Abstract
Ischemic insults on neurons trigger excessive, pathological glutamate release that causes Ca2+ overload resulting in neuronal cell death (excitotoxicity). The Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of physiological excitatory glutamate signals underlying neuronal plasticity and learning. Glutamate stimuli trigger autophosphorylation of CaMKII at T286, a process that makes the kinase “autonomous” (partially active independent from Ca2+ stimulation) and that is required for forms of synaptic plasticity. Recent studies suggested autonomous CaMKII activity also as potential drug target for post-insult neuroprotection, both after glutamate insults in neuronal cultures and after focal cerebral ischemia in vivo. However, CaMKII and other members of the CaM kinase family have been implicated in regulation of both neuronal death and survival. Here, we discuss past findings and possible mechanisms of CaM kinase functions in excitotoxicity and cerebral ischemia, with a focus on CaMKII and its regulation.
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Introduction
Global cerebral ischemia is caused by a general loss of oxygen supply to the brain (during drowning, suffocation, or cardiac arrest). While the increased availability of automated external defibrillators has dramatically increased the survival rate after cardiac arrest, survivors may suffer from neuronal damage caused by the oxygen deficit in the brain. Despite increased demand for a neuroprotective treatment of global cerebral ischemia, no effective therapy has been developed to date. Focal cerebral ischemia (stroke) also involves decreased oxygen supply to parts of the brain. However, focal cerebral ischemia is caused by regional lack of blood supply after clotting or hemorrhage of blood vessels in the brain. The focal core area of the stroke is generally considered to be beyond rescue, however, neurons in the surrounding penumbra (where secondary cell loss occurs) are potential targets for therapeutic intervention1 (Figure 1). Currently, the only available FDA approved pharmacological treatment is haemolytic therapy with tissue plasminogen activator (tPA). However, tPA is contra-indicated in hemorrhagic stroke, and by the time most patients receive diagnostic evaluation, tPA is no longer effective (and may even do more harm than good)2, 3, 4. Thus, less than 2% of stroke patients actually receive tPA5, leaving a significant void in therapies that are more universally applicable for stroke treatment. Two independent studies recently implicated CaMKII as a promising drug target for post-insult neuroprotection6, 7. Here, we discuss the CaM kinase family and its possible involvement in the regulation of neuronal cell death, with a focus on CaMKII and its regulation.
Glutamate excitotoxicity causes neuronal cell death after cerebral ischemia
Glutamate is the major excitatory neurotransmitter in the mammalian brain. However, excessive glutamate also leads to neuronal cell death via “glutamate excitotoxicity”, a process that involves Ca2+ overload8 and that was first demonstrated in retinal neurons9, and subsequently in the brain10, 11. Ischemic conditions trigger anoxic depolarization of neurons, which in turn triggers massive release of glutamate1, 12, 13. It is thought that the excitotoxic effect of such glutamate overstimulation causes much of the neuronal cell death seen following cerebral ischemia13, 14, 15, 16. Additionally, excitotoxic glutamate signaling is thought to contribute to neuronal cell death after traumatic brain injury and in neurodegenerative diseases16, 17, 18. Excitotoxicity can cause neuronal death with apoptotic or necrotic appearance19, 20, but both may involve similar signaling pathways despite a different morphological end state14, 21, 22. Overstimulation of most glutamate receptors can cause neuronal cell death, but the Ca2+ conducting NMDA-type glutamate receptors appear to be the most sensitive “death receptors”23, 24, 25, 26: Even relatively brief (∼5 min) application of glutamate or NMDA to cultured neurons can trigger signaling events that cause significant cell death within 24 h. Consequently, extensive efforts were made to develop NMDA-receptor inhibitors into stroke therapies, unfortunately without success27, 28. Alternative strategies include targeting signaling molecules downstream of the NMDA-receptor, such as CaMKII, which has been shown to mediate key effects of physiological NMDA-receptor stimulation in neuronal plasticity and learning and memory29, 30, 31.
