RESUMO
Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) is widely expressed in the brain and is involved in various functions, including memory formation, mood and sleep. We previously reported that CaMKIIα is involved in the circadian molecular clock. Mice lacking functional CaMKIIα (K42R mice) exhibited a gradual increase in activity time (α decompression) of running-wheel (RW) activity due to a lengthened circadian period (τ) of activity offset under constant darkness (DD). In the present study, to investigate the functional roles of CaMKIIα in behavioural rhythms, we measured RW and general movements simultaneously under prolonged DD. Tau became longer as the relative intensity of behaviour activity within an activity time shifted from activity onset towards activity offset. In some K42R mice, α was gradually expanded with a marked reduction of RW activity, while general movements persisted without noticeable decline, which was followed by an abrupt shortening of α (α compression) with differential phase shifts of the activity onset and offset and recovery of RW activity. These results suggest that an internal coupling between the oscillators controlling activity onset and offset is bidirectional but with different strengths. The α compression occurred recurrently in 38% of K42R mice examined with an average interval of 37 days in association with attenuation of RW activity but never in the wild-type (WT) mice. Consistent with behavioural rhythms, the circadian period of the PER2::LUC rhythm in the cultured suprachiasmatic nucleus (SCN) slice was significantly longer in K42R than in WT. These findings are best interpreted by assuming that a loss of functional CaMKIIα attenuates the coupling between the onset and offset oscillators.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Ritmo Circadiano , Núcleo Supraquiasmático , Animais , Masculino , Camundongos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Camundongos Endogâmicos C57BL , Atividade Motora/fisiologia , Proteínas Circadianas Period/metabolismo , Proteínas Circadianas Period/genética , Núcleo Supraquiasmático/fisiologia , Núcleo Supraquiasmático/metabolismoRESUMO
Neurodevelopmental disorders, such as intellectual disability (ID), epilepsy, and autism, involve altered synaptic transmission and plasticity. Functional characterization of their associated genes is vital for understanding physio-pathological brain functions. LGI3 is a recently recognized ID-associated gene encoding a secretory protein related to an epilepsy-gene product, LGI1. Here, we find that LGI3 is uniquely secreted from oligodendrocytes in the brain and enriched at juxtaparanodes of myelinated axons, forming nanoscale subclusters. Proteomic analysis using epitope-tagged Lgi3 knockin mice shows that LGI3 uses ADAM23 as a receptor and selectively co-assembles with Kv1 channels. A lack of Lgi3 in mice disrupts juxtaparanodal clustering of ADAM23 and Kv1 channels and suppresses Kv1-channel-mediated short-term synaptic plasticity. Collectively, this study identifies an extracellular organizer of juxtaparanodal Kv1 channel clustering for finely tuned synaptic transmission. Given the defective secretion of the LGI3 missense variant, we propose a molecular pathway, the juxtaparanodal LGI3-ADAM23-Kv1 channel, for understanding neurodevelopmental disorders.
Assuntos
Epilepsia , Proteômica , Animais , Camundongos , Axônios/metabolismo , Epilepsia/metabolismo , Plasticidade Neuronal , Oligodendroglia/metabolismo , Proteínas/metabolismoRESUMO
Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) is a key mediator of activity-dependent neuronal modifications and has been implicated in the molecular mechanisms of learning and memory. Indeed, several types of CaMKIIα knock-in (KI) and knock-out (KO) mice revealed impairments in hippocampal synaptic plasticity and behavioral learning. On the other hand, a similar role for CaMKIIα has been implicated in amygdala-dependent memory, but detailed analyses have not much been performed yet. To better understand its involvement in amygdala-dependent memory as compared to hippocampus-dependent memory, here we performed biochemical analyses and behavioral memory tests using the kinase-dead CaMKIIα (K42R)-KI mouse. In the Morris water maze tasks, homozygous mutants performed well in the visible platform trials, while they failed to form spatial memory in the hippocampus-dependent hidden platform trials. In fear conditioning, these mice were impaired but showed a certain level of amygdala-dependent cued fear memory, which lasted four weeks, while they showed virtually no hippocampus-dependent context discrimination. Neither stronger stimulation nor repetitive stimulation compensated for their memory deficits. The differential outcome of hippocampus- and amygdala-dependent memory in the mutant mouse was not due to differential expression of CaMKIIα between the hippocampus and the amygdala, because biochemical analyses revealed that both kinase activity and protein levels of CaMKII were indistinguishable between the two brain regions. These results indicate that kinase activity of CaMKIIα is indispensable for hippocampus-dependent memory, but not necessarily for amygdala-dependent memory. There may be a secondary, CaMKIIα activity-independent pathway, in addition to the CaMKIIα activity-dependent pathway, in the acquisition of amygdala-dependent memory.
