LTP induction by structural rather than enzymatic functions of CaMKII.

Learning and memory are thought to require hippocampal long-term potentiation (LTP), and one of the few central dogmas of molecular neuroscience that has stood undisputed for more than three decades is that LTP induction requires enzymatic activity of the Ca2+/calmodulin-dependent protein kinase II (CaMKII)1-3. However, as we delineate here, the experimental evidence is surprisingly far from conclusive. All previous interventions inhibiting enzymatic CaMKII activity and LTP4-8 also interfere with structural CaMKII roles, in particular binding to the NMDA-type glutamate receptor subunit GluN2B9-14. Thus, we here characterized and utilized complementary sets of new opto-/pharmaco-genetic tools to distinguish between enzymatic and structural CaMKII functions. Several independent lines of evidence demonstrated LTP induction by a structural function of CaMKII rather than by its enzymatic activity. The sole contribution of kinase activity was autoregulation of this structural role via T286 autophosphorylation, which explains why this distinction has been elusive for decades. Directly initiating the structural function in a manner that circumvented this T286 role was sufficient to elicit robust LTP, even when enzymatic CaMKII activity was blocked.

Stimulating ß-adrenergic receptors promotes synaptic potentiation by switching CaMKII movement from LTD to LTP mode.

Learning, memory, and cognition are thought to require synaptic plasticity, specifically including hippocampal long-term potentiation and depression (LTP and LTD). LTP versus LTD is induced by high-frequency stimulation versus low-frequency, but stimulating ß-adrenergic receptors (ßARs) enables LTP induction also by low-frequency stimulation (1 Hz) or theta frequencies (∼5 Hz) that do not cause plasticity by themselves. In contrast to high-frequency stimulation-LTP, such ßAR-LTP requires Ca2+-flux through L-type voltage-gated Ca2+-channels, not N-methyl-D-aspartate-type glutamate receptors. Surprisingly, we found that ßAR-LTP still required a nonionotropic scaffolding function of the N-methyl-D-aspartate-type glutamate receptor: the stimulus-induced binding of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) to its GluN2B subunit that mediates CaMKII movement to excitatory synapses. In hippocampal neurons, ß-adrenergic stimulation with isoproterenol (Iso) transformed LTD-type CaMKII movement to LTP-type movement, resulting in CaMKII movement to excitatory instead of inhibitory synapses. Additionally, Iso enabled induction of a major cell-biological feature of LTP in response to LTD stimuli: increased surface expression of GluA1 fused with super-ecliptic pHluorein. Like for ßAR-LTP in hippocampal slices, the Iso effects on CaMKII movement and surface expression of GluA1 fused with super-ecliptic pHluorein involved L-type Ca2+-channels and specifically required ß2-ARs. Taken together, these results indicate that Iso transforms LTD stimuli to LTP signals by switching CaMKII movement and GluN2B binding to LTP mode.

Short-term CaMKII inhibition with tatCN19o does not erase pre-formed memory in mice and is neuroprotective in pigs.

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a central regulator of learning and memory, which poses a problem for targeting it therapeutically. Indeed, our study supports prior conclusions that long-term interference with CaMKII signaling can erase pre-formed memories. By contrast, short-term pharmacological CaMKII inhibition with the neuroprotective peptide tatCN19o interfered with learning in mice only mildly and transiently (for less than 1 h) and did not at all reverse pre-formed memories. These results were obtained with ≥500-fold of the dose that protected hippocampal neurons from cell death after a highly clinically relevant pig model of transient global cerebral ischemia: ventricular fibrillation followed by advanced life support and electrical defibrillation to induce the return of spontaneous circulation. Of additional importance for therapy development, our preliminary cardiovascular safety studies in mice and pig did not indicate any concerns with acute tatCN19o injection. Taken together, although prolonged interference with CaMKII signaling can erase memory, acute short-term CaMKII inhibition with tatCN19o did not cause such retrograde amnesia that would pose a contraindication for therapy.

CaMKII T286 phosphorylation has distinct essential functions in three forms of long-term plasticity.

