK5, addition of Ca2+/CaM causes a substrate-directed doubling of the rate and stoichiometry of phosphorylation of synGAP by Plk2, targeting residues also phosphorylated by CaMKII. In summary, phosphorylation by Plk2, like CDK5, shifts the ratio of GAP activity of synGAP to produce a greater decrease in active Ras than in active Rap, which would produce a shift toward a decrease in the number of surface AMPA receptors in neuronal dendrites.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas de Unión al GTP rap/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Animales , Células COS , Chlorocebus aethiops , Humanos , Espectrometría de Masas , FosforilaciónAsunto(s)
Transición de Fase , Densidad Postsináptica , Cristales Líquidos , Modelos Químicos , Fase S , Temperatura , TermodinámicaRESUMEN
synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAP's HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Quinasa 5 Dependiente de la Ciclina , Proteínas Activadoras de GTPasa , Proteínas Oncogénicas , Proteínas Proto-Oncogénicas p21(ras) , Sinapsis/enzimología , Proteínas de Unión al GTP rap , Proteínas de Unión al GTP rap1 , Proteínas Activadoras de ras GTPasa , Proteínas ras , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Células Cultivadas , Quinasa 5 Dependiente de la Ciclina/química , Quinasa 5 Dependiente de la Ciclina/genética , Quinasa 5 Dependiente de la Ciclina/metabolismo , Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Humanos , Neuronas/citología , Neuronas/enzimología , Proteínas Oncogénicas/química , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Ratas , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas de Unión al GTP rap1/química , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/metabolismo , Proteínas Activadoras de ras GTPasa/química , Proteínas Activadoras de ras GTPasa/genética , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas ras/química , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Multi-state modeling of biomolecules refers to a series of techniques used to represent and compute the behavior of biological molecules or complexes that can adopt a large number of possible functional states. Biological signaling systems often rely on complexes of biological macromolecules that can undergo several functionally significant modifications that are mutually compatible. Thus, they can exist in a very large number of functionally different states. Modeling such multi-state systems poses two problems: the problem of how to describe and specify a multi-state system (the "specification problem") and the problem of how to use a computer to simulate the progress of the system over time (the "computation problem"). To address the specification problem, modelers have in recent years moved away from explicit specification of all possible states and towards rule-based formalisms that allow for implicit model specification, including the κ-calculus, BioNetGen, the Allosteric Network Compiler, and others. To tackle the computation problem, they have turned to particle-based methods that have in many cases proved more computationally efficient than population-based methods based on ordinary differential equations, partial differential equations, or the Gillespie stochastic simulation algorithm. Given current computing technology, particle-based methods are sometimes the only possible option. Particle-based simulators fall into two further categories: nonspatial simulators, such as StochSim, DYNSTOC, RuleMonkey, and the Network-Free Stochastic Simulator (NFSim), and spatial simulators, including Meredys, SRSim, and MCell. Modelers can thus choose from a variety of tools, the best choice depending on the particular problem. Development of faster and more powerful methods is ongoing, promising the ability to simulate ever more complex signaling processes in the future.
Asunto(s)
Algoritmos , Modelos Biológicos , Modelos Moleculares , Biología de Sistemas/métodos , Bacterias , Simulación por Computador , ADN/química , ADN/metabolismo , ADN/fisiología , Humanos , Proteínas/química , Proteínas/metabolismo , Proteínas/fisiologíaRESUMEN
PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise â¼90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and ß-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins.
