Severn B. Churn

Temperature modulation of ischemic neuronal death and inhibition of calcium/calmodulin-dependent protein kinase II in gerbils.

We used brief bilateral carotid artery occlusion in gerbils to examine the effects of temperature on ischemia-induced inhibition of calcium/calmodulin-dependent protein kinase II activity and neuronal death. In normothermic (36 degrees C) gerbils, ischemia induced a severe loss of hippocampal CA1 pyramidal neurons measured 7 days after ischemia (28.4 neurons/mm, n = 10; control density in 10 naive gerbils 262.1 neurons/mm) and a significant decrease in forebrain calcium/calmodulin-dependent protein kinase II autophosphorylation measured 2 hours after ischemia (12.9 fmol/min, n = 6; control phosphorylation in six naive gerbils 23.5 fmol/min). The effect of temperature on these indicators of ischemic damage was examined by adjusting intracerebral temperature before and during the ischemic insult. Hyperthermic (39 degrees C) gerbils showed almost complete loss of neurons in the CA1 region (3.0 neurons/mm, n = 11) and extension of neuronal death into the CA2, CA3, and CA4 regions. In addition, hyperthermia exacerbated ischemia-induced inhibition of calcium/calmodulin-dependent protein kinase II activity (4.2 fmol/min, n = 6). Hypothermia (32 degrees C) protected against ischemia-induced CA1 pyramidal cell damage (257.0 neurons/mm, n = 20) and inhibition of calcium/calmodulin-dependent protein kinase II activity (26.0 fmol/min, n = 6). Our results are consistent with the hypothesis that loss of calcium/calmodulin-dependent protein kinase II activity may be a critical event in the development of ischemia-induced cell death.

Inability to restore resting intracellular calcium levels as an early indicator of delayed neuronal cell death

The hippocampus is especially vulnerable to excitotoxicity and delayed neuronal cell death. Chronic elevations in free intracellular calcium concentration ([Ca2+]i) following glutamate-induced excitotoxicity have been implicated in contributing to delayed neuronal cell death. However, no direct correlation between delayed cell death and prolonged increases in [Ca2+]i has been determined in mature hippocampal neurons in culture. This investigation was initiated to determine the statistical relationship between delayed neuronal cell death and prolonged increases in [Ca2+]i in mature hippocampal neurons in culture. Using indo-1 confocal fluorescence microscopy, we observed that glutamate induced a rapid increase in [Ca2+]i that persisted after the removal of glutamate. Following excitotoxic glutamate exposure, neurons exhibited prolonged increases in [Ca2+]i, and significant delayed neuronal cell death was observed. The N-methyl-D-aspartate (NMDA) channel antagonist MK-801 blocked the prolonged increases in [Ca2+]i and cell death. Depolarization of neurons with potassium chloride (KCl) resulted in increases in [Ca2+]i, but these increases were buffered immediately upon removal of the KCl, and no cell death occurred. Linear regression analysis revealed a strong correlation (R = 0.973) between glutamate-induced prolonged increases in [Ca2+]i and delayed cell death. These data suggest that excitotoxic glutamate exposure results in an NMDA-induced inability to restore resting [Ca2+]i (IRRC) that is a statistically significant indicator of delayed neuronal cell death.

Global Forebrain Ischemia Induces a Posttranslational Modification of Multifunctional Calcium‐ and Calmodulin‐Dependent Kinase II

Abstract: The activity of multifunctional calcium/calmodulin‐dependent protein kinase II (CaM kinase II) has recently been shown to be inhibited by transient global ischemia. To investigate the nature of ischemia‐induced inhibition of the enzyme, CaM kinase II was purified to > 1,000‐fold from brains of control and ischemic gerbils. The characteristics of CaM kinase II from control and ischemic preparations were compared by numerous parameters. Kinetic analysis of purified control and ischemic CaM kinase II was performed for autophosphorylation properties, ATP, magnesium, calcium, and calmodulin affinity, immunoreactivity, and substrate recognition. Ischemia induced a reproducible inhibition of CaM kinase II activity, which could not be overcome by increasing the concentration of any of the reaction parameters. Ischemic CaM kinase II was not different from control enzyme in affinity for calmodulin, Ca2+, Mg2+, or exogenously added substrate or rate of autophosphorylation. CaM kinase II isolated from ischemic gerbils displayed decreased immunoreactivity with a monoclonal antibody (immunoglobulin G3) directed toward the β subunit of the enzyme. In addition, ischemia caused a significant decrease in affinity of CaM kinase II for ATP when measured by extent of autophosphorylation. To characterize further the decrease in ATP affinity of CaM kinase II, the covalent‐binding ATP analog 8‐azidoadenosine‐5′‐[α‐32P]triphosphate was used. Covalent binding of 25 μM azido‐ATP was decreased 40.4 ± 12.3% in ischemic CaM kinase II when compared with control enzyme (n = 5; p < 0.01 by paired Students t test). Thus, CaM kinase II levels for ischemia and control fractions were equivalent by protein staining, percent recovery, and calmodulin binding but were significantly different by immunoreactivity and ATP binding. The data are consistent with the hypothesis that ischemia induces a posttranslational modification that alters ATP binding in CaM kinase II and that results in an apparent decrease in enzymatic activity.