CaMKII and the CaM kinase family
CaM kinases (Figure 2) are a large family of Ser/Thr protein kinases that include kinases with broad substrate spectrum (ie multifunctional kinases such as CaMKI, CaMKII, and CaMKIV) and with high substrate selectivity [ie dedicated kinases such as myosin light chain kinases (MLCKs) and phosphorylase kinase (PhK)]. As the name implies, CaM kinases are generally activated by binding of Ca2+/CaM to their regulatory region. However, several CaM kinases (including DAPK3 and AMPKs) lack a CaM-binding regulatory region, but are included in the CaM kinase family based on high homology of their core kinase domain. Regulation by Ca2+/CaM does not automatically classify a kinase in the CaM kinase family. For instance, CaMKIII (now termed eEF2 kinase), which is also activated by Ca2+/CaM, is not closely related to the other CaM kinases32 and is instead grouped with the family of atypical protein kinases33. Like many other kinases (including PKA, PKB/Akt, and PKC), several CaM kinase family members require phosphorylation within the activation loop of their core kinase domain for full activity (including CaMKI T177, CaMKIV T196, and AMPKs T172). Interestingly, an upstream kinase that phosphorylates the activation loop of CaMKI, CaMKIV, and AMPK (as well as PKB/Akt, which is not a CaM kinase) is CaMKK, which is itself a Ca2+/CaM-stimulated kinase34. However, other CaM kinases (including CaMKII and DAPKs) do not even have a phosphorylatable residue at the homologous activation loop position, even though their activity can be regulated by other phosphorylation events outside of their core kinase domains (such as T286 of CaMKII, which makes the kinase partially Ca2+/CaM-independent35, 36, 37, 38, and S735 of DAPK1, which further enhances Ca2+/CaM stimulated activity39).
Functionally, DAPKs (death associated protein kinases) are associated with regulation of cell death40, MLCKs (myosin light chain kinases) regulate smooth muscle contraction41, and AMPKs (AMP activated kinases) are regulators of energy metabolism42. CaMKI, CaMKII, and CaMKIV have been implicated in various neuronal functions, including plasticity30, 31, 43. CaMKII comprises a family of closely related kinases, with four isoforms (α, β, γ, and δ) encoded by different genes, and alternative splicing gives rise to additional diversity31. At least one CaMKII isoform was found to be expressed in every cell type examined, with CaMKIIγ and CaMKIIδ being the most ubiquitous isoforms44, 45. CaMKIIα is almost exclusively expressed in brain, where it is also extremely abundant, making up more than 1% of total protein in some brain regions, such as the hippocampus46. Notably, the hippocampus, specifically its CA1 region, is also the brain area that is most sensitive to damage following global cerebral ischemia47. The hippocampus is important in memory formation, and the CaMKIIα knockout mice were the first knockout mice described to show impaired neuronal plasticity and learning48, 49.
CaMKII structure and regulation
The relationship between CaMKII structure and regulation has been reviewed previously in detail31. However, there have since been several significant advances, especially regarding CaMKII structure50, 51, 52, 53 (Figure 3). CaMKII forms 12meric holoenzymes, with the C-terminal association domains forming a central hub and the N-terminal kinase domains radiating outwards like spokes or petals (Figure 3A). Between the core kinase domain and the association domain, the CaMKII subunits contain a Ca2+/CaM-binding autoregulatory region followed by a variable region that is subject to extensive alternative splicing. The largest described splice variant is CaMKII βM (72 kDa)54; the dominant isoforms in brain are α (50 kDa) and β (60 kDa)55, 56. Holoenzymes can be homomeric or heteromeric (ie formed by subunits of the same or different isoforms)54, 56, 57, 58, 59, 60, with a molecular weight of ∼600–750 kDa and a diameter of ∼20 nm38, 50, 51, 52, 61, 62.