Asunto(s)
Cromatografía de Afinidad/métodos , Proteínas/química , Proteínas/aislamiento & purificación , Cromatografía de Afinidad/instrumentación , Humanos , Ligandos , Dominios PDZ , Ingeniería de Proteínas , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Long-term potentiation (LTP) is a biochemical process in excitatory glutamatergic synapses in the Central Nervous System (CNS). It is initiated by a bout of synaptic activation that is strong enough to contribute to production of an action potential in the axon of the postsynaptic neuron, and it results in an increase in the size of postsynaptic depolarization during subsequent activity. The first step leading to LTP is activation and autophosphorylation of an abundant postsynaptic enzyme, Ca 2+ /calmodulin-dependent protein kinase II (CaMKII). We use simulation of activation of CaMKII holoenzymes in a realistic spatial model of a spine synapse, created in MCell4, to test three hypotheses about how the autophosphorylation response of CaMKII is shaped during a repeated high-frequency stimulus. First, the simulation results indicate that autophosphorylation of CaMKII does not constitute a bistable switch under biologically realistic conditions. Instead, prolonged autophosphorylation of CaMKII may contribute to a biochemical "kinetic proof-reading" mechanism that controls induction of synaptic plasticity. Second, concentration of CaMKII near the postsynaptic membrane increases the local concentration of kinase activity. However, neither localization nor "Ca 2+ -calmodulin-trapping (CaM-trapping)" increase the proportion of autophosphorylated subunits in holoenzymes after a complex stimulus, as previously hypothesized. Finally, we show that, as hypothesized, the amplitude of autophosphorylation in the first 30 seconds after a stimulus is extremely sensitive to the level and location of PP1 activity when PP1 is present in biologically accurate amounts. We further show that prolonged steric hindrance of dephosphorylation of CaMKII, caused by CaM-trapping, can increase the amplitude of autophosphorylation after a complex stimulus. These simulation results sharpen our quantitative understanding of the early events leading to LTP at excitatory synapses. Author Summary: Neurons in the brain are interconnected in an organized fashion by synapses that transmit neuronal activity from one neuron to another. Most of the billions of neurons in the brain have about 10,000 synapses spread over the neuronal membrane. Information is stored in the brain when the ability of specific synapses to pass along neuronal activity is strengthened resulting in formation of new networks. The increase in strength of a synapse is tightly controlled by the frequency and amplitude of its activity, and by neurohormonal signals, which, in combination, can cause long-lasting biochemical changes at the synapse that underlie learning and memory. Defects in these biochemical pathways cause mental and neurological diseases. To develop treatments, we need to understand the precise choreography of these critical biochemical changes. However, the tiny size of the synaptic compartment makes precise measurements of the biochemical reactions impossible. We have used computer simulation techniques and information gathered from experiments on purified synaptic proteins to simulate, within a single synapse, the choreography of the first biochemical step in synaptic strengthening: activation of the enzyme Ca 2+ / calmodulin-dependent protein kinase II. Our results provide insights that can be used in future studies to develop treatments for neuronal diseases.
RESUMEN
The synaptic Ras/Rap-GTPase-activating protein (SynGAP1) plays a unique role in regulating specific downstream intracellular events in response to N-methyl-D-aspartate receptor (NMDAR) activation. Constitutive heterozygous loss of SynGAP1 disrupts NMDAR-mediated physiological and behavioral processes, but the disruptions might be of developmental origin. Therefore, the precise role of SynGAP1 in the adult brain, including its relative functional significance within specific brain regions, remains unexplored. The present study constitutes the first attempt in achieving adult hippocampal-specific SynGAP1 knockout using the Cre/loxP approach. Here, we report that this manipulation led to a significant numerical increase in both small and large GluA1 and NR1 immunoreactive clusters, many of which were non-opposed to presynaptic terminals. In parallel, the observed marked decline in the amplitude of spontaneous excitatory currents (sEPSCs) and inter-event intervals supported the impression that SynGAP1 loss might facilitate the accumulation of extrasynaptic glutamatergic receptors. In addition, SynGAP1-mediated signaling appears to be critical for the proper integration and survival of newborn neurons. The manipulation impaired reversal learning in the probe test of the water maze and induced a delay-dependent impairment in spatial recognition memory. It did not significantly affect anxiety or reference memory acquisition but induced a substantial elevation in spontaneous locomotor activity in the open field test. Thus, the present study demonstrates the functional significance of SynGAP1 signaling in the adult brain by capturing several changes that are dependent on NMDAR and hippocampal integrity.