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) mediates long-term potentiation or depression (LTP or LTD) after distinct stimuli of hippocampal NMDA-type glutamate receptors (NMDARs). NMDAR-dependent LTD prevails in juvenile mice, but a mechanistically different form of LTD can be readily induced in adults by instead stimulating metabotropic glutamate receptors (mGluRs). However, the role that CaMKII plays in the mGluR-dependent form of LTD is not clear. Here we show that mGluR-dependent LTD also requires CaMKII and its T286 autophosphorylation (pT286), which induces Ca2+-independent autonomous kinase activity. In addition, we compared the role of pT286 among three forms of long-term plasticity (NMDAR-dependent LTP and LTD, and mGluR-dependent LTD) using simultaneous live imaging of endogenous CaMKII together with synaptic marker proteins. We determined that after LTP stimuli, pT286 autophosphorylation accelerated CaMKII movement to excitatory synapses. After NMDAR-LTD stimuli, pT286 was strictly required for any movement to inhibitory synapses. Similar to NMDAR-LTD, we found the mGluR-LTD stimuli did not induce CaMKII movement to excitatory synapses. However, in contrast to NMDAR-LTD, we demonstrate that the mGluR-LTD did not involve CaMKII movement to inhibitory synapses and did not require additional T305/306 autophosphorylation. Thus, despite its prominent role in LTP, we conclude that CaMKII T286 autophosphorylation is also required for both major forms of hippocampal LTD, albeit with differential requirements for the heterosynaptic communication of excitatory signals to inhibitory synapses.

Young DAPK1 knockout mice have altered presynaptic function.

The death-associated protein kinase 1 (DAPK1) has recently been shown to have a physiological function in long-term depression (LTD) of glutamatergic synapses: acute inhibition of DAPK1 blocked the LTD that is normally seen at the hippocampal CA1 synapse in young mice, and a pharmacogenetic combination approach showed that this specifically required DAPK1-mediated suppression of postsynaptic Ca2+/calmodulin-dependent protein kinase II binding to the NMDA-type glutamate receptor (NMDAR) subunit GluN2B during LTD stimuli. Surprisingly, we found here that genetic deletion of DAPK1 (in DAPK1-/- mice) did not reduce LTD. Paired pulse facilitation experiments indicated a presynaptic compensation mechanism: in contrast to wild-type mice, LTD stimuli in DAPK1-/- mice decreased presynaptic release probability. Basal synaptic strength was normal in young DAPK1-/- mice, but basal glutamate release probability was reduced, an effect that normalized with maturation.NEW & NOTEWORTHY Young death-associated protein kinase 1 (DAPK1) knockout mice have reduced basal glutamate release probability, an effect that normalized with maturation. This provided a compensatory mechanism that may have prevented a reduction of long-term depression in the young DAPK1 knockout mice.

CaMKII regulates the depalmitoylation and synaptic removal of the scaffold protein AKAP79/150 to mediate structural long-term depression.

Both long-term potentiation (LTP) and depression (LTD) of excitatory synapse strength require the Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) and its autonomous activity generated by Thr-286 autophosphorylation. Additionally, LTP and LTD are correlated with dendritic spine enlargement and shrinkage that are accompanied by the synaptic accumulation or removal, respectively, of the AMPA-receptor regulatory scaffold protein A-kinase anchoring protein (AKAP) 79/150. We show here that the spine shrinkage associated with LTD indeed requires synaptic AKAP79/150 removal, which in turn requires CaMKII activity. In contrast to normal CaMKII substrates, the substrate sites within the AKAP79/150 N-terminal polybasic membrane-cytoskeletal targeting domain were phosphorylated more efficiently by autonomous compared with Ca2+/CaM-stimulated CaMKII activity. This unusual regulation was mediated by Ca2+/CaM binding to the substrate sites resulting in protection from phosphorylation in the presence of Ca2+/CaM, a mechanism that favors phosphorylation by prolonged, weak LTD stimuli versus brief, strong LTP stimuli. Phosphorylation by CaMKII inhibited AKAP79/150 association with F-actin; it also facilitated AKAP79/150 removal from spines but was not required for it. By contrast, LTD-induced spine removal of AKAP79/150 required its depalmitoylation on two Cys residues within the N-terminal targeting domain. Notably, such LTD-induced depalmitoylation was also blocked by CaMKII inhibition. These results provide a mechanism how CaMKII can indeed mediate not only LTP but also LTD through regulated substrate selection; however, in the case of AKAP79/150, indirect CaMKII effects on palmitoylation are more important than the effects of direct phosphorylation. Additionally, our results provide the first direct evidence for a function of the well-described AKAP79/150 trafficking in regulating LTD-induced spine shrinkage.

