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- MATERIALS AND METHODS
Summary: Purpose: This study was conducted to characterize the early cellular changes in CaM kinase II activity that occur during the induction of status epilepticus (SE).
Methods: The pilocarpine model of SE was characterized both behaviorally and electrographically. At specific time points after the first discrete seizure, specific brain regions were isolated for biochemical study. Phosphate incorporation into a CaM kinase II–specific substrate, autocamtide III, was used to determine kinase activity.
Results: After the development of SE, the data show an immediate inhibition of both cortical and hippocampal CaM kinase II activity in homogenate, but a delayed inhibition in synaptic kinase activity. The maintenance of synaptic kinase activity was due to a translocation of CaM kinase II protein to the synapse. However, despite the translocation of functional kinase, CaM kinase II activity was not maintained, membrane potential was not restored, and the newly translocated CaM kinase II did not terminate the SE event. Unlike the homogenate samples, in the crude synaptoplasmic membrane (SPM) subcellular fractions, a positive correlation is found between the duration of SE and the inhibition of CaM kinase II activity in both the cortex and hippocampus.
Conclusions: The data support the hypothesis that alterations of CaM kinase II activity are involved in the early events of SE pathology.
Status epilepticus (SE) is a life-threatening emergency affecting >100,000 people annually in the United States (1–4) and is associated with a significant mortality rate (4–6). SE is commonly defined as continuous seizures lasting ≥30 min without recovery of consciousness (2). Recently, Lowenstein et al. (7,8) defined SE in adults and children older than 5 years as ≥5 min of continuous seizures or two or more discrete seizures between which incomplete recovery of consciousness occurs (7,8). This definition is justified by the fact that most tonic–clonic seizures cease within 5 min (9). Additionally, a loss of efficacy of front-line medications is found, including benzodiazepines (BZDs), as seizure duration increases (3). This decreased efficacy is observed both clinically and in experimental models. For this reason, we sought to elucidate the early cellular mechanisms that are involved in the loss of membrane-potential regulation and drug efficacy during SE.
Calcium/calmodulin-dependent protein kinase II (CaM kinase II) is a neuronally enriched protein that makes up the majority of the total protein in the postsynaptic density (10). CaM kinase II has been shown to modulate positively both glutamatergic (11–16) and γ-aminobutyric acid (GABA)ergic channels (17–19). For instance, an increase in CaM kinase II activity has been associated with induction of long-term potentiation (LTP) (16,20–26). Activation of CaM kinase II results in this increased phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, which causes an increase in AMPA function, and is a critical step in the induction of LTP. Injection of CaM kinase II-α subunit also has been shown to modulate GABAergic channels. Activation of CaM kinase II has been shown to increase agonist-evoked inhibitory currents (17) and to increase both agonist (18) and allosteric modulator binding (19). Because CaM kinase II positively modulates both excitability and inhibitory synaptic receptor function, alteration of CaM kinase II activity would alter neuronal membrane excitability.
CaM kinase II has been well studied, and its activity is altered in many central nervous system pathologies (27). It has been shown that CaM kinase II is significantly inhibited after stroke (28). Additionally, multiple laboratories have demonstrated that epileptiform activity also causes an inhibition of CaM kinase II (29–31). One hour of SE has been shown to inhibit CaM kinase II (32–34) activity in both the short (35,36) and the long term (32) after a single SE episode. However, little research has looked at the early cellular changes that occur during SE induction, and no research has been done to characterize the temporal relation between the induction of SE and the inhibition of CaM kinase II that occurs.
If loss of CaM kinase II activity is involved in the early pathological events in SE, then it is important to determine at what time point during/immediately following the initiation of SE that the inhibition of CaM kinase II occurs. This study used EEG monitoring in the Pilo-SE model and behavioral observations to track the temporal profile of specific cellular changes occurring as the pathology is developing. Coincident with the development of SE, the data show an immediate inhibition of CaM kinase II activity in homogenate, but a delayed inhibition in synaptic activity. The data suggest that the inhibition of CaM kinase II activity is involved in the early cellular responses during the induction of SE.
- Top of page
- MATERIALS AND METHODS
This study used electrographic mapping of seizure progression in the Pilo-SE model. By mapping seizure progression in the Pilo-SE model, we demonstrated an immediate inhibition of total CaM kinase II activity that was coincident with SE onset, despite no overall decrease in CaM kinase II protein levels. One possible mechanism to explain this observation is that a posttranslational modification occurred early in the course of SE that resulted in a rapid decrease in kinase activity. The results also show a delayed inhibition in synaptic CaM kinase II activity. The synaptic kinase activity was maintained by translocation of additional enzyme to the SPM. These observations suggest that the brain may be attempting to preserve synaptic kinase function; however, as SE duration increases, the attempt to maintain kinase activity fails. The data support the hypothesis that alterations of CaM kinase II activity are involved in the early events of SE pathology.
At least two scenarios exist whereby modulation of CaM kinase II activity is involved in the series of events of SE induction. One possibility for the initial increase in synaptic CaM kinase II activity is an extension of a pharmacologic response. On a synaptic level, high-frequency stimulation has been shown induce LTP, which is associated with an increase in synaptic CaM kinase II activity (25,26,47–49), autophosphorylation level (50), and translocation of additional enzyme to the synaptic region (51–53). One hypothesis for this is that the increase in CaM kinase II causes an enhancement of glutamate receptors (AMPA and NMDA), which has been shown to increase synaptic strength. This increases the function of the circuit by increasing the signal-to-noise ratio, relative to neighboring neuronal circuits.
