Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of physiological glutamate signaling involved in higher brain functions. Here, we show CaMKII involvement in ...pathological glutamate signaling relevant in stroke. The novel inhibitor tatCN21 was neuroprotective even when added hours after glutamate insults. By contrast, the "traditional" inhibitor KN93 attenuated excitotoxicity only when present during the insult. Both inhibitors efficiently blocked Ca(2+)/CaM-stimulated CaMKII activity, CaMKII interaction with NR2B and aggregation of CaMKII holoenzymes. However, only tatCN21 but not KN93 blocked the Ca(2+)-independent "autonomous" activity generated by Thr-286 autophosphorylation, the hallmark feature of CaMKII regulation. Mutational analysis further validated autonomous CaMKII activity as the drug target crucial for post-insult neuroprotection. Overexpression of CaMKII wild type but not the autonomy-deficient T286A mutant significantly increased glutamate-induced neuronal death. Maybe most importantly, tatCN21 also significantly reduced infarct size in a mouse stroke model (middle cerebral arterial occlusion) when injected (1 mg/kg intravenously) 1 h after onset of arterial occlusion. Together, these data demonstrate that inhibition of autonomous CaMKII activity provides a promising therapeutic avenue for post-insult neuro-protection after stroke.
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.
Higher brain functions are thought to require synaptic frequency decoding that can lead to long-term potentiation (LTP) or depression (LTD). We show that the LTP versus LTD decision is determined by ...complex cross-regulation of T286 and T305/306 autophosphorylation within the 12meric CaMKII holoenzyme, which enabled molecular computation of stimulus frequency, amplitude, and duration. Both LTP and LTD require T286 phosphorylation, but T305/306 phosphorylation selectively promoted LTD. In response to excitatory LTP versus LTD stimuli, the differential T305/306 phosphorylation directed CaMKII movement to either excitatory or inhibitory synapses, thereby coordinating plasticity at both synapse types. Fast T305/306 phosphorylation required prior T286 phosphorylation and then curbed CaMKII activity by two mechanisms: (i) a cis-subunit reaction reduced both Ca
stimulation and autonomous activity and (ii) a trans-subunit reaction enabled complete activity shutdown and feed-forward inhibition of further T286 phosphorylation. These are fundamental additions to the long-studied CaMKII regulation and function in neuronal plasticity.
Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of physiological glutamate signaling involved in higher brain functions. Here, we show CaMKII involvement in ...pathological glutamate signaling relevant in stroke. The novel inhibitor tatCN21 was neuroprotective even when added hours after glutamate insults. By contrast, the “traditional” inhibitor KN93 attenuated excitotoxicity only when present during the insult. Both inhibitors efficiently blocked Ca2+/CaM-stimulated CaMKII activity, CaMKII interaction with NR2B and aggregation of CaMKII holoenzymes. However, only tatCN21 but not KN93 blocked the Ca2+-independent “autonomous” activity generated by Thr-286 autophosphorylation, the hallmark feature of CaMKII regulation. Mutational analysis further validated autonomous CaMKII activity as the drug target crucial for post-insult neuroprotection. Overexpression of CaMKII wild type but not the autonomy-deficient T286A mutant significantly increased glutamate-induced neuronal death. Maybe most importantly, tatCN21 also significantly reduced infarct size in a mouse stroke model (middle cerebral arterial occlusion) when injected (1 mg/kg intravenously) 1 h after onset of arterial occlusion. Together, these data demonstrate that inhibition of autonomous CaMKII activity provides a promising therapeutic avenue for post-insult neuro-protection after stroke.
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.
•NO-induced autonomous CaMKII activity requires C280 in the α but not β isoform.•NO-induced CaMKIIβ autonomy requires S-nitrosylation of C290.•Oxidation-induced CaMKIIα autonomy requires C289 in ...addition to C280/M281.
The Ca2+/calmodulin-dependent protein kinase II (CaMKII) mediates physiological and pathological functions by its Ca2+-independent autonomous activity. Two novel mechanisms for generating CaMKII autonomy include oxidation and S-nitrosylation, the latter requiring both Cys280 and Cys289 amino acid residues in the brain-specific isoform CaMKIIα. Even though the other CaMKII isoforms have a different amino acid in the position homologous to Cys280, we show here that nitric oxide (NO)-signaling generated autonomy also for the CaMKIIβ isoform. Furthermore, although oxidation of the Met280/281 residues is sufficient to generate autonomy for most CaMKII isoforms, oxidation-induced autonomy was also prevented by a Cys289-mutation in the CaMKIIα isoform. Thus, all CaMKII isoforms can be regulated by physiological NO-signaling, but CaMKIIα regulation by oxidation and S-nitrosylation is more stringent.