The α-helical autoregulatory region is responsible for holding CaMKII in an inactive state under basal conditions (Figure 3B). Its N-terminal part surrounding T286 interacts with the core kinase domain at a region termed the T286-binding site (T-site)51, 63, 64. When the autoregulatory region is bound to the T-site, access to the adjacent substrate binding S-site is blocked, thus preventing CaMKII activity (Figure 3B). Each kinase subunit within a holoenzyme is activated separately by direct binding of Ca2+/CaM to the autoregulatory region (“stimulated activity”). This Ca2+/CaM-binding displaces the autoregulatory region from the T-site, allowing the kinase to make other protein-protein interactions via the T-site (see below). Importantly, displacement of the autoregulatory region also allows access to the S-site, thereby activating the kinase. Additionally, releasing T286 from the T-site makes T286 accessible for auto-phosphorylation by neighboring subunits within the holoenzyme, provided these neighboring subunits are also activated with an exposed S-site65, 66. This auto-phosphorylation of T286 prevents complete re-association of the autoregulatory region with the T- and S-sites, leaving CaMKII partially active even after dissociation of Ca2+/CaM (“autonomous activity”)35, 36, 37, 67. This autonomous activity has been described as a molecular memory mechanism and is indeed important for synaptic plasticity and learning67, 68. More recently, autonomous CaMKII activity has also been identified as a drug target for post-insult neuroprotection6, 7.
An additional regulatory auto-phosphorylation at T305/306 can occur as an intra-subunit reaction and prevents subsequent Ca2+/CaM binding69, 70, 71, 72 (Figure 3B). Vice versa, Ca2+/CaM binding prevents this inhibitory auto-phosphorylation. Thus, efficient T305/306 auto-phosphorylation occurs only after generation of autonomous activity by T286 auto-phosphorylation and subsequent Ca2+/CaM dissociation. Such a triple-phosphorylated CaMKII would be locked in a partially active state that cannot be further stimulated67, but it would be completely inactivated only after selective T286 dephosphorylation.
Glutamate-induced translocation of CaMKII
Ca2+-influx through NMDA-type glutamate receptors stimulates CaMKII activity and T286 auto-phosphorylation. Both are required for induction of long-term potentiation of synaptic strength68, 73, 74, a form of synaptic plasticity underlying learning and memory29, 30, 75. Additionally, glutamate-induced Ca2+-influx causes two forms of CaMKII translocation: (i) to post-synaptic sites64, 76, 77, 78, and (ii) to extra-synaptic clusters79, 80, 81, 82. Synaptic translocation occurs after physiological glutamate stimuli and is involved in synaptic plasticity83, 84. Several synaptic proteins have been described as binding partners for CaMKII, but the NMDA receptor subunit GluN2B (formerly known as NR2B) appears to be most important for CaMKII translocation to the synapse64, 78, 85, 86, 87, 88, 89. Extra-synaptic clustering has been described after pathological glutamate stimuli and ischemic conditions79, 80, 81, 82, and is likely mediated by self-association of multiple CaMKII holoenzymes into large insoluble complexes79, 90, 91, a process here termed aggregation. Both types of translocation require protein-interactions via the CaMKII T-site, specifically T-site binding to the GluN2B region around S130364, 85, 92 or the region around T286 of a CaMKII subunit within another holoenzyme82, 90, 91. Accordingly, both interactions require Ca2+/CaM-binding, in order to make the T-site accessible. Both interactions are also enhanced by nucleotide. However, holoenzyme aggregation in vitro requires mimicking ischemic conditions, ie low ATP/ADP ratio and low pH. High ATP concentrations favor T286 auto-phosphorylation, which enhances CaMKII binding to GluN2B64, 85, but inhibits CaMKII aggregation (as it inhibits interaction of T286 with the T-site)91. The molecular basis for requirement of a pH below 6.8 for CaMKII aggregation is unclear, but may involve protonation of His282, which is located at the hinge of the auto-regulatory α-helix (Figure 3B). CaMKII aggregation causes a reduction in the degree of activity that can be stimulated by Ca2+/CaM79, an effect also observed in brain after ischemia93, 94, 95. For this reason, it has been speculated that CaMKII aggregation may be a neuroprotective mechanism, as it would limit aberrant CaMKII activity during disregulated, pathological Ca2+ signaling79. However, the potential neuroprotective functions of CaMKII aggregation have yet to be determined.