Assuntos
Tonsila do Cerebelo/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Condicionamento Clássico/fisiologia , Medo/fisiologia , Hipocampo/fisiologia , Aprendizagem em Labirinto/fisiologia , Memória Espacial/fisiologia , Animais , Comportamento Animal/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
The roles of calcium-calmodulin-dependent protein kinase II-alpha (CaMKIIα) in the expression of long-term synaptic plasticity in the adult brain have been extensively studied. However, how increased CaMKIIα activity controls the maturation of neuronal circuits remains incompletely understood. Herein, we show that pyramidal neurons without CaMKIIα activity upregulate the rate of spine addition, resulting in elevated spine density. Genetic elimination of CaMKIIα activity specifically eliminated the observed maturation-dependent suppression of spine formation. Enhanced spine formation was associated with the stabilization of actin in the spine and could be reversed by increasing the activity of the small GTPase Rap1. CaMKIIα activity was critical in the phosphorylation of synaptic Ras GTPase-activating protein (synGAP), the dispersion of synGAP from postsynaptic sites, and the activation of postsynaptic Rap1. CaMKIIα is already known to be essential in learning and memory, but our findings suggest that CaMKIIα plays an important activity-dependent role in restricting spine density during postnatal development.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Diferenciação Celular/genética , Espinhas Dendríticas/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Proteínas rap1 de Ligação ao GTP/genética , Animais , Biomarcadores , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Imunofluorescência , Hipocampo , Camundongos , Modelos Biológicos , Plasticidade Neuronal , Fosforilação , Ligação Proteica , Células Piramidais/citologia , Células Piramidais/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
Extracellular signal-regulated kinase 1/2 (ERK1/2) plays diverse roles in the central nervous system. Activation of ERK1/2 has been observed in various types of neuronal excitation, including seizure activity in vivo and in vitro. However, studies examining ERK1/2 activity and its substrate phosphorylation in parallel are scarce especially in seizure models. We have been studying the phosphorylation state of the presynaptic protein, synapsin I at ERK1/2-dependent and -independent sites in various types of seizure models and showed that ERK1/2-dependent phosphorylation of synapsin I was indeed under control of ERK1/2 activity in vivo. To further expand our study, here we examined the effects of prolonged seizure activity on ERK1/2 activity and synapsin I phosphorylation by using status epilepticus induced by kainic acid (KA-SE) in rats in vivo. In KA-SE, robust ERK1/2 activation was observed in the hippocampus, a representative limbic structure, with lesser activation in the parietal cortex, a representative non-limbic structure. In contrast, the phosphorylation level of synapsin I at ERK1/2-dependent phospho-site 4/5 was profoundly decreased, the extent of which was much larger in the hippocampus than in the parietal cortex. In addition, phosphorylation at other ERK1/2-independent phospho-sites in synapsin I also showed an even larger decrease. All these changes disappeared after recovery from KA-SE. These results indicate that the phosphorylation state of synapsin I is dynamically regulated by the balance between kinase and phosphatase activities. The contrasting features of robust ERK1/2 activation yet synapsin I dephosphorylation may be indicative of an irreversible pathological outcome of the epileptic state in vivo.
Assuntos
Encéfalo/metabolismo , Agonistas de Aminoácidos Excitatórios/toxicidade , Ácido Caínico/toxicidade , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Estado Epiléptico/induzido quimicamente , Sinapsinas/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Fosforilação/efeitos dos fármacos , Ratos , Ratos Wistar , Estado Epiléptico/patologiaRESUMO
Daily behavioral rhythms in mammals are governed by the central circadian clock, located in the suprachiasmatic nucleus (SCN). The behavioral rhythms persist even in constant darkness, with a stable activity time due to coupling between two oscillators that determine the morning and evening activities. Accumulating evidence supports a prerequisite role for Ca(2+) in the robust oscillation of the SCN, yet the underlying molecular mechanism remains elusive. Here, we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity is essential for not only the cellular oscillation but also synchronization among oscillators in the SCN. A kinase-dead mutation in mouse CaMKIIα weakened the behavioral rhythmicity and elicited decoupling between the morning and evening activity rhythms, sometimes causing arrhythmicity. In the mutant SCN, the right and left nuclei showed uncoupled oscillations. Cellular and biochemical analyses revealed that Ca(2+)-calmodulin-CaMKII signaling contributes to activation of E-box-dependent gene expression through promoting dimerization of circadian locomotor output cycles kaput (CLOCK) and brain and muscle Arnt-like protein 1 (BMAL1). These results demonstrate a dual role of CaMKII as a component of cell-autonomous clockwork and as a synchronizer integrating circadian behavioral activities.
