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In this review, we discuss the poorly explored role of calcium/calmodulin‐dependent protein kinase II (CaMKII) in memory maintenance, and its influence on memory destabilization. After a brief review on CaMKII and memory destabilization, we present critical pieces of evidence suggesting that CaMKII activity increases retrieval‐induced memory destabilization. We then proceed to propose two potential molecular pathways to explain the association between CaMKII activation and increased memory destabilization. This review will pinpoint gaps in our knowledge and discuss some 'controversial' observations, establishing the basis for new experiments on the role of CaMKII in memory reconsolidation. The role of CaMKII in memory destabilization is of great clinical relevance. Still, because of the lack of scientific literature on the subject, more basic science research is necessary to pursue this pathway as a clinical tool. Calcium/calmodulin‐dependent protein kinase II (CaMKII) is a kinase highly expressed at synapses in the brain. This enzyme has been shown to be necessary for memory formation, but its role on memory maintenance is still a matter of debate. Here, we discuss a new and unexplored role for CaMKII in memory maintenance. We present evidences indicating that after memory retrieval CaMKII is activated and contributes to memory destabilization. We propose two molecular pathways by which CaMKII may induce memory destabilization, involving protein degradation and/or increase in the synaptic levels of GluN2B. We also discuss the possibility that an endogenous CaMKII inhibitor protein controls retrieval‐induced memory destabilization. [ABSTRACT FROM AUTHOR]

Glutamate clearance by astrocytes is an essential part of normal excitatory neurotransmission. Failure to adapt or maintain low levels of glutamate in the central nervous system is associated with multiple acute and chronic neurodegenerative diseases. The primary excitatory amino acid transporters in human astrocytes are EAAT1 and EAAT2 ( GLAST and GLT-1, respectively, in rodents). While the inhibition of calcium/calmodulin-dependent kinase (Ca MKII), a ubiquitously expressed serine/threonine protein kinase, results in diminished glutamate uptake in cultured primary rodent astrocytes (Ashpole et al. 2013), the molecular mechanism underlying this regulation is unknown. Here, we use a heterologous expression model to explore Ca MKII regulation of EAAT1 and EAAT2. In transiently transfected HEK293T cells, pharmacological inhibition of Ca MKII (using KN-93 or tat- CN21) reduces [3H]-glutamate uptake in EAAT1 without altering EAAT2-mediated glutamate uptake. While over-expressing the Thr287Asp mutant to enhance autonomous Ca MKII activity had no effect on either EAAT1 or EAAT2-mediated glutamate uptake, over-expressing a dominant-negative version of Ca MKII (Asp136Asn) diminished EAAT1 glutamate uptake. SPOTS peptide arrays and recombinant glutathione S-transferase-fusion proteins of the intracellular N- and C-termini of EAAT1 identified two potential phosphorylation sites at residues Thr26 and Thr37 in the N-terminus. Introducing an Ala (a non-phospho mimetic) at Thr37 diminished EAAT1-mediated glutamate uptake, suggesting that the phosphorylation state of this residue is important for constitutive EAAT1 function. Our study is the first to identify a glutamate transporter as a direct Ca MKII substrate and suggests that Ca MKII signaling is a critical driver of constitutive glutamate uptake by EAAT1. [ABSTRACT FROM AUTHOR]

Learning leads to changes in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor ( AMPAR)-mediated synaptic excitation. The mechanisms for maintaining such alterations needed for memory persistence remain to be clarified. Here, we report a novel molecular mechanism for maintaining learning-induced AMPAR-mediated enhancement of synaptic excitation. We show that training rats in a complex olfactory discrimination task, such that requires rule learning, leads to the enhancement of averaged amplitude of AMPAR-mediated miniature excitatory post-synaptic currents (m EPSCs) in piriform cortex pyramidal neurons for days after learning. Inhibiting calcium/calmodulin-dependent kinase II (Ca MKII) using KN93 or tat CN21 days after learning, reduced the averaged m EPSC amplitude in neurons in piriform cortex of trained rats to the level where they are not significantly different from m EPSC of control animals. Ca MKII inhibition leads to a decrease in single channel conductance and not to changes in the number of synaptic-activated channels. We conclude that the maintenance of learning-induced enhancement of AMPAR-mediated synaptic excitation requires the activity of calcium/calmodulin-dependent kinase II. [ABSTRACT FROM AUTHOR]

