Yoko Yamagata

Kinase-Dead Knock-In Mouse Reveals an Essential Role of Kinase Activity of Ca2+/Calmodulin-Dependent Protein Kinase IIα in Dendritic Spine Enlargement, Long-Term Potentiation, and Learning

Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) 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 CaMKIIα, respectively, in the induction of long-term potentiation (LTP) and hippocampus-dependent learning. However, kinase activity of CaMKIIα, the most essential enzymatic function, has not been genetically dissected yet. Here, we generated a novel CaMKIIα 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 CaMKIIα (K42R) mice, kinase activity was reduced to the same level as in CaMKIIα-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 CaMKIIα was preserved. In addition, CaMKIIα (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 CaMKIIα is a common critical gate controlling structural, functional, and behavioral expression of synaptic memory.

Bidirectional changes in synapsin I phosphorylation at MAP kinase-dependent sites by acute neuronal excitation in vivo.

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.

CaMKII is essential for the cellular clock and coupling between morning and evening behavioral rhythms

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.

Dynamic Regulation of the Activated, Autophosphorylated State of Ca2+/Calmodulin-Dependent Protein Kinase II by Acute Neuronal Excitation In Vivo

Abstract: Ca2+/calmodulin‐dependent protein kinase II (CaMKII) has been implicated in various neuronal functions, including synaptic plasticity. To examine the physiological regulation of its activated, autophosphorylated state in relation to acute neuronal excitation in vivo, we studied the effect of electroconvulsive treatment in rats on CaMKII activity and in situ autophosphorylation levels. As early as 30 s after the electrical stimulation, a profound but transient decrease in its Ca2+/calmodulin‐independent activity, as well as in the level of its autophosphorylation at Thr286 (α)/Thr287 (β) measured by using phosphorylation state‐specific antibodies, was observed in homogenate from hippocampus and parietal cortex, which was reversible in 5 min. In the later time course, a moderate, reversible increase, which peaked at around 60 min after the electrical stimulation, was observed in parietal cortex but not in hippocampus. The early‐phase decrease was found to occur exclusively in the soluble fraction. In addition, partial translocation of CaMKII from the soluble to the particulate fraction seems to have occurred in this early phase. Thus, the activated, autophosphorylated state of CaMKII is under dynamic and precise regulation in vivo, and its regulatory mechanisms seem to have regional specificity.

Ca2+/calmodulin-dependent protein Kinase II in septally kindled rat brains : changes in protein level, activity and subcellular distribution in hippocampus and cerebral cortex

The protein level and the activity of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in homogenate from septally kindled rat brains were quantitatively compared with those from paired controls 2 weeks after the final stimulation. The major alpha subunit level was decreased, while the activity was increased in crude homogenate from hippocampus and parietal cortex of kindled animals, indicating an apparent increase in the specific activity of CaM kinase II in these regions of the kindled brains. No such changes were observed in cerebellum. After the separation of crude homogenate into the soluble and particulate fractions, the ratio of CaM kinase II activity recovered in the soluble fraction was increased in hippocampus and parietal cortex, indicating a change in subcellular distribution of CaM kinase II in the kindled state.

Regulation of ERK1/2 mitogen-activated protein kinase by NMDA-receptor-induced seizure activity in cortical slices

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.

Ca2+/calmodulin-dependent protein kinase II is reversibly autophosphorylated, inactivated and made sedimentable by acute neuronal excitation in rats in vivo

Ca2+/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(α)/287(β) 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.

Contrasting features of ERK1/2 activity and synapsin I phosphorylation at the ERK1/2-dependent site in the rat brain in status epilepticus induced by kainic acid in vivo

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.

Differential Involvement of Kinase Activity of Ca2+/Calmodulin-Dependent Protein Kinase IIα in Hippocampus- and Amygdala-Dependent Memory Revealed by Kinase-Dead Knock-In Mouse

Abstract 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.

Developmental stage-dependent regulation of spine formation by calcium-calmodulin-dependent protein kinase IIα and Rap1

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.