The current study shows that at least two events associated with LTP also are observed in the Pilo-SE model: an initial increase in total synaptic CaM kinase II activity as well as a translocation. However, unlike LTP, the observed increase in SPM CaM kinase II occurs over the whole structures of both the cortex and the hippocampus, not just in specific synapses. This suggests that, unlike the increase in efficacy for specific synaptic complexes, an increase is found in whole-brain synaptic efficacy. That is, the two to three discrete seizures may be equivalent to whole-brain tetanic stimulation, which should result in an increase in whole-brain synaptic CaM kinase II. Instead of increasing the signal-to-noise ratio, as observed in LTP, an increase in whole-brain synaptic efficacy would increase overall brain responsiveness in the form of seizure activity. It also is possible that the increase in synaptic efficacy increases neuronal responsiveness enough to overwhelm the mechanisms that normally terminate seizure activity. Thus the translocation and increase in CaM kinase II activity may be involved in the mechanisms that induce SE in this model.
Support for this theory is that a transient increase takes place in the synaptic CaM kinase II activity at 10 min after the first discrete seizure as well as a translocation in the crude SPM. Both of these observations occur coincident with SE onset. Additionally, animals that have discrete seizures, but no SE, do not experience a translocation, nor do they have an inhibition of CaM kinase II activity after 1 h of SE (36). It has been noted and is currently being studied that rats that have only one discrete seizure typically do not develop SE, whereas rats that have two to three discrete seizures within 10 min of each other typically progress into SE. Therefore future studies will characterize CaM kinase II modulation in animals that display discrete seizures but do not develop SE. This would require the ability to segregate nonresponder animals accurately (animals that display discrete seizure activity but do not develop SE) from rats that develop SE. Preliminary data from our laboratory suggest that a measurable difference exists between the discrete seizure phase for these two groups that can predict which rats will enter SE (Kumar et al., unpublished data).
Another scenario is that the inhibition of CaM kinase II activity may be a surrogate marker for pathology in the Pilo-SE model. Supporting this theory is that, in the homogenate isolated from SE animals, an immediate inhibition of CaM kinase II activity is found. However, in rats that have discrete seizures, but do not develop SE, no inhibition occurs (36). Whereas a delayed inhibition of CaM kinase II activity was found in the crude SPM, an increase in calcium-independent activity was not observed. In many learning and memory models, autophosphorylation of the Thr286 residue in CaM kinase II results in significant calcium-independent activity (54–56). Because no increase in calcium-independent activity was observed in the present study, it is likely that other mechanisms are involved in the translocation of CaM kinase II to synaptic structures. For instance, the translocation may be an attempt by the neuron to maintain or regain synaptic function during SE. However, despite the translocation of functional kinase, CaM kinase II activity is not maintained, membrane potential is not restored, and the newly translocated CaM kinase II does not terminate the SE event.
Conversely, another possibility is that the translocation of CaM kinase II to the SPM is an epiphenomenon. It has been shown that cellular mechanisms are in place that transport CaM kinase II to the synapse in multiple models (57–60). In the crude SPM, increased protein levels maintain CaM kinase II activity toward an exogenously applied substrate. However, activity toward endogenous substrates must be determined to confirm whether a functional translocation of CaM kinase II exists in SE. It is possible that the kinase does not reach the appropriate endogenous targets. This could be due to the newly translocated CaM kinase II not being able to gain access to synaptic targets for the kinase because of the inhibited CaM kinase II blocking access to the endogenous substrate. To test this theory, endogenous substrates must be studied to determine CaM kinase II incorporation.
A more likely possibility is that all of these mechanisms are involved in the observed effects of CaM kinase II. During the discrete seizure phase, the initial translocation of the CaM kinase II is in response to a whole-brain LTP-like event. However, as SE progresses, even the SPM kinase is inhibited. At this point, loss of kinase activity may actually be a mechanism for induction of epilepsy, a process termed epileptogenesis. Once the inhibition of CaM kinase II activity is induced, it has been shown to be long-lasting (32). In other models of chronic kinase inhibition, significant seizure activity is observed. Butler et al. (61) showed significant seizure activity in a chronic knockdown mouse model. In addition, knockdown of CaM kinase II α-subunit protein expression also results in seizure activity in hippocampal neuronal cultures (44), possibly because of loss of GABAergic modulation (46). Thus inhibition of CaM kinase II activity has significant implications for regulation of neuronal excitability.
Because the inhibition in total CaM kinase II activity was observed within 10 min, it is not probable that the SE-induced inhibition is due to a change in transcription levels. Additionally, the inhibition was not due to a decrease in protein level. These two observations suggest that the inhibition of CaM kinase II is due to a posttranslational modification. Additionally, the inhibition of CaM kinase II is not likely due to autophosphorylation at the threonine305 residue because no change in phosphatase activity has been reported in SE. Support for this theory comes from studies in cerebral ischemia in which addition of exogenous phosphatases did not reverse the inhibition of CaM kinase II activity. In addition, SE has been shown to increase calcineurin activity (62). Although calcineurin does not directly act on CaM kinase II, increasing calcineurin activity should remove inhibition of phosphatase 1 by inhibitor 1. The overall result would be increased phosphatase activity, which should reduce Thr305 phosphorylation. Future studies will be directed at the cellular changes due to the inhibition of CaM Kinase II as SE progresses.
The findings in this article characterize the temporal aspects of both the electrographic and neurochemical changes during the induction of SE in the rat pilocarpine model. A significant inhibition of CaM kinase II activity was observed coincident with the induction of SE in both cortical and hippocampal homogenates. Thus CaM kinase II activity provides a neuronal marker for the aggressive treatment of SE (7,8). Future studies will be to directed at the cellular changes due to the inhibition of CaM kinase II as SE progresses.