Inhibitors of CaMKII
The traditional small molecule CaMKII inhibitors KN62 and KN9396, 97 have proved to be very useful tools for studying CaMKII functions in cells, as they are membrane penetrating and their selectivity for CaMKII is relatively good96, 97, 98. However, the KN inhibitors cannot distinguish between CaMKII and CaMKIV99. Through an unusual affect on the scaffolding protein AKAP79/150100, KN inhibitors can also inhibit PKC action at the synapse101. Maybe more importantly, KN inhibitors additionally affect voltage gated Ca2+ and K+ channels102, 103. Another limitation is that both KN62 and KN93 are competitive with CaM and inhibit only stimulated but not autonomous CaMKII activity6, 96, 97. Peptide inhibitors derived from the CaMKII autoregulatory region, such as AC3-I or AIP7, 104, 105, are generally thought to be more selective than the KN compounds. However, they can also inhibit other kinases, including PKC, MLCK, and PKD106, 107, 108. Additionally, some studies indicated a relatively low potency (IC50 of ∼3 μmol/L)109, 110.
More recently, an alternative was provided by CN21, a potent and selective CaMKII inhibitor111. CN21 is a 21mer peptide derived from the natural CaMKII inhibitory protein CaM-KIIN112. Peptides and even proteins can be made cell penetrating by fusion with sequence motifs such as ant/penetratin or tat113, 114, 115. In initial attempts to make CN peptides cell penetrating, ant fusion was used116, 117. However, it was subsequently shown that the ant/penetratin sequence directly binds CaM, an effect further enhanced by fusion with CN2174. Thus, while fusion of CN to ant added an additional CaMKII-inhibitory mechanism, selectivity for CaMKII was compromised. However, alternative fusion of CN21 to tat still allowed cell-penetration without the caveat of binding CaM74. The resulting tatCN21 is a potent (IC50 of ∼50 nmol/L), selective, and cell penetrating peptide inhibitor of both stimulated and autonomous CaMKII activity6, 74, 111. Consistent with previous reports that tat fusion peptides can cross the blood brain barrier26, 118, tatCN21 inhibited CaMKII functions in brain even after systemic application6, 74.
CaMKII autonomy as a drug target for post-insult neuroprotection
Inhibiting stimulated and autonomous CaMKII activity with tatCN21 attenuated neuronal cell death induced by glutamate insult in primary cultures as well as in a mouse model of stroke (middle cerebral arterial occlusion; MCAO), even when administered significantly after the insult6. The longest post-insult time periods tested in this study were 6 h in hippocampal cultures, 1 h in cortical cultures, and 1 h in vivo6. A recent study independently confirmed post-insult neuroprotection by CaMKII inhibition, using both tatCN21 and tatAIP7. This study found significant neuroprotection in cortical cultures also when tatCN21 was administered 2 h after the insult7. By contrast, the traditional CaMKII inhibitor KN93 was neuroprotective only when administered during but not after the insult in both studies6, 7. During an insult, tatCN21, tatAIP, and KN93 can inhibit CaMKII activation and/or generation of autonomous activity. However, after the insult, tatCN21 and tatAIP but not KN93 can inhibit autonomous CaMKII activity that has already been generated. Thus, these findings indicated that autonomous CaMKII activity is the relevant drug target for post-insult neuroprotection by tatCN21. Indeed, overexpression of CaMKII wild-type or the constitutively autonomous T286D mutant (which mimics T286 phosphorylation) increased glutamate-induced neuronal death significantly more than overexpression of the autonomy-incompetent T286A mutant6. Consistent with these recent results6, 7, neuroprotection by CaMKII inhibition had been described previously, although only for inhibition during or prior to excitotoxic insults119, 120, 121, 122, 123. Protection was seen in cortical cultures6, 7, 119, 121, hippocampal cultures6, 123 and retinal cells120, 122. However, other studies indicated an opposite effect, ie that abolishing CaMKII activity can promote neuronal cell death124, 125, 126, 127, 128. Some of these results may be explained by different death-inducing stimuli and different culture systems, as cerebellar granule cells and spiral ganglion cells depend on depolarization-induced Ca2+ signals for survival126, 127, 128. Additionally, different results using the inhibitor KN93 may depend on the balance between death- and survival-promoting signals mediated by CaMKII and CaMKIV, respectively125. Notably, however, genetic knock-out of CaMKIIα resulted in increased infarct size in a mouse model of stroke124, the opposite effect from the observation after acute CaMKII inhibition with tatCN216. This difference could be explained by developmental effects caused by the absence of CaMKIIα. Indeed, the CaMKIIα knock-out mice are epileptic129, and hyper-excitability may contribute to higher susceptibility to ischemic insults. Additionally, inhibition of CaMKII and complete removal of CaMKIIα protein may have profoundly different effects on glutamate-induced neuronal death. Thus, it will be interesting to compare CaMKII knock down by RNAi with CaMKII inhibition in neuronal cultures and to compare CaMKIIα knock-out mice with existing knock-in mice that carry the inactive CaMKIIα K42R mutation130. However, a recent study indicated that long-term (8–24 h) inhibition of CaMKII activity (without loss of CaMKII protein) is sufficient to increase vulnerability to subsequent excitotoxic insults7, even though acute CaMKII inhibition during or after the insults reduced neuronal cell death (Table 1)6, 7.
