Both signaling by nitric oxide (NO) and by the Ca2+/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 Ca2+-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 Ca2+/CaM was present during NO exposure. Thus, generation of this form of CaMKIIα autonomy requires simultaneous signaling by NO and Ca2+. 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.
Background: Ca2+-independent autonomous CaMKII activity and nitric oxide (NO) signaling regulate neuronal function and death.
Results: NO generated autonomous CaMKII activity by Ca2+/CaM-dependent S-nitrosylation, and CaMKII inhibition protected from NO-induced neuronal cell death.
Conclusion: NO-mediated regulation of CaMKII contributes to its pathological functions.
Significance:S-Nitrosylation is a novel path to CaMKII autonomy that connects Ca2+- and NO signaling.
CaMKII is an important mediator of forms of synaptic plasticity that are thought to underly learning and memory. The CaMKII mutants K42M and K42R have been used interchangeably as research tools, ...although some reported phenotypic differences suggest that they may differ in the extent to which they impair ATP binding. Here, we directly compared the two mutations at the high ATP concentrations that exist within cells (~4 mM). We found that both mutations equally blocked GluA1 phosphorylation in vitro and GluN2B binding within cells. Both mutations also reduced but did not completely abolish CaMKII T286 autophosphorylation in vitro or CaMKII movement to excitatory synapses in neurons. Thus, despite previously suggested differences, both mutations appear to interfere with ATP binding to the same extent.
Abstract Excitotoxic insults such as cerebral ischemia are thought to enhance neuronal autophagy, which is then thought to promote neuronal cell death. Excitotoxic insults indeed increase autophagy ...markers. Notably, however, autophagy markers can be increased either by autophagy induction (as this enhances their production) or by late-stage autophagy inhibition (as this prevents their degradation during autophagic flux). By comparing each condition with and without protease inhibitors that prevent autophagic degradation of the autophagy markers, the results of this study show that excitotoxic glutamate increases autophagy markers by a late-stage block of autophagy. Initially, this study set out to test if the CaMKII inhibitor tatCN21 mediates its post-insult neuroprotection by regulating autophagy. While tatCN21 partially inhibited basal autophagy in hippocampal neurons, it had no effects on the already blocked autophagy after excitotoxic glutamate insults, indicating that autophagy inhibition is not its neuroprotective mechanism. Additionally, while the autophagy inhibitor chloroquine had no effect, significant neuroprotection was seen instead with two drugs that enhance autophagy induction by different mechanisms, rapamycin (mTOR-dependent) and trehalose (mTOR-independent). This suggests that therapeutic approaches should seek to enhance rather than inhibit autophagy, not only in neurodegenerative diseases (where such approach is widely accepted) but also after acute excitotoxic insults. Together, these findings significantly reshape the current view on the mutual cross-regulation of autophagy and excitotoxicity.
Calcium- and calmodulin-dependent protein kinase II (CaMKII) and glutamate receptors are integrally involved in forms of synaptic plasticity that may underlie learning and memory. In the simplest ...model for long-term potentiation, CaMKII is activated by Ca2+ influx through NMDA (N-methyl-d-aspartate) receptors and then potentiates synaptic efficacy by inducing synaptic insertion and increased single-channel conductance of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. Here we show that regulated CaMKII interaction with two sites on the NMDA receptor subunit NR2B provides a mechanism for the glutamate-induced translocation of the kinase to the synapse in hippocampal neurons. This interaction can lead to additional forms of potentiation by: facilitated CaMKII response to synaptic Ca2+; suppression of inhibitory autophosphorylation of CaMKII; and, most notably, direct generation of sustained Ca2+/calmodulin (CaM)-independent (autonomous) kinase activity by a mechanism that is independent of the phosphorylation state. Furthermore, the interaction leads to trapping of CaM that may reduce down-regulation of NMDA receptor activity. CaMKII-NR2B interaction may be prototypical for direct activation of a kinase by its targeting protein.
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.