The Ca2+/calmodulin-dependent protein kinase II (Ca MKII) forms 12meric holoenzymes. These holoenzymes cluster into larger aggregates within neurons under ischemic conditions and in vitro when ischemic conditions are mimicked. This aggregation is thought to be mediated by interaction between the regulatory domain of one kinase subunit with the T-site of another kinase subunit in a different holoenzyme, an interaction that requires stimulation by Ca2+/CaM and nucleotide for its induction. This model makes several predictions that were verified here: Aggregation in vitro was reduced by the Ca MKII inhibitors tat CN21 and tat CN19o (which block the T-site) as well as by KN93 (which is CaM-competitive). Notably, these and previously tested manipulations that block Ca MKII activation all reduced aggregation, suggesting an alternative mechanism that instead requires kinase activity. However, experiments with the nucleotide-competitive broad-spectrum kinase inhibitors staurosporin and H7 showed that this is not the case. In vitro, staurosporine and H7 enabled Ca MKII aggregation even in the absence of nucleotide. Within rat hippocampal neurons, an intra-body enabled live monitoring of endogenous Ca MKII aggregation. This aggregation was blocked by tat CN21, but not by staurosporine, even though both effectively inhibit Ca MKII activity. These results support the mechanistic model for Ca MKII aggregation and show that kinase activity is not required. [ABSTRACT FROM AUTHOR]

Metabotropic GABAB receptors are known to modulate the activity of voltage-dependent calcium channels. Previously, we have shown that GABAB receptors couple to a non-Gi/o G-protein to enhance calcium influx through L-type calcium channels by activating protein kinase C in neonatal rat hippocampal neurons. In this study, the components of this signaling pathway were investigated further. Gαq was knocked down using morpholino oligonucleotides prior to examining GABAB-mediated enhancement of calcium influx. When Gαq G-proteins were eliminated using morpholino-mediated knockdown, the enhancing effects of the GABAB receptor agonist baclofen (10 μM) on calcium current or entry were eliminated. These data suggest that GABAB receptors couple to Gαq to regulate calcium influx. Confocal imaging analysis illustrating colocalization of GABAB receptors with Gαq supports this hypothesis. Furthermore, baclofen treatment caused translocation of PKCα (protein kinase C α) but not PKCβ or PKCε, suggesting that it is the α isoform of PKC that mediates calcium current enhancement. Inhibition of calcium/calmodulin-dependent kinase II did not affect the baclofen-mediated enhancement of calcium levels. In summary, activation of GABAB receptors during development leads to increased calcium in a subset of neurons through Gαq signaling and PKCα activation without the involvement of calcium/calmodulin-dependent kinase II. [ABSTRACT FROM AUTHOR]

Although the excitatory action of GABA has been shown to activate the expression of brain-derived neurotrophic factor (BDNF), its molecular mechanisms remain unclear. Using cultured rat cortical cells, we here demonstrated that GABA induced Bdnf mRNA expression mainly via L-type voltagedependent Ca2+ channels (L-VDCC) at the early stage and inhibited it at the late stage of the culture, which corresponded to the excitatory and inhibitory states of cortical cells. The excitatory GABA-induced Bdnf mRNA expression was controlled by multiple Ca2+ signaling pathways including Ca2+/ calmodulin-dependent protein kinase (CaMK), mitogen-activated protein kinase (MAPK) and calcineurin (CN). The Bdnfpromoter IV (Bdnf-pIV) was activated by GABA, mainly via cAMP-response element (CRE)/CREB, and this was prevented by the over-expression of a dominant negative CREB. The nuclear translocation of CREB-regulated transcriptional coactivator 1 (CRTC1) was selectively induced by the GABA-induced CN pathway to activate Bdnf-pIV. On the other hand, GABA-induced Gal4-CREB-dependent transcription, which was controlled by multiple Ca2+ signaling pathways, was prevented when the serine at position 133 of Gal4-CREB was mutated to alanine. Taken together, the excitatory action of GABA transcriptionally activated Bdnf expression through the combination of nuclear-localized CRTC1 and phosphorylated CREB in immature cortical cells, and may be the molecular mechanisms underlying Bdnf expression to control neuronal development. [ABSTRACT FROM AUTHOR]