Other CaM kinases in neuronal death and survival
CaMKII is not the only CaM kinase family member implicated in the regulation of neuronal survival and cell death. For example, CaMKIV activity has been described to be neuroprotective in multiple systems, likely by its phosphorylation and activation of the transcription factor CREB127, 131, 132, 133. CaMKI may have a similar effect, as it can activate Mek/Erk, which, in turn, also activates CREB134. CaMKK activity is also neuroprotective, as its inhibition by STO-609135 increases neuronal cell death6. CaMKK is an upstream activator of at least two survival kinases, CaMKIV (see above) and Akt/PKB (which is not a member of the CaM kinase family)136. However, CaMKK is also an upstream activator of AMPK, a non-CaM-binding CaM kinase family member that has been implicated in promoting stroke-related neuronal death137, 138. Interestingly, acute treatment with the AMPK activator metformin during stroke insults increases neuronal death, while previous treatment with metformin before the insult reduces cell death139. This is similar to the phenomenon of ischemic preconditioning, in which previous mild insults can partially protect from the effect of subsequent stronger insults140, 141, 142, 143, indicating that AMPK may be part of a mechanism for this ischemic preconditioning. Thus, AMPK is also a possible target for stroke treatment, however, the effect of therapeutically relevant post-insult AMPK inhibition has not yet been tested144.
Death-associated protein kinases (DAPKs) are another branch of the CaM kinases family associated with the regulation of cell death and survival40. DAPK1 has specifically been implicated in mediating excitotoxicity and stroke related death of neurons145. Interestingly, this required an interaction between DAPK1 and the NMDA-receptor subunit GluN2B145 at the same site that also interacts with CaMKII64, 92. This raises the possibility that some of the neuroprotective treatments designed to target DAPK1 or CaMKII may act in part through effects on the other kinase; in fact, targeting both kinases with the same compound may be desirable for maximal therapeutic effect. However, molecular manipulations indicated that targeting each kinase separately is also neuroprotective independent from effects on the other kinase6, 145. Interestingly, manipulations that dissociate the scaffolding protein PSD95 from GluN2B also protect neurons from excitotoxic and stroke induced death26, 146. It has been proposed that this neuroprotection is due to the resulting dissociation of neuronal nitric oxide synthase (nNOS) from the NMDA-receptor26, 146. It will be interesting to test if this manipulation additionally indirectly affects GluN2B interaction with CaMKII and/or DAPK1.
What are the downstream targets for CaMKII after excitotoxic insults?