The CaMKII/GluN2B Protein Interaction Maintains Synaptic Strength.

Learning, memory, and cognition are thought to require normal long-term potentiation (LTP) of synaptic strength, which in turn requires binding of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) to the NMDA-type glutamate receptor (NMDAR) subunit GluN2B. For LTP induction, many additional required players are known. Here we tested the hypothesis that CaMKII/GluN2B binding also mediates the more elusive maintenance of synaptic strength. Intriguingly, the CaMKII inhibitor tatCN21 reduces synaptic strength only at high concentrations necessary for CaMKII/NMDAR disruption (20 µm) but not at lower concentrations sufficient for kinase inhibition (5 µm). However, increased concentration also causes unrelated effects. Thus, to distinguish between correlation and causality, we used a pharmacogenetic approach. In a mouse with a mutant NMDAR GluN2B subunit that is CaMKII binding-incompetent, any tatCN21 effects that are specific to the CaMKII/GluN2B interaction should be abolished, and any remaining tatCN21 effects have to be nonspecific (i.e. mediated by other targets). The results showed that the persistent reduction of synaptic strength by transient application of 20 µm tatCN21 had a nonspecific presynaptic component (on fiber volley amplitude) that was unrelated to the CaMKII/GluN2B interaction or CaMKII activity. However, the remaining component of the persistent tatCN21 effect was almost completely abolished in the GluN2B mutant mouse. These results highlight the requirement for stringent pharmacogenetic approaches to separate specific on-target effects from nonspecific off-target effects. Importantly, they also demonstrate that the CaMKII/GluN2B interaction is required not only for normal LTP induction but also for the maintenance of synaptic strength.

Multiple CaMKII Binding Modes to the Actin Cytoskeleton Revealed by Single-Molecule Imaging.

Localization of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) to dendritic spine synapses is determined in part by the actin cytoskeleton. We determined binding of GFP-tagged CaMKII to tag-RFP-labeled actin cytoskeleton within live cells using total internal reflection fluorescence microscopy and single-molecule tracking. Stepwise photobleaching showed that CaMKII formed oligomeric complexes. Photoactivation experiments demonstrated that diffusion out of the evanescent field determined the track lifetimes. Latrunculin treatment triggered a coupled loss of actin stress fibers and the colocalized, long-lived CaMKII tracks. The CaMKIIα (α) isoform, which was previously thought to lack F-actin interactions, also showed binding, but this was threefold weaker than that observed for CaMKIIß (ß). The ßE' splice variant bound more weakly than α, showing that binding by ß depends critically on the interdomain linker. The mutations ßT287D and αT286D, which mimic autophosphorylation states, also abolished F-actin binding. Autophosphorylation triggers autonomous CaMKII activity, but does not impair GluN2B binding, another important synaptic protein interaction of CaMKII. The CaMKII inhibitor tatCN21 or CaMKII mutations that inhibit GluN2B association by blocking binding of ATP (ßK43R and αK42M) or Ca(2+)/calmodulin (ßA303R) had no effect on the interaction with F-actin. These results provide the first rationale for the reduced synaptic spine localization of the αT286D mutant, indicating that transient F-actin binding contributes to the synaptic localization of the CaMKIIα isoform. The track lifetime distributions had a stretched exponential form consistent with a heterogeneously diffusing population. This heterogeneity suggests that CaMKII adopts different F-actin binding modes, which is most easily rationalized by multiple subunit contacts between the CaMKII dodecamer and the F-actin cytoskeleton that stabilize the initial weak (micromolar) monovalent interaction.

Developmental restoration of LTP deficits in heterozygous CaMKIIα KO mice.