This article is part of a mini review series: “Synaptic Function and Dysfunction in Brain Diseases”. “Synaptic Function and Dysfunction in Brain Diseases” is a review series with contributions by speakers at the 7th ISN Special Neurochemistry conference, held in Coimbra (Portugal) in June 2016. The authors have surveyed important developments in research on mechanisms of synapse formation, neurotransmitter release, post‐synaptic receptor trafficking, synaptic plasticity, neuronal circuits and cognition and behavior. Changes in these processes underlying neurodegenerative and neurodevelopmental disorders, in addition to their treatment, are also addressed in some of the reviews in the hope of shedding new light on these problems. The paper of this Review Series by Bariselli et al. (DOI: 10.1111/jnc.13779) summarizes current knowledge about neuronal heterogeneity in the Ventral Tegmental Area, a brain structure implicated in processing rewarding and aversive experiences. Figure design by Dominique Fernandes. This article is part of a mini review series: “Synaptic Function and Dysfunction in Brain Diseases”. [ABSTRACT FROM AUTHOR]

Because the cholinergic system is down-regulated in the brain of Alzheimer's disease patients, cognitive deficits in Alzheimer's disease patients are significantly improved by rivastigmine treatment. To address the mechanism underlying rivastigmine-induced memory improvements, we chronically treated olfactory bulbectomized ( OBX) mice with rivastigmine. The chronic rivastigmine treatments for 12-13 days starting at 10 days after OBX operation significantly improved memory-related behaviors assessed by Y-maze task, novel object recognition task, passive avoidance task, and Barnes maze task, whereas the single rivastigmine treatment failed to improve the memory. Consistent with the improved memory-related behaviors, long-term potentiation in the hippocampal CA1 region was markedly restored by rivastigmine treatments. In immunoblotting analyses, the reductions of calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation and calcium/calmodulin-dependent protein kinase IV (CaMKIV) phosphorylation in the CA1 region in OBX mice were significantly restored by rivastigmine treatments. In addition, phosphorylation of AMPAR subunit glutamate receptor 1 (GluA1) (Ser-831) and cAMP-responsive element-binding protein (Ser-133) as downstream targets of CaMKII and CaMKIV, respectively, in the CA1 region was also significantly restored by chronic rivastigmine treatments. Finally, we confirmed that rivastigmine-induced improvements of memory-related behaviors and long-term potentiation were not obtained in CaMKIIα+/− mice. On the other hand, CaMKIV−/− mice did not exhibit the cognitive impairments. Taken together, the stimulation of CaMKII activity in the hippocampus is essential for rivastigmine-induced memory improvement in OBX mice. [ABSTRACT FROM AUTHOR]

Prenatal ethanol exposure causes persistent neurodevelopmental deficits by inducing apoptosis within neuronal progenitors including the neural crest. The cellular signaling events underlying this apoptosis are unclear. Using an established chick embryo model, we previously identified ethanol's activation of calmodulin-dependent protein kinase II (CaMKII) as a crucial early step in this pathway. Here, we report that CaMKII is pro-apoptotic because it mediates the loss of transcriptionally active β-catenin, which normally provides trophic support to these cells. β-catenin over-expression normalized cell survival in ethanol's presence. CaMKII inhibition similarly restored β-catenin content and transcriptional activity within ethanol-treated cells and prevented their cell death. In contrast, inhibition of alternative effectors known to destabilize β-catenin, including glycogen synthase kinase-3β, Protein Kinase C, JNK, and calpain, failed to normalize cell survival and β-catenin activity in ethanol's presence. Importantly, we found that purified CaMKII can directly phosphorylate β-catenin. Using targeted mutagenesis we identified CaMKII phosphorylation sites within human β-catenin at T332, T472, and S552. This is the first demonstration that β-catenin is a phosphorylation target of CaMKII and represents a novel mechanism by which calcium signals could regulate β-catenin-dependent transcription. These results inform ethanol's neurotoxicity and offer unexpected insights into other neurodevelopmental and neurodegenerative disorders having dysregulated calcium or β-catenin signaling. [ABSTRACT FROM AUTHOR]