Assuntos
Relógios Biológicos/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Ritmo Circadiano/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Comportamento Animal , Relógios Biológicos/efeitos dos fármacos , Proteínas CLOCK/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Ritmo Circadiano/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Células NIH 3T3 , Neurônios/enzimologia , Fosforilação , Ratos , Transdução de SinaisRESUMO
Extracellular signal-regulated kinase 1/2 (ERK1/2) that belongs to a subfamily of mitogen-activated protein kinases (MAPKs) plays diverse roles in the central nervous system. Activation of ERK1/2 has been observed in various types of neuronal excitation, including seizure activity in vivo and in vitro, as well as in NMDA-receptor (NMDA-R)-dependent long-term potentiation in the hippocampus. On the other hand, recent studies in cultured neurons have shown that NMDA-R stimulation could result in either ERK1/2 activation or non-activation, depending on the pharmacological manipulations. To assess NMDA-R-dependent regulation of ERK1/2 activity in vivo, here we examined the effect of NMDA-R-induced seizure activity on ERK1/2 activation by using rat cortical slice preparations. NMDA-R-dependent seizure activity introduced by Mg2+ -free condition did not cause ERK1/2 activation. On the other hand, when picrotoxin was added to concurrently suppress GABAA-receptor-mediated inhibition, profound ERK1/2 activation occurred, which was accompanied by strong phospho-ERK1/2-staining in the superficial and deep cortical layer neurons. In this case, prolonged membrane depolarization and enhanced burst action potential firings, both of which were much greater than those in Mg2+ -free condition alone, were observed. Differential ERK1/2 activation was supported by the concurrent selective increase in phosphorylation of a substrate protein, phospho-site 4/5 of synapsin I. These results indicate that NMDA-R activation through a release from Mg2+ -blockade, which accompanies enhancement of both excitatory and inhibitory synaptic transmission, was not enough, but concurrent suppression of GABAergic inhibition, which leads to a selective increase in excitatory synaptic transmission, was necessary for robust ERK1/2 activation to occur within the cortical network.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Piramidais/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Convulsões/enzimologia , Córtex Somatossensorial/fisiologia , Animais , Antagonistas GABAérgicos/farmacologia , Técnicas In Vitro , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Magnésio/farmacologia , Masculino , Picrotoxina/farmacologia , Células Piramidais/enzimologia , Células Piramidais/metabolismo , Ratos , Ratos Wistar , Convulsões/induzido quimicamente , Convulsões/metabolismo , Convulsões/fisiopatologia , Córtex Somatossensorial/efeitos dos fármacosRESUMO
Ca2+/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha) is an essential mediator of activity-dependent synaptic plasticity that possesses multiple protein functions. So far, the autophosphorylation site-mutant mice targeted at T286 and at T305/306 have demonstrated the importance of the autonomous activity and Ca2+/calmodulin-binding capacity of CaMKIIalpha, respectively, in the induction of long-term potentiation (LTP) and hippocampus-dependent learning. However, kinase activity of CaMKIIalpha, the most essential enzymatic function, has not been genetically dissected yet. Here, we generated a novel CaMKIIalpha knock-in mouse that completely lacks its kinase activity by introducing K42R mutation and examined the effects on hippocampal synaptic plasticity and behavioral learning. In homozygous CaMKIIalpha (K42R) mice, kinase activity was reduced to the same level as in CaMKIIalpha-null mice, whereas CaMKII protein expression was well preserved. Tetanic stimulation failed to induce not only LTP but also sustained dendritic spine enlargement, a structural basis for LTP, at the Schaffer collateral-CA1 synapse, whereas activity-dependent postsynaptic translocation of CaMKIIalpha was preserved. In addition, CaMKIIalpha (K42R) mice showed a severe impairment in inhibitory avoidance learning, a form of memory that is dependent on the hippocampus. These results demonstrate that kinase activity of CaMKIIalpha is a common critical gate controlling structural, functional, and behavioral expression of synaptic memory.