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a major mediator of long-term potentiation (LTP) and depression (LTD), two opposing forms of synaptic plasticity underlying learning, memory and cognition. The heterozygous CaMKIIα isoform KO (CaMKIIα+/-) mice have a schizophrenia-related phenotype, including impaired working memory. Here, we examined synaptic strength and plasticity in two brain areas implicated in working memory, hippocampus CA1 and medial prefrontal cortex (mPFC). Young CaMKIIα+/- mice (postnatal days 12-16; corresponding to a developmental stage well before schizophrenia manifestation in humans) showed impaired hippocampal CA1 LTP. However, this LTP impairment normalized over development and was no longer detected in older CaMKIIα+/- mice (postnatal weeks 9-11; corresponding to young adults). By contrast, the CaMKIIα+/- mice failed to show the developmental increase of basal synaptic transmission in the CA1 seen in wild-type (WT) mice, resulting in impaired basal synaptic transmission in the older CaMKIIα+/- mice. Other electrophysiological parameters were normal, including mPFC basal transmission, LTP, and paired-pulse facilitation, as well as CA1 LTD, depotentiation, and paired-pulse facilitation at either age tested. Hippocampal CaMKIIα levels were ∼60% of WT in both the older CaMKIIα+/- mice and in the younger WT mice, resulting in ∼30% of adult WT expression in the younger CaMKIIα+/- mice; levels in frontal cortex were the same as in hippocampus. Thus, in young mice, ∼30% of adult CaMKIIα expression is sufficient for normal LTD and depotentiation, while normal LTP requires higher levels, with ∼60% of CaMKIIα expression sufficient for normal LTP in adult mice.

Nitric oxide induces Ca2+-independent activity of the Ca2+/calmodulin-dependent protein kinase II (CaMKII).

Both signaling by nitric oxide (NO) and by the Ca(2+)/calmodulin (CaM)-dependent protein kinase II α isoform (CaMKIIα) are implicated in two opposing forms of synaptic plasticity underlying learning and memory, as well as in excitotoxic/ischemic neuronal cell death. For CaMKIIα, these functions specifically involve also Ca(2+)-independent autonomous activity, traditionally generated by Thr-286 autophosphorylation. Here, we demonstrate that NO-induced S-nitrosylation of CaMKIIα also directly generated autonomous activity, and that CaMKII inhibition protected from NO-induced neuronal cell death. NO induced S-nitrosylation at Cys-280/289, and mutation of either site abolished autonomy, indicating that simultaneous nitrosylation at both sites was required. Additionally, autonomy was generated only when Ca(2+)/CaM was present during NO exposure. Thus, generation of this form of CaMKIIα autonomy requires simultaneous signaling by NO and Ca(2+). Nitrosylation also significantly reduced subsequent CaMKIIα autophosphorylation specifically at Thr-286, but not at Thr-305. A previously described reduction of CaMKII activity by S-nitrosylation at Cys-6 was also observed here, but only after prolonged (>5 min) exposure to NO donors. These results demonstrate a novel regulation of CaMKII by another second messenger system and indicate its involvement in excitotoxic neuronal cell death.

Live imaging of endogenous Ca²âº/calmodulin-dependent protein kinase II in neurons reveals that ischemia-related aggregation does not require kinase activity.

The Ca(2+) /calmodulin-dependent protein kinase II (CaMKII) 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 Ca(2+) /CaM and nucleotide for its induction. This model makes several predictions that were verified here: Aggregation in vitro was reduced by the CaMKII inhibitors tatCN21 and tatCN19o (which block the T-site) as well as by KN93 (which is CaM-competitive). Notably, these and previously tested manipulations that block CaMKII 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 CaMKII aggregation even in the absence of nucleotide. Within rat hippocampal neurons, an intra-body enabled live monitoring of endogenous CaMKII aggregation. This aggregation was blocked by tatCN21, but not by staurosporine, even though both effectively inhibit CaMKII activity. These results support the mechanistic model for CaMKII aggregation and show that kinase activity is not required. CaMKII aggregation is prevented by inhibiting kinase activity with mutations (red italics; shown previously) or inhibitors (red bold; shown here), indicating requirement of kinase activity. However, we show here that nucleotide-competitive inhibitors (green) allow CaMKII aggregation (including endogenous CaMKII within neurons), demonstrating that kinase activity is not required and supporting the current mechanistic model for CaMKII aggregation.