A hallmark feature of Ca2+/calmodulin (CaM)‐dependent protein kinase II (CaMKII) is generation of autonomous (Ca2+‐independent) activity by T286 autophosphorylation. Biochemical studies have shown ...that “autonomous” CaMKII is ~5‐fold further stimulated by Ca2+/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 Ca2+/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 Ca2+/CaM binding and was not mimicked by T305/306D mutation. Thus, even autonomous CaMKII activity must be further stimulated by Ca2+/CaM for enhancement of synaptic strength.—Barcomb, K., Buard, I., Coultrap, S. J., Kulbe, J. R., O'Leary, H., Benke, T. A., Bayer, K. U. Autonomous CaMKII requires further stimulation by Ca2+/calmodulin for enhancing synaptic strength. FASEB J. 28, 3810–3819 (2014). www.fasebj.org
Learning and memory requires coordinated activity between different regions of the brain. Here we studied the interaction between infralimbic medial prefrontal cortex (mPFC) and hippocampal dorsal ...CA1 during associative odorant discrimination learning in the mouse. We found that as the animal learns to discriminate odorants in a go-no go task, the coupling of high frequency neural oscillations to the phase of theta oscillations (theta-referenced phase-amplitude coupling or tPAC) changes in a manner that results in divergence between rewarded and unrewarded odorant-elicited changes in the theta-phase referenced power (tPRP) for beta and gamma oscillations. In addition, in the proficient animal there was a decrease in the coordinated oscillatory activity between CA1 and mPFC in the presence of the unrewarded odorant. Furthermore, the changes in tPAC resulted in a marked increase in the accuracy for decoding contextual odorant identity from tPRP when the animal became proficient. Finally, we studied the role of Ca
/calmodulin-dependent protein kinase II α (CaMKIIα), a protein involved in learning and memory, in oscillatory neural processing in this task. We find that the accuracy for decoding the contextual odorant identity from tPRP decreases in CaMKIIα knockout mice and that this accuracy correlates with behavioral performance. These results implicate a role for tPAC and CaMKIIα in olfactory go-no go associative learning in the hippocampal-prefrontal circuit.
Coupling of neural oscillations within and between hippocampal CA1 and medial prefrontal cortex (mPFC) is involved in spatial learning and memory, but the role of oscillation coupling for other learning tasks is not well understood. Here we performed local field potential recording in CA1 and mPFC in mice learning to differentiate rewarded from unrewarded odorants in an associative learning task. We find that odorant-elicited changes in the power of bursts of gamma oscillations at distinct phases of theta oscillations become divergent as the animal becomes proficient allowing decoding of contextual odorant identity. Finally, we find that the accuracy to decode contextual odorant identity decreases in mice deficient for the expression of Ca
/calmodulin-dependent protein kinase II α, a protein involved in synaptic plasticity.
Addictive drugs such as cocaine induce synaptic plasticity in discrete regions of the reward circuit. The aim of the present study is to investigate whether cocaine-evoked synaptic plasticity in the ...ventral tegmental area (VTA) and nucleus accumbens (NAc) is causally linked. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is a central regulator of long-term synaptic plasticity, learning, and drug addiction. We examined whether blocking CaMKII activity in the VTA affected cocaine conditioned place preference (CPP) and cocaine-evoked synaptic plasticity in its target brain region, the NAc. TatCN21 is a CaMKII inhibitory peptide that blocks both stimulated and autonomous CaMKII activity with high selectivity. We report that intra-VTA microinjections of tatCN21 before cocaine conditioning blocked the acquisition of cocaine CPP, whereas intra-VTA microinjections of tatCN21 before saline conditioning did not significantly affect cocaine CPP, suggesting that the CaMKII inhibitor blocks cocaine CPP through selective disruption of cocaine-cue-associated learning. Intra-VTA tatCN21 before cocaine conditioning blocked cocaine-evoked depression of excitatory synaptic transmission in the shell of the NAc slices ex vivo. In contrast, intra-VTA microinjection of tatCN21 just before the CPP test did not affect the expression of cocaine CPP and cocaine-induced synaptic plasticity in the NAc shell. These results suggest that CaMKII activity in the VTA governs cocaine-evoked synaptic plasticity in the NAc during the time window of cocaine conditioning.
The Ca
/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.
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