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.

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.

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.

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.

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

The Ca 2+ /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 tatCN19o interfered with learning in mice only mildly and transiently (for less than 1 h) and did not at all reverse pre-formed memories. This was at ≥500fold 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 return of spontaneous circulation. Of additional importance for therapeutic development, cardiovascular safety studies in mice and pig did not indicate any concerns with acute tatCN19o injection. Taken together, even though 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.

Aß-induced synaptic impairments require CaMKII activity that is stimulated by indirect signaling events.

Aß bears homology to the CaMKII regulatory domain, and peptides derived from this domain can bind and disrupt the CaMKII holoenzyme, suggesting that Aß could have a similar effect. Notably, Aß impairs the synaptic CaMKII accumulation that is mediated by GluN2B binding, which requires CaMKII assembly into holoenzymes. Furthermore, this Aß-induced impairment is prevented by CaMKII inhibitors that should also inhibit the putative direct Aß binding. However, our study did not find any evidence for direct effects of Aß on CaMKII: Aß did not directly disrupt CaMKII holoenzymes, GluN2B binding, T286 autophosphorylation, or kinase activity in vitro. Most importantly, in neurons, the Aß-induced impairment of CaMKII synaptic accumulation was prevented by an ATP-competitive CaMKII inhibitor that would not interfere with the putative direct Aß binding. Together, our results indicate that synaptic Aß effects are not mediated by direct binding to CaMKII, but instead require CaMKII activation via indirect signaling events.

GluN2B S1303 phosphorylation by CaMKII or DAPK1: no indication for involvement in ischemia or LTP.

Binding of two different CaM kinases, CaMKII and DAPK1, to the NMDA-type glutamate receptor (NMDAR) subunit GluN2B near S1303 has been implicated in excitotoxic/ischemic neuronal cell death. The GluN2BΔCaMKII mutation (L1298A, R1300Q) is neuroprotective but abolishes only CaMKII but not DAPK1 binding. However, both kinases can additionally phosphorylate GluN2B S1303. Thus, we here tested S1303 phosphorylation for possible contribution to neuronal cell death. The GluN2BΔCaMKII mutation completely abolished phosphorylation by CaMKII and DAPK1, suggesting that the mutation could mediate neuroprotection by disrupting phosphorylation. However, S1303 phosphorylation was not increased by excitotoxic insults in hippocampal slices or by global cerebral ischemia induced by cardiac arrest and cardiopulmonary resuscitation in vivo. In hippocampal cultures, S1303 phosphorylation was induced by chemical LTD but not LTP stimuli. These results indicate that the additional effect of the GluN2BΔCaMKII mutation on phosphorylation needs to be considered only in LTD but not in LTP or ischemia/excitotoxicity.

CaMKIIα knockout protects from ischemic neuronal cell death after resuscitation from cardiac arrest.

CaMKIIα plays a dual role in synaptic plasticity, as it can mediate synaptic changes in opposing directions. We hypothesized that CaMKIIα plays a similar dual role also in neuronal cell death and survival. Indeed, the CaMKII inhibitor tatCN21 is neuroprotective when added during or after excitotoxic/ischemic insults, but was described to cause sensitization when applied long-term prior to such insult. However, when comparing long-term CaMKII inhibition by several different inhibitors in neuronal cultures, we did not detect any sensitization. Likewise, in a mouse in vivo model of global cerebral ischemia (cardiac arrest followed by cardiopulmonary resuscitation), complete knockout of the neuronal CaMKIIα isoform did not cause sensitization but instead significant neuroprotection.

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.

The CaMKII K42M and K42R mutations are equivalent in suppressing kinase activity and targeting.

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.

CaMKII versus DAPK1 Binding to GluN2B in Ischemic Neuronal Cell Death after Resuscitation from Cardiac Arrest.

DAPK1 binding to GluN2B was prominently reported to mediate ischemic cell death in vivo. DAPK1 and CaMKII bind to the same GluN2B region, and their binding is mutually exclusive. Here, we show that mutating the binding region on GluN2B (L1298A/R1300Q) protected against neuronal cell death induced by cardiac arrest followed by resuscitation. Importantly, the GluN2B mutation selectively abolished only CaMKII, but not DAPK1, binding. During ischemic or excitotoxic insults, CaMKII further accumulated at excitatory synapses, and this accumulation was mediated by GluN2B binding. Interestingly, extra-synaptic GluN2B decreased after ischemia, but its relative association with DAPK1 increased. Thus, ischemic neuronal death requires CaMKII binding to synaptic GluN2B, whereas any potential role for DAPK1 binding is restricted to a different, likely extra-synaptic population of GluN2B.