Autonomous CaMKII requires further stimulation by Ca2+/calmodulin for enhancing synaptic strength.

A hallmark feature of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) is generation of autonomous (Ca(2+)-independent) activity by T286 autophosphorylation. Biochemical studies have shown that "autonomous" CaMKII is ∼5-fold further stimulated by Ca(2+)/CaM, but demonstration of a physiological function for such regulation within cells has remained elusive. In this study, CaMKII-induced enhancement of synaptic strength in rat hippocampal neurons required both autonomous activity and further stimulation. Synaptic strength was decreased by CaMKIIα knockdown and rescued by reexpression, but not by mutants impaired for autonomy (T286A) or binding to NMDA-type glutamate receptor subunit 2B (GluN2B; formerly NR2B; I205K). Full rescue was seen with constitutively autonomous mutants (T286D), but only if they could be further stimulated (additional T305/306A mutation), and not with two other mutations that additionally impair Ca(2+)/CaM binding. Compared to rescue with wild-type CaMKII, the CaM-binding-impaired mutants even had reduced synaptic strength. One of these mutants (T305/306D) mimicked an inhibitory autophosphorylation of CaMKII, whereas the other one (Δstim) abolished CaM binding without introducing charged residues. Inhibitory T305/306 autophosphorylation also reduced GluN2B binding, but this effect was independent of reduced Ca(2+)/CaM binding and was not mimicked by T305/306D mutation. Thus, even autonomous CaMKII activity must be further stimulated by Ca(2+)/CaM for enhancement of synaptic strength.

Persistent reversal of enhanced amphetamine intake by transient CaMKII inhibition.

Amphetamine exposure transiently increases Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) α expression in the nucleus accumbens (NAcc) shell and this persistently increases local GluA1 S831 phosphorylation and enhances behavioral responding to the drug. Here we assessed whether transiently interfering with CaMKII signaling using a dominant-negative CaMKIIα mutant delivered to the NAcc shell with herpes simplex viral vectors could reverse these long-lasting biochemical and behavioral effects observed following exposure to amphetamine. As expected, transient expression of CaMKIIα K42M in the NAcc shell produced a corresponding transient increase in CaMKIIα and decrease in pCaMKIIα (T286) protein levels in this site. Remarkably, this transient inhibition of CaMKII activity produced a long-lasting reversal of the increased GluA1 S831 phosphorylation levels in NAcc shell and persistently blocked the enhanced locomotor response to and self-administration of amphetamine normally observed in rats previously exposed to the drug. Together, these results indicate that even transient interference with CaMKII signaling may confer long-lasting benefits in drug-sensitized individuals and point to CaMKII and its downstream pathways as attractive therapeutic targets for the treatment of stimulant addiction.

IKK regulates the deubiquitinase CYLD at the postsynaptic density.

K63-linked polyubiquitination of proteins regulates their trafficking into specific cellular pathways such as endocytosis and autophagy. CYLD, a deubiquitinase specific for K63-linked polyubiquitins, is present in high quantities at the postsynaptic density (PSD). It was previously shown that, under excitatory conditions, CaMKII activates CYLD in a Ca(2+)-dependent manner. The observation that CYLD can also be phosphorylated in the absence of Ca(2+) in isolated PSDs led us to further explore the regulation of CYLD under basal conditions. A possible involvement of the autonomous form of CaMKII and IKK, both kinases known to be localized at the PSD, was examined. A CaMKII inhibitor CN21 had no effect on CYLD phosphorylation in the absence of Ca(2+), but two different IKK inhibitors, IKK16 and tatNEMO, inhibited its phosphorylation. Immuno-electron microscopy on hippocampal cultures, using an antibody for CYLD phosphorylated at S-418, revealed that the phosphorylated form of CYLD is present at the PSD under basal conditions. Phosphorylation of CYLD under basal conditions was inhibited by IKK16. NMDA treatment further promoted phosphorylation of CYLD at the PSD, but IKK16 failed to block the NMDA-induced effect. In vitro experiments using purified proteins demonstrated direct phosphorylation and activation of CYLD by the beta catalytic subunit of IKK. Activation of IKK in isolated PSDs also promoted phosphorylation of CYLD and an increase in endogenous deubiquitinase activity for K63-linked polyubiquitins. Altogether, the results suggest that in the absence of excitatory conditions, constitutive IKK activity at the PSD regulates CYLD and maintains basal levels of K63-linkage specific deubiquitination at the synapse.