The death-associated protein kinase 1 (DAPK1) is a potent mediator of neuronal cell death. Here, we find that DAPK1 also functions in synaptic plasticity by regulating the Ca2+/calmodulin ...(CaM)-dependent protein kinase II (CaMKII). CaMKII and T286 autophosphorylation are required for both long-term potentiation (LTP) and depression (LTD), two opposing forms of synaptic plasticity underlying learning, memory, and cognition. T286-autophosphorylation induces CaMKII binding to the NMDA receptor (NMDAR) subunit GluN2B, which mediates CaMKII synaptic accumulation during LTP. We find that the LTP specificity of CaMKII synaptic accumulation is due to its LTD-specific suppression by calcineurin (CaN)-dependent DAPK1 activation, which in turn blocks CaMKII binding to GluN2B. This suppression is enabled by competitive DAPK1 versus CaMKII binding to GluN2B. Negative regulation of DAPK1/GluN2B binding by Ca2+/CaM results in synaptic DAPK1 removal during LTP but retention during LTD. A pharmacogenetic approach showed that suppression of CaMKII/GluN2B binding is a DAPK1 function required for LTD.
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•DAPK1 binding to GluN2B competes with CaMKII binding and is disrupted by Ca2+/CaM•DAPK1 is removed from synapses during LTP but retained during LTD•DAPK1 activation and GluN2B binding prevents synaptic CaMKII accumulation during LTD•DAPK1-mediated suppression of CaMKII/GluN2B binding is required for LTD
Goodell et al. find that calcineurin (CaN)-dependent activation of the death-associated protein kinase 1 (DAPK1) is required for a form of long-term synaptic plasticity, LTD. Specifically, DAPK1 suppresses Ca2+/calmodulin-dependent protein kinase II (CaMKII) synaptic accumulation and GluN2B binding during LTD, thus making these CaMKII mechanisms LTP specific.
CaMKIIα is a central mediator of bidirectional synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). To study how CaMKIIα movement during plasticity is affected ...by soluble amyloid-β peptide oligomers (Aβ), we used FingR intrabodies to simultaneously image endogenous CaMKIIα and markers for excitatory versus inhibitory synapses in live neurons. Aβ blocks LTP-stimulus-induced CaMKIIα accumulation at excitatory synapses. This block requires CaMKII activity, is dose and time dependent, and also occurs at synapses without detectable Aβ; it is specific to LTP, as CaMKIIα accumulation at inhibitory synapses during LTD is not reduced. As CaMKII movement to excitatory synapses is required for normal LTP, its impairment can mechanistically explain Aβ-induced impairment of LTP. CaMKII movement during LTP requires binding to the NMDA receptor, and Aβ induces internalization of NMDA receptors. However, surprisingly, this internalization does not cause the block in CaMKIIα movement and is observed for extrasynaptic, but not synaptic, NMDA receptors.
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•FingR intrabodies enabled simultaneous live imaging of three endogenous proteins•Aβ inhibits CaMKII trafficking after LTP-, but not LTD-inducing, stimuli•The Aβ-induced block of CaMKII trafficking requires CaMKII activity•Aβ induced internalization of the extrasynaptic, but not synaptic, NMDA receptor
Cook et al. used simultaneous live imaging of multiple endogenous proteins to probe LTP- versus LTD-induced CaMKII trafficking to excitatory versus inhibitory synapses. They show that Aβ inhibits LTP by blocking LTP-induced CaMKII trafficking via signaling mechanisms that require CaMKII activity.
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.
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•Basal synaptic DAPK1 localization requires F-actin•cLTD stimuli promote actin-independent DAPK1 synaptic retention•This indicates two distinct modes of synaptic DAPK1 localization•Both DAPK1 modes act together to suppress CaMKII movement during LTD
Molecular neuroscience; Cellular neuroscience
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 β-adrenergic receptors (βARs) 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 βAR-LTP requires Ca2+-flux through L-type voltage-gated Ca2+-channels, not N-methyl-D-aspartate–type glutamate receptors. Surprisingly, we found that βAR-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, β-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 βAR-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 β2-ARs. Taken together, these results indicate that Iso transforms LTD stimuli to LTP signals by switching CaMKII movement and GluN2B binding to LTP mode.
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 Ca
/calmodulin-dependent protein kinase II (CaMKII)
. However, as we delineate here, the experimental evidence is surprisingly far from conclusive. All previous interventions inhibiting enzymatic CaMKII activity and LTP
also interfere with structural CaMKII roles, in particular binding to the NMDA-type glutamate receptor subunit GluN2B
. 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.
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