Asunto(s)
Hipocampo/citología , Discapacidades para el Aprendizaje/genética , Neuronas/fisiología , Transmisión Sináptica/genética , Proteínas Activadoras de ras GTPasa/deficiencia , Análisis de Varianza , Animales , Reacción de Prevención/fisiología , Proteínas de Dominio Doblecortina , Regulación de la Expresión Génica/genética , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Hipocampo/metabolismo , Integrasas/genética , Integrasas/metabolismo , Aprendizaje por Laberinto/fisiología , Potenciales de la Membrana/genética , Trastornos de la Memoria/genética , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Actividad Motora/genética , Neuronas/citología , Neuronas/efectos de los fármacos , Neuropéptidos/metabolismo , Técnicas de Placa-Clamp , Tiempo de Reacción/genética , Receptores AMPA/genética , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Conducta Espacial/fisiología , Transmisión Sináptica/efectos de los fármacos , Transducción Genética , Proteínas Activadoras de ras GTPasa/metabolismoRESUMEN
Densin is an abundant scaffold protein in the postsynaptic density (PSD) that forms a high-affinity complex with αCaMKII and α-actinin. To assess the function of densin, we created a mouse line with a null mutation in the gene encoding it (LRRC7). Homozygous knock-out mice display a wide variety of abnormal behaviors that are often considered endophenotypes of schizophrenia and autism spectrum disorders. At the cellular level, loss of densin results in reduced levels of α-actinin in the brain and selective reduction in the localization of mGluR5 and DISC1 in the PSD fraction, whereas the amounts of ionotropic glutamate receptors and other prominent PSD proteins are unchanged. In addition, deletion of densin results in impairment of mGluR- and NMDA receptor-dependent forms of long-term depression, alters the early dynamics of regulation of CaMKII by NMDA-type glutamate receptors, and produces a change in spine morphology. These results indicate that densin influences the function of mGluRs and CaMKII at synapses and contributes to localization of mGluR5 and DISC1 in the PSD fraction. They are consistent with the hypothesis that mutations that disrupt the organization and/or dynamics of postsynaptic signaling complexes in excitatory synapses can cause behavioral endophenotypes of mental illness.
Asunto(s)
Regulación de la Expresión Génica/genética , Trastornos Mentales/genética , Proteínas del Tejido Nervioso/metabolismo , Densidad Postsináptica/metabolismo , Receptores de Ácido Kaínico/metabolismo , Sialoglicoproteínas/deficiencia , Actinas/metabolismo , Agresión/fisiología , Animales , Conducta Animal/fisiología , Bicuculina/farmacología , Peso Corporal/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Células Cultivadas , Espinas Dendríticas/metabolismo , Modelos Animales de Enfermedad , Embrión de Mamíferos , Endofenotipos , Conducta Exploratoria/fisiología , Femenino , Antagonistas del GABA/farmacología , Antagonistas de Receptores de GABA-A/farmacología , Genotipo , Proteína Ácida Fibrilar de la Glía/metabolismo , Glicina/farmacología , Proteínas Fluorescentes Verdes/genética , Hipocampo/citología , Técnicas In Vitro , Inhibición Psicológica , Potenciación a Largo Plazo/genética , Depresión Sináptica a Largo Plazo/genética , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Memoria a Corto Plazo/fisiología , Trastornos Mentales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/genética , Fuerza Muscular/genética , Mutación/genética , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Picrotoxina/farmacología , Desempeño Psicomotor/fisiología , Receptores AMPA/genética , Receptores de Ácido Kaínico/genética , Reconocimiento en Psicología/fisiología , Prueba de Desempeño de Rotación con Aceleración Constante , Estadísticas no Paramétricas , Factores de TiempoRESUMEN
Expansion of a polyglutamine (polyQ) tract in the Huntingtin (Htt) protein causes Huntington's disease (HD), a fatal inherited neurodegenerative disorder. Loss of the normal function of Htt is thought to be an important pathogenetic component of HD. However, the function of wild-type Htt is not well defined. Htt is thought to be a multifunctional protein that plays distinct roles in several biological processes, including synaptic transmission, intracellular transport and neuronal transcription. Here, we show with biochemical and live cell imaging studies that wild-type Htt stimulates the transport of nuclear factor κ light-chain-enhancer of activated B cells (NF-κB) out of dendritic spines (where NF-κB is activated by excitatory synaptic input) and supports a high level of active NF-κB in neuronal nuclei (where NF-κB stimulates the transcription of target genes). We show that this novel function of Htt is impaired by the polyQ expansion and thus may contribute to the etiology of HD.