Two glutamate receptors, metabotropic glutamate receptor 5 ( mGluR5), and ionotropic NMDA receptors (NMDAR), functionally interact with each other to regulate excitatory synaptic transmission in the mammalian brain. In exploring molecular mechanisms underlying their interactions, we found that Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) may play a central role. The synapse-enriched CaMKIIα directly binds to the proximal region of intracellular C terminal tails of mGluR5 in vitro. This binding is state-dependent: inactive CaMKIIα binds to mGluR5 at a high level whereas the active form of the kinase (following Ca2+/calmodulin binding and activation) loses its affinity for the receptor. Ca2+ also promotes calmodulin to bind to mGluR5 at a region overlapping with the CaMKIIα-binding site, resulting in a competitive inhibition of CaMKIIα binding to mGluR5. In rat striatal neurons, inactive CaMKIIα constitutively binds to mGluR5. Activation of mGluR5 Ca2+-dependently dissociates CaMKIIα from the receptor and simultaneously promotes CaMKIIα to bind to the adjacent NMDAR GluN2B subunit, which enables CaMKIIα to phosphorylate GluN2B at a CaMKIIα-sensitive site. Together, the long intracellular C-terminal tail of mGluR5 seems to serve as a scaffolding domain to recruit and store CaMKIIα within synapses. The mGluR5-dependent Ca2+ transients differentially regulate CaMKIIα interactions with mGluR5 and GluN2B in striatal neurons, which may contribute to cross-talk between the two receptors. [ABSTRACT FROM AUTHOR]

The non-selective cationic transient receptor canonical 6 ( TRPC6) channels are involved in synaptic plasticity changes ranging from dendritic growth, spine morphology changes and increase in excitatory synapses. We previously showed that the TRPC6 activator hyperforin, the active antidepressant component of St. John's wort, induces neuritic outgrowth and spine morphology changes in PC12 cells and hippocampal CA1 neurons. However, the signaling cascade that transmits the hyperforin-induced transient rise in intracellular calcium into neuritic outgrowth is not yet fully understood. Several signaling pathways are involved in calcium transient-mediated changes in synaptic plasticity, ranging from calmodulin-mediated Ras-induced signaling cascades comprising the mitogen-activated protein kinase, PI3K signal transduction pathways as well as Ca2+/calmodulin-dependent protein kinase II (CAMKII) and CAMKIV. We show that several mechanisms are involved in TRPC6-mediated synaptic plasticity changes in PC12 cells and primary hippocampal neurons. Influx of calcium via TRPC6 channels activates different pathways including Ras/ mitogen-activated protein kinase/extracellular signal-regulated kinases, phosphatidylinositide 3-kinase/protein kinase B, and CAMKIV in both cell types, leading to cAMP-response element binding protein phosphorylation. These findings are interesting not only in terms of the downstream targets of TRPC6 channels but also because of their potential to facilitate further understanding of St. John's wort extract-mediated antidepressant activity. [ABSTRACT FROM AUTHOR]