The mechanisms by which inhibition of autonomous CaMKII activity may mediate post-insult neuroprotection are currently unclear. Possible pathways by which CaMKII may participate in the regulation of neuronal death and survival are described below and summarized in Figure 4. Potentially death promoting effects of CaMKII activity include increase of AMPA-receptor single channel conductance by phosphorylating its GluR1 subunit at S831147, 148. This effect is especially pronounced for GluR1-homomeric AMPA-receptors149, which are (in contrast to GluR2 containing channels) Ca2+-conducting150, 151, and could thus further promote death-inducing Ca2+ overload. Indeed, such Ca2+-conducting AMPA-receptors have been implicated in ischemic injury152. CaMKII could also further increase the Ca2+ overload by facilitating or potentiating L-type voltage dependent Ca2+ channels (VDCCs) via their α or β subunits153, 154. Additionally, CaMKII can directly interact with connexin hemichannels155, which are important in neuronal homeostasis and for neuron-glia communication and have been implicated in glutamate-induced cell death156, 157, 158. CaMKII activity may also contribute to neuronal cell death through phosphorylation of acid-sensing ion channels, which enhances the ischemia-induced activation of the ion channel123. Recently, CaMKII activity was also shown to be required for ischemia-induced shuttling of cytoplasmic polyadenylation element binding 4 (CPEB4) into the nucleus159. CPEBs regulate cytoplasmic polyadenylation and translation in neurons160, 161, and some cross-talk between CPEB1 and CaMKII signaling has been described162, 163. CPEB4 knock down induces neuronal death, which can be rescued by re-expression of CPEB4 wild type, but not to the same extent by its nuclear export-incompetent mutant159. Thus, nuclear retention of CPEB4 by CaMKII activity may contribute to ischemia-induced neuronal death. Another protein that induces neuronal death upon nuclear shuttling is apoptosis inducing factor (AIF)164. However, involvement of CaMKII in nuclear shuttling of AIF has not yet been tested.
On the other hand, several downstream effects of CaMKII activity could instead promote neuronal survival. Maybe most prominently, CaMKII can phosphorylate and inhibit nNOS165, 166, 167, 168, thereby reducing production of nitric oxide, which is neuro-toxic14, 169, 170, 171, 172. CaMKII can also regulate several ion channels in a way that may afford neuroprotection from excitotoxic insults: It promotes desensitization NMDA-receptors173, and it enhances surface expression of the inhibitory GABAA-receptors174, 175. Additionally, CaMKII can activate pro-survival proteins such as Erk117, 176 and CREB177, inhibit death-promoting proteins such GSK-3178 and Bad127. CaMKII can also promote neuronal survival by inhibiting HDAC5126, a CaMKII substrate that inhibits the pro-survival transcription factor MEF179. These possible mechanisms may explain why the effect of CaMKII inhibition on cell survival can depend on the cell type and/or the death-inducing stimulus. Maybe more importantly, it may also explain why prolonged reduction of CaMKII activity increases vulnerability to subsequent excitotoxic insults7, 124. However, there is ample evidence that acute CaMKII inhibition protects hippocampal, cortical, and retinal neurons from glutamate excitotoxicity6, 7, 119, 120, 121, 122, 123, consistent with the neuroprotection seen in a mouse model of stroke6.
CaMKII regulation of apoptotic cell death: lessons from non-neuronal systems?
Apoptosis is a common mechanism of regulated cell death that involves caspase activation, nuclear condensation, and DNA fragmentation180, 181, 182, 183. Ca2+ signaling and CaMKII have been linked to the regulation of apoptosis also in non-neuronal systems. However, as for the neuronal CaMKII targets discussed above, some of these links to apoptosis can promote cell death while others promote survival. CaMKII has been shown to be involved in mediating apoptosis induced by TNFα and UV irradiation184 microcystein (phosphatase inhibitor)185, 186, GW7845 (PPARγ agonist)187, ER stress188, TRAIL189, and -in cardiac myocytes- by isoproterenol (β adrenergic agonist)190, 191, ouabain (Na+/K+-ATPase inhibitor)192, oxidative stress (induced by H2O2 or angiotensin II)193, 194, 195, and cardiac ischemia196, 197, 198. In these cases, CaMKII inhibition attenuated apoptotic death. However, CaMKII inhibition has also been described to enhance apoptosis, indicating that CaMKII can also suppress apoptosis in other systems199, 200, 201, 202, 203, 204. Anti-apoptotic CaMKII mechanisms included inhibition of caspase 2199, enhancing expression of Bcl-xL203, and promoting Akt-mediated inhibition of Bad204. Interestingly, CaMKII-mediated activation of Akt has been linked to both pro-189 and anti-apoptotic200, 201, 204 functions.