Asunto(s)
Núcleo Celular/metabolismo , Mutación , FN-kappa B/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Sinapsis/metabolismo , Animales , Transporte Biológico/genética , Núcleo Celular/genética , Núcleo Celular/patología , Técnicas de Sustitución del Gen , Proteína Huntingtina , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Ratones , Ratones Noqueados , FN-kappa B/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Péptidos/metabolismo , Ratas , Ratas Sprague-Dawley , Sinapsis/genética , Sinapsis/patologíaRESUMEN
During the acquisition of memories, influx of Ca2+ into the postsynaptic spine through the pores of activated N-methyl-D-aspartate-type glutamate receptors triggers processes that change the strength of excitatory synapses. The pattern of Ca2+influx during the first few seconds of activity is interpreted within the Ca2+-dependent signaling network such that synaptic strength is eventually either potentiated or depressed. Many of the critical signaling enzymes that control synaptic plasticity,including Ca2+/calmodulin-dependent protein kinase II (CaMKII), are regulated by calmodulin, a small protein that can bindup to 4 Ca2+ ions. As a first step toward clarifying how the Ca2+-signaling network decides between potentiation or depression, we have created a kinetic model of the interactions of Ca2+, calmodulin, and CaMKII that represents our best understanding of the dynamics of these interactions under conditions that resemble those in a postsynaptic spine. We constrained parameters of the model from data in the literature, or from our own measurements, and then predicted time courses of activation and autophosphorylation of CaMKII under a variety of conditions. Simulations showed that species of calmodulin with fewer than four bound Ca2+ play a significant role in activation of CaMKII in the physiological regime,supporting the notion that processing of Ca2+ signals in a spine involves competition among target enzymes for binding to unsaturated species of CaM in an environment in which the concentration of Ca2+ is fluctuating rapidly. Indeed, we showed that dependence of activation on the frequency of Ca2+ transients arises from the kinetics of interaction of fluctuating Ca2+with calmodulin/CaMKII complexes. We used parameter sensitivity analysis to identify which parameters will be most beneficial to measure more carefully to improve the accuracy of predictions. This model provides a quantitative base from which to build more complex dynamic models of postsynaptic signal transduction during learning.
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Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Calcio/química , Calmodulina/química , Complejos Multiproteicos/química , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calmodulina/metabolismo , Cinética , Modelos Químicos , Simulación de Dinámica Molecular , Complejos Multiproteicos/metabolismo , Fosforilación , Unión Proteica , TermodinámicaRESUMEN
SynGAP is a postsynaptic density (PSD) protein that binds to PDZ domains of the scaffold protein PSD-95. We previously reported that heterozygous deletion of Syngap1 in mice is correlated with increased steady-state levels of other key PSD proteins that bind PSD-95, although the level of PSD-95 remains constant (Walkup et al., 2016). For example, the ratio to PSD-95 of Transmembrane AMPA-Receptor-associated Proteins (TARPs), which mediate binding of AMPA-type glutamate receptors to PSD-95, was increased in young Syngap1+/-mice. Here we show that only females and not males show a highly significant correlation between an increase in TARP and a decrease in synGAP in the PSDs of Syngap1+/-rodents. The data reveal a sex difference in the adaptation of the PSD scaffold to synGAP haploinsufficiency.
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Proteínas Activadoras de GTPasa/metabolismo , Haploinsuficiencia , Densidad Postsináptica/metabolismo , Animales , Sistemas CRISPR-Cas , Femenino , Proteínas Activadoras de GTPasa/genética , Masculino , Ratones , Ratones Noqueados , Fosforilación , Factores SexualesRESUMEN
SynGAP, a prominent Ras/Rap GTPase-activating protein in the postsynaptic density, regulates the timing of spine formation and trafficking of glutamate receptors in cultured neurons. However, the molecular mechanisms by which it does this are unknown. Here, we show that synGAP is a key regulator of spine morphology in adult mice. Heterozygous deletion of synGAP was sufficient to cause an excess of mushroom spines in adult brains, indicating that synGAP is involved in steady-state regulation of actin in mature spines. Both Ras- and Rac-GTP levels were elevated in forebrains from adult synGAP(+/-) mice. Rac is a well known regulator of actin polymerization and spine morphology. The steady-state level of phosphorylation of cofilin was also elevated in synGAP(+/-) mice. Cofilin, an F-actin severing protein that is inactivated by phosphorylation, is a downstream target of a pathway regulated by Rac. We show that transient regulation of cofilin by treatment with NMDA is also disrupted in synGAP mutant neurons. Treatment of wild-type neurons with 25 mum NMDA triggered transient dephosphorylation and activation of cofilin within 15 s. In contrast, neurons cultured from mice with a homozygous or heterozygous deletion of synGAP lacked the transient regulation by the NMDA receptor. Depression of EPSPs induced by a similar treatment of hippocampal slices with NMDA was disrupted in slices from synGAP(+/-) mice. Our data show that synGAP mediates a rate-limiting step in steady-state regulation of spine morphology and in transient NMDA-receptor-dependent regulation of the spine cytoskeleton.
Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Espinas Dendríticas/metabolismo , Espinas Dendríticas/ultraestructura , Proteínas Activadoras de ras GTPasa/metabolismo , Animales , Western Blotting , Encéfalo/metabolismo , Encéfalo/ultraestructura , Citoesqueleto , Espinas Dendríticas/efectos de los fármacos , Electroforesis en Gel de Poliacrilamida , Agonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Depresión Sináptica a Largo Plazo/efectos de los fármacos , Depresión Sináptica a Largo Plazo/fisiología , Ratones , Ratones Noqueados , Microscopía Confocal , N-Metilaspartato/farmacología , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Fosforilación , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteínas Activadoras de ras GTPasa/genética , Proteínas ras/metabolismoRESUMEN
The NR2B subunit of the NMDA receptor interacts with several prominent proteins in the postsynaptic density, including calcium/calmodulin-dependent protein kinase II (CaMKII). To determine the function of these interactions, we derived transgenic mice expressing a ligand-activated carboxy-terminal NR2B fragment (cNR2B) by fusing this fragment to a tamoxifen (TAM)-dependent mutant of the estrogen receptor ligand-binding domain LBD(G521R). Here, we show that induction by TAM allows the transgenic cNR2B fragment to bind to endogenous CaMKII in neurons. Activation of the LBD(G521R)-cNR2B transgenic protein in mice leads to the disruption of CaMKII/NR2B interactions at synapses. The disruption decreases Thr286 phosphorylation of alphaCaMKII, lowers phosphorylation of a key CaMKII substrate in the postsynaptic membrane (AMPA receptor subunit glutamate receptor 1), and produces deficits in hippocampal long-term potentiation and spatial learning. Together our results demonstrate the importance of interactions between CaMKII and NR2B for CaMKII activity, synaptic plasticity, and learning.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Aprendizaje por Laberinto/fisiología , Plasticidad Neuronal/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/fisiología , Animales , Ligandos , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/fisiología , Depresión Sináptica a Largo Plazo/efectos de los fármacos , Depresión Sináptica a Largo Plazo/fisiología , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/fisiología , Técnicas de Cultivo de Órganos , Fosforilación , Receptores de Estrógenos/genética , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tamoxifeno/farmacologíaRESUMEN
By combining biochemical experiments with computer modelling of biochemical reactions we elucidated some of the currently unresolved aspects of calcium-calmodulin-dependent protein kinase II (CaMKII) activation and autophosphorylation that might be relevant for its physiological function and provided a model that incorporates in detail the mechanism of CaMKII activation and autophosphorylation at T286 that is based on experimentally determined binding constants and phosphorylation rates. To this end, we developed a detailed state model of CaMKII activation and autophosphorylation based on the currently available literature, and constrained it with data from CaMKII autophosphorylation essays. Our model takes exact phosphorylation patterns of CaMKII holoenzymes into account, and is valid at physiologically relevant conditions where the concentrations of calcium and calmodulin are not saturating. Our results strongly suggest that even when bound to less than fully calcium-bound calmodulin, CaMKII is in the active state, and indicate that the autophosphorylation of T286 by an active non-phosphorylated CaMKII subunit is significantly faster than by an autophosphorylated CaMKII subunit. These results imply that CaMKII can be efficiently activated at significantly lower calcium concentrations than previously thought, which may explain how CaMKII gets activated at calcium concentrations existing at synapses in vivo. We also investigated the significance of CaMKII holoenzyme structure on CaMKII autophosphorylation and obtained estimates of previously unknown binding constants.
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Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/ultraestructura , Modelos Químicos , Modelos Moleculares , Simulación por Computador , Activación Enzimática , FosforilaciónRESUMEN
Shortly before he died in October 2017, John Lisman submitted an invited review to Molecular Brain on 'Criteria for identifying the molecular basis of the engram (CaMKII, PKMζ)'. John had no opportunity to read the referees' comments, and as a mark of the regard in which he was held by the neuroscience community the Editors decided to publish his review as submitted. This obituary takes the form of a series of commentaries on Lisman's review. At the same time we are publishing as a separate article a longer response by Todd Sacktor and André Fenton entitled 'What does LTP tell us about the roles of CaMKII and PKMζ in memory?' which presents the case for a rival memory molecule, PKMζ.