NMDA-induced accumulation of Shank at the postsynaptic density is mediated by CaMKII.

Shank is a specialized scaffold protein present in high abundance at the postsynaptic density (PSD). Using pre-embedding immunogold electron microscopy on cultured hippocampal neurons, we had previously demonstrated further accumulation of Shank at the PSD under excitatory conditions. Here, using the same experimental protocol, we demonstrate that a cell permeable CaMKII inhibitor, tatCN21, blocks NMDA-induced accumulation of Shank at the PSD. Furthermore we show that NMDA application changes the distribution pattern of Shank at the PSD, promoting a 7-10 nm shift in the median distance of Shank labels away from the postsynaptic membrane. Inhibition of CaMKII with tatCN21 also blocks this shift in the distribution of Shank. Altogether these results imply that upon activation of NMDA receptors, CaMKII mediates accumulation of Shank, preferentially at the distal regions of the PSD complex extending toward the cytoplasm.

Induction of LTP mechanisms in dually innervated dendritic spines.

Dendritic spines are the postsynaptic compartments of excitatory synapses, however, a substantial subset of spines additionally receives inhibitory input. In such dually innervated spines (DiSs), excitatory long-term potentiation (LTP) mechanisms are suppressed, but can be enabled by blocking tonic inhibitory GABAB receptor signaling. Here we show that LTP mechanisms at DiSs are also enabled by two other excitatory LTP stimuli. In hippocampal neurons, these chemical LTP (cLTP) stimuli induced robust movement of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) to DiSs. Such synaptic CaMKII accumulation is an essential LTP mechanism at singly innervated spines (SiSs). Indeed, CaMKII accumulation at DiSs was also accompanied by other readouts for successful LTP induction: spine growth and surface insertion of GluA1. Thus, DiSs are capable of the same LTP mechanisms as SiSs, although induction of these mechanism additionally requires either reduced inhibitory signaling or increased excitatory stimulation. This additional regulation may provide further computational control.

Enzymatic activity of CaMKII is not required for its interaction with the glutamate receptor subunit GluN2B.

Binding of the Ca²âº/calmodulin (CaM)-dependent protein kinase II (CaMKII) to the NMDA-type glutamate receptor subunit GluN2B is an important control mechanism for the regulation of synaptic strength. CaMKII binding to GluN2B and CaMKII translocation to synapses are induced by an initial Ca²âº/CaM stimulus, which also activates the kinase. Indeed, several mechanistically different CaMKII inhibitors [tatCN21 and KN-93 (N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide)] and inactivating mutations (K42M, A302R, and T305/T306D) impair this interaction, suggesting that it requires CaMKII enzymatic activity. However, this study shows that two general kinase inhibitors, H7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine] and staurosporine (Sta), which inhibit CaMKII activity by yet another mechanism, did not interfere with GluN2B binding in vitro or within cells. In contrast to a previous report, we found that Sta, like H7, inhibited CaMKII in an ATP-competitive manner. Nucleotide binding significantly enhances CaMKII/GluN2B binding in vitro, but the nucleotide competition by H7 or Sta did not prevent this effect and instead even mimicked it. H7 (700 µM) and Sta (2 µM) efficiently blocked enzymatic activity of CaMKII, both in vitro and within cells. However, neither H7 nor Sta prevented Ca²âº-induced translocation of CaMKII to GluN2B in heterologous cells or to synapses in hippocampal neurons. Thus, activity of CaMKII (or of any other kinase inhibited by H7 or Sta) is not required for stimulation-induced GluN2B-binding or synaptic translocation of CaMKII, despite previous indication to the contrary. This shows that results with inhibitors and inhibiting mutants can be caused by structural effects independent from catalytic activity, and that detailed understanding of the mechanisms is required for their interpretation.