In the heart, CaMKII contributes to cell death in response to a variety of insults, and CaMKII inhibition consistently promoted cell survival205 (see above). However, even in the heart, the situation is more complicated: While cytoplasmic CaMKII isoforms were consistently found to promote cell death, nuclear isoforms (ie CaMKII δB)206 may instead promote survival194, 207.
Clearly, the mechanisms by which CaMKII can regulate cell viability are complex and in need of further elucidation. However, the dual role for CaMKII in mediating both cell survival and death may not be that surprising after all, given that its principal activator, Ca2+, is already well established to mediate both208, 209. Even Ca2+ signaling mediated by NMDA receptors can promote not only death but also survival210, 211. Generally, CaMKII appears likely to have pro-survival function in systems and situations in which Ca2+ is required for maintaining cell viability (such as activity-mediated neuronal survival). By contrast, in situations where Ca2+ acts instead as a trigger of death signaling, CaMKII is likely to be involved as a major mediator of cell death (such as in cerebral ischemia and exitotoxicity).
Concluding remarks and therapeutic potential
Recent results implicated that autonomous activity of CaMKII, induced by T286 auto-phosphorylation, provides a promising drug target for post-insult neuroprotection after cerebral ischemia and possibly other conditions involving glutamate excitotoxicity6, 7. In the future, therapy development may include identification of selective small-molecule inhibitors of autonomous CaMKII. However, the peptide inhibitor tatCN21 itself also holds promise, as it was effective even after systemic delivery in an animal model6, 74. The toxicology for tatCN21 is still lacking, but no immediately obvious toxic effects were observed in mice. TatCN21 did interfere with learning in mice, which could be a potential contraindication for chronic treatment in humans74. However, for acute treatment after stroke, a temporary learning impairment would be more than acceptable, especially since tatCN21 did not interfere with memory storage or retrieval74, and is thus unlikely to induce retrograde amnesia. Potency of tatCN21 is very reasonable, with an IC50 of ∼50 nmol/L74, and further improvements of potency may be possible. However, further studies are needed in order to elucidate the downstream mechanisms linking CaMKII to the neuronal cell death that is induced by ischemic conditions. Additionally, the window of therapeutic opportunity (at least 1 h in a mouse model of stroke6) needs to be evaluated more closely after different types of ischemic insults in vivo. After glutamate insults in culture, hippocampal neurons were significantly protected by tatCN21 application even 6 h after the insult6, while protection of cortical neurons was only seen when tatCN21 was applied less than 3 h after the insult7. This difference may be caused by the type of glutamate insult (400 μmol/L for 5 min6, versus 100 μmol/L for 60 min7). However, if the difference is instead inherent to the neuronal cell type, tatCN21 may have an even longer window of therapeutic opportunity after global cerebral ischemia, which particularly affects the hippocampus47, compared to focal cerebral ischemia (stroke). Additionally, the therapeutic potential in other acute conditions involving glutamate excitotoxicity, such as traumatic brain injury, should be evaluated.
Financial disclosure
The University of Colorado is currently seeking patent protection for tatCN21, its derivatives, and its uses (including the ones outlined here).
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Acknowledgements
The structural model of the CaMKII holoenzyme was kindly provided by Drs Luke CHAO and John KURIYAN (University of California, Berkeley). Support was provided by NIH training grants T32GM007635 and F31NS061584 (RSV), Ralph W and Grace M Showalter (AH), Indiana State Department of Health, Spinal Cord Brain Injury research grant ISDH/A70-0-0-079212 (AH), and NIH R01NS052644 (KUB).
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Coultrap, S., Vest, R., Ashpole, N. et al. CaMKII in cerebral ischemia. Acta Pharmacol Sin 32, 861–872 (2011). https://doi.org/10.1038/aps.2011.68
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DOI: https://doi.org/10.1038/aps.2011.68
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