The pre-Bötzinger complex (pre-BötC) in the ventrolateral medulla oblongata is a presumed kernel of respiratory rhythmogenesis. Ca2+-activated non-selective cationic current is an essential cellular mechanism for shaping inspiratory drive potentials. Ca2+/calmodulin-dependent protein kinase II (CaMKII), an ideal 'interpreter' of diverse Ca2+ signals, is highly expressed in neurons in mediating various physiological processes. Yet, less is known about Ca MKII activity in the pre-BötC. Using neurokinin-1 receptor as a marker of the pre-BötC, we examined phospho (P)-Ca MKII subcellular distribution, and found that P-Ca MKII was extensively expressed in the region. P-Ca MKII-ir neurons were usually oval, fusiform, or pyramidal in shape. P-Ca MKII immunoreactivity was distributed within somas and dendrites, and specifically in association with the post-synaptic density. In dendrites, most synapses (93.1%) examined with P-Ca MKII expression were of asymmetric type, occasionally with symmetric type (6.9%), whereas in somas, 38.1% were of symmetric type. P-Ca MKII asymmetric synaptic identification implicates that Ca MKII may sense and monitor Ca2+ activity, and phosphorylate post-synaptic proteins to modulate excitatory synaptic transmission, which may contribute to respiratory modulation and plasticity. In somas, Ca MKII acts on both symmetric and asymmetric synapses, mediating excitatory and inhibitory synaptic transmission. P-Ca MKII was also localized to the perisynaptic and extrasynaptic regions in the pre-BötC. [ABSTRACT FROM AUTHOR]

NMDA-type glutamate receptors mediate both trophic and excitotoxic signalling in CNS neurons. We have previously shown that blocking NMDAR- post-synaptic density-95 ( PSD95) interactions provides significant protection from excitotoxicity and in vivo ischaemia; however, the mechanism of neuroprotection is unclear. Here, we report that blocking PSD-95 interactions with the Tat- NR2B9c peptide enhances a Ca2 + -dependent protective pathway converging on cAMP Response Element binding protein ( CREB) activation. We provide evidence that Tat- NR2B9c neuroprotection from oxygen glucose deprivation and NMDA toxicity occurs in parallel with the activation of calmodulin kinase signalling and is dependent on a sustained phosphorylation of the CREB transcription factor and its activator Ca MKIV. Tat- NR2B9c-dependent neuroprotection and CREB phosphorylation are blocked by coapplication of CaM kinase ( KN93 and STO-609) or CREB ( KG-501) inhibitors, and by si RNA knockdown of Ca MKIV. These results are mirrored in vivo in a rat model of permanent focal ischaemia. Tat- NR2B9c application significantly reduces infarct size and causes a selective and sustained elevation in Ca MKIV phosphorylation; effects which are blocked by coadministration of KN93. Thus, calcium-dependent nuclear signalling via Ca MKIV and CREB is critical for neuroprotection via NMDAR- PSD95 blockade, both in vitro and in vivo. This study highlights the importance of maintaining neuronal function following ischaemic injury. Future stroke research should target neurotrophic and pro-survival signal pathways in the development of novel neuroprotective strategies. [ABSTRACT FROM AUTHOR]

ATP-gated P2X3 receptors of sensory ganglion neurons are important transducers of painful stimuli and are modulated by extracellular algogenic substances, via changes in the receptor phosphorylation state. The present study investigated the role of calcium/calmodulin-dependent serine protein kinase ( CASK) in interacting and controlling P2X3 receptor expression and function in mouse trigeminal ganglia. Most ganglion neurons in situ or in culture co-expressed P2X3 and CASK. CASK was immunoprecipitated with P2X3 receptors from trigeminal ganglia and from P2X3/ CASK-cotransfected human embryonic kidney ( HEK) cells. Recombinant P2X3/ CASK expression in HEK cells increased serine phosphorylation of P2X3 receptors, typically associated with receptor upregulation. CASK deletion mutants also enhanced P2X3 subunit expression. After silencing CASK, cell surface P2X3 receptor expression was decreased, which is consistent with depressed P2X3 currents. The reduction in P2X3 expression levels was reversed by the proteasomal inhibitor MG-132. Moreover, neuronal CASK/P2X3 interaction was up-regulated by nerve growth factor ( NGF) signaling and down-regulated by P2X3 agonist-induced desensitization. These data suggest a novel interaction between CASK and P2X3 receptors with positive outcome for receptor stability and function. As CASK-mediated control of P2X3 receptors was dependent on the receptor activation state, CASK represents an intracellular gateway to regulate purinergic nociceptive signaling. [ABSTRACT FROM AUTHOR]