Assuntos
Aprendizagem da Esquiva/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Espinhas Dendríticas/enzimologia , Hipocampo/enzimologia , Potenciação de Longa Duração/fisiologia , Neurônios/enzimologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Células Cultivadas , Espinhas Dendríticas/fisiologia , Espinhas Dendríticas/ultraestrutura , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Técnicas de Introdução de Genes , Hipocampo/fisiologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação de Sentido Incorreto , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Sinapses/enzimologia , Sinapses/fisiologiaRESUMO
Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in the central nervous system, and is proposed to play important roles in activity-dependent modifications of neuronal functions. We reported previously on the dynamic regulation of the autonomous CaMKII in homogenates from hippocampus and parietal cortex by acute neuronal excitation induced by electroconvulsive treatment (ECT) in rats in vivo. In the present study, we examined in more detail the biochemical changes in CaMKII under such conditions. We unexpectedly found a concurrent increase in autophosphorylation at Thr286(alpha)/287(beta) and decrease in the specific activity of CaMKII in the particulate fraction in either hippocampus or parietal cortex during ECT-induced acute, brief seizure activity. On the other hand, the soluble CaMKII showed a marked decrease in autophosphorylation with unchanged or rather increased specific activity. Increased autophosphorylation and decreased CaMKII activity were associated with the detergent-insoluble particulate fraction. All these changes disappeared soon after the termination of seizure activity. The reversible formation of such an autophosphorylated, inactivated and sedimentable form of CaMKII during acute neuronal excitation may indicate the existence of a novel regulatory mechanism of CaMKII that may be important for normal functioning of the brain.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Hipocampo/enzimologia , Neurônios/enzimologia , Neurônios/fisiologia , Lobo Parietal/enzimologia , Convulsões/enzimologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Progressão da Doença , Eletrochoque , Estabilidade Enzimática/fisiologia , Masculino , Fosforilação , Ratos , Ratos Wistar , Recuperação de Função Fisiológica , Solubilidade , Frações Subcelulares/enzimologia , Sinapsinas/metabolismoRESUMO
Synapsin I is a synaptic vesicle-associated protein that is phosphorylated at multiple sites by various protein kinases. It has been proposed to play an important role in the regulation of neurotransmitter release and the organization of cytoskeletal architecture in the presynaptic terminal. In the present minireview, I describe the dynamic changes in synapsin I phosphorylation induced by acute neuronal excitation in vivo, and discuss its regulation by protein kinases and phosphatases and its functional significance in vivo. When acute neuronal excitation was induced by electroconvulsive treatment (ECT) in rats, phosphorylation of synapsin I at multiple sites was decreased during brief seizure activity in hippocampal and parieto-cortical homogenates. After termination of the seizure activity, phosphorylation at mitogen-activated protein kinase-dependent sites was increased dramatically. Phosphorylation at a Ca(2+)/calmodulin-dependent protein kinase II-dependent site was also increased moderately afterwards. The dynamic and differential changes in synapsin I phosphorylation induced by acute neuronal excitation may be involved in plastic changes induced by ECT and may have some role in its effectiveness for the treatment of psychiatric diseases in humans.
Assuntos
Exocitose/fisiologia , Neurônios/fisiologia , Neurotransmissores/metabolismo , Sinapsinas/metabolismo , Animais , Eletrochoque/métodos , Humanos , Neurônios/metabolismo , Fosforilação , Ratos , Sinapsinas/fisiologiaRESUMO
Synapsin I is a synaptic vesicle-associated protein which is phosphorylated at multiple sites by various kinases. It has been proposed to play a role in the regulation of neurotransmitter release and the organization of cytoskeletal architecture in the presynaptic terminal. To better understand the physiological regulation of its phosphorylation in vivo, we induced acute, reversible neuronal excitation by electroconvulsive treatment (ECT) in rats, and studied its effects on synapsin I phosphorylation at sites 3, 4/5 and 6 by immunoblot analyses of homogenates from hippocampus and parietal cortex using phospho-site-specific antibodies. A decrease in phosphorylation at all sites was observed soon after the electrical stimulation, followed by a large increase in phosphorylation at site 4/5 peaking at 5 min and a moderate increase in phosphorylation at site 6 peaking at 20 min. Systemic injection of SL327, a mitogen-activated protein kinase (MAPK) kinase inhibitor, prior to ECT, suppressed the increase in phospho-site 4/5 level, as well as that in MAPK activity, but not that in phospho-site 6 level. Thus, phosphorylation at site 4/5 of synapsin I has been shown to be regulated by MAPK in vivo.