Selective translocation of Ca2+/calmodulin protein kinase IIalpha (CaMKIIalpha) to inhibitory synapses.

Ca(2+)/calmodulin protein kinase IIα (CaMKIIα) has a central role in regulating neuronal excitability. It is well established that CaMKIIα translocates to excitatory synapses following strong glutamatergic stimuli that induce NMDA-receptor (NMDAR)-dependent long-term potentiation in CA1 hippocampal neurons. We now show that CaMKIIα translocates to inhibitory but not excitatory synapses in response to more moderate NMDAR-activating stimuli that trigger GABA(A)-receptor (GABA(A)R) insertion and enhance inhibitory transmission. Such moderate NMDAR activation causes Thr286 autophosphorylation of CaMKIIα, which our results demonstrate is necessary and sufficient, under basal conditions, to localize CaMKIIα at inhibitory synapses and enhance surface GABA(A)R expression. Although stronger glutamatergic stimulation coupled to AMPA receptor insertion also elicits Thr286 autophosphorylation, accumulation of CaMKIIα at inhibitory synapses is prevented under these conditions by the phosphatase calcineurin. This preferential targeting of CaMKIIα to glutamatergic or GABAergic synapses provides neurons with a mechanism whereby activity can selectively potentiate excitation or inhibition through a single kinase mediator.

Distinct synaptic pools of DAPK1 differentially regulate activity-dependent synaptic CaMKII accumulation.

The death-associated protein kinase 1 (DAPK1) regulates the synaptic movement of the Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII). Synaptic CaMKII accumulation is mediated via binding to the NMDA-receptor subunit GluN2B and is required for long-term potentiation (LTP). By contrast, long-term depression (LTD) instead requires specific suppression of this movement, which is mediated by competitive DAPK1 binding to GluN2B. We find here that DAPK1 localizes to synapses via two distinct mechanisms: basal localization requires F-actin, but retention of DAPK1 at synapses during LTD requires an additional binding mode, likely to GluN2B. While F-actin binding mediates DAPK1 enrichment at synapses, it is not sufficient to suppress synaptic CaMKII movement. However, it is a prerequisite that enables the additional LTD-specific binding mode of DAPK1, which in turn mediates suppression of the CaMKII movement. Thus, both modes of synaptic DAPK1 localization work together to regulate synaptic CaMKII localization and thereby synaptic plasticity.

Decreased nitrosylation of CaMKII causes aging-associated impairments in memory and synaptic plasticity in mice.

CaMKII has molecular memory functions because transient calcium ion stimuli can induce long-lasting increases in its synaptic localization and calcium ion-independent (autonomous) activity, thereby leaving memory traces of calcium ion stimuli beyond their duration. The synaptic effects of two mechanisms that induce CaMKII autonomy are well studied: autophosphorylation at threonine-286 and binding to GluN2B. Here, we examined the neuronal functions of additional autonomy mechanisms: nitrosylation and oxidation of the CaMKII regulatory domain. We generated a knock-in mouse line with mutations that render the CaMKII regulatory domain nitrosylation/oxidation-incompetent, CaMKIIΔSNO, and found that it had deficits in memory and synaptic plasticity that were similar to those in aged wild-type mice. In addition, similar to aged wild-type mice, in which CaMKII was hyponitrosylated, but unlike mice with impairments of other CaMKII autonomy mechanisms, CaMKIIΔSNO mice showed reduced long-term potentiation (LTP) when induced by theta-burst stimulation but not high-frequency stimulation (HFS). As in aged wild-type mice, the HFS-LTP in the young adult CaMKIIΔSNO mice required L-type voltage-gated calcium ion channels. The effects in aged mice were likely caused by the loss of nitrosylation because no decline in CaMKII oxidation was detected. In hippocampal neurons, nitrosylation of CaMKII induced its accumulation at synapses under basal conditions in a manner mediated by GluN2B binding, like after LTP stimuli. However, LTP-induced synaptic CaMKII accumulation did not require nitrosylation. Thus, an aging-associated decrease in CaMKII nitrosylation may cause impairments by chronic synaptic effects, such as the decrease in basal synaptic CaMKII.