Status epilepticus results in an N-methyl-d-aspartate receptor-dependent inhibition of Ca2+/calmodulin-dependent kinase II activity in the rat

Status epilepticus is a major medical emergency that results in significant alteration of neuronal function. Status epilepticus involves seizure activity recurring frequently enough to induce a sustained alteration in brain function. This study was initiated to investigate how status epilepticus affects the activity of calcium and calmodulin-dependent kinase II in the brain. Calcium and calmodulin-dependent kinase II is a neuronally enriched signal transducing system involved in the regulation of neurotransmitter synthesis and release, cytoskeletal function, gene transcription, neurotransmitter receptor function and neuronal excitability. Therefore, alteration of this signal transduction system would have significant physiological effects. Status epilepticus was induced in rats by pilocarpine injection, allowed to progress for 60 min and terminated by repeated diazepam injections. Animals were killed at specific time-points and examined for calcium and calmodulin-dependent kinase II activity. Calcium and calmodulin-dependent kinase II activity was significantly reduced in cerebral cortex and hippocampal homogenates obtained from status epilepticus rats when compared with control animals. Once established, the status epilepticus-induced inhibition of calcium and calmodulin-dependent kinase II activity was observed at all time-points tested following the termination of seizure activity. However, calcium and calmodulin-dependent kinase II activity was not significantly decreased in thalamus and cerebellar homogenates. In addition, status epilepticus-induced inhibition of calcium and calmodulin-dependent kinase II activity was dependent upon activation of N-methyl-D-aspartate subtype of glutamatergic receptors. Thus, status epilepticus induced a significant inhibition of calcium and calmodulin-dependent kinase II activity that involves N-methyl-D-aspartate receptor activation. The data support the hypothesis that inhibition of calcium and calmodulin-dependent kinase II activity may be involved in the alteration of neuronal function following status epilepticus.

Traumatic Brain Injury Causes an FK506-Sensitive Loss and an Overgrowth of Dendritic Spines in Rat Forebrain

Traumatic brain injury (TBI) causes both an acute loss of tissue and a progressive injury through reactive processes such as excitotoxicity and inflammation. These processes may worsen neural dysfunction by altering neuronal circuitry beyond the focally-damaged tissue. One means of circuit alteration may involve dendritic spines, micron-sized protuberances of dendritic membrane that support most of the excitatory synapses in the brain. This study used a modified Golgi-Cox technique to track changes in spine density on the proximal dendrites of principal cells in rat forebrain regions. Spine density was assessed at 1 h, 24 h, and 1 week after a lateral fluid percussion TBI of moderate severity. At 1 h after TBI, no changes in spine density were observed in any of the brain regions examined. By 24 h after TBI, however, spine density had decreased in ipsilateral neocortex in layer II and III and dorsal dentate gyrus (dDG). This apparent loss of spines was prevented by a single, post-injury administration of the calcineurin inhibitor FK506. These results, together with those of a companion study, indicate an FK506-sensitive mechanism of dendritic spine loss in the TBI model. Furthermore, by 1 week after TBI, spine density had increased substantially above control levels, bilaterally in CA1 and CA3 and ipsilaterally in dDG. The apparent overgrowth of spines in CA1 is of particular interest, as it may explain previous reports of abnormal and potentially epileptogenic activity in this brain region.

Chronic inhibition of Ca2+/calmodulin kinase II activity in the pilocarpine model of epilepsy

The development of symptomatic epilepsy is a model of long-term plasticity changes in the central nervous system. The rat pilocarpine model of epilepsy was utilized to study persistent alterations in calcium/calmodulin-dependent kinase II (CaM kinase II) activity associated with epileptogenesis. CaM kinase II-dependent substrate phosphorylation and autophosphorylation were significantly inhibited for up to 6 weeks following epileptogenesis in both the cortex and hippocampus, but not in the cerebellum. The net decrease in CaM kinase II autophosphorylation and substrate phosphorylation was shown to be due to decreased kinase activity and not due to increased phosphatase activity. The inhibition in CaM kinase II activity and the development of epilepsy were blocked by pretreating seizure rats with MK-801 indicating that the long-lasting decrease in CaM kinase II activity was dependent on N-methyl-D-aspartate receptor activation. In addition, the inhibition of CaM kinase II activity was associated in time and regional localization with the development of spontaneous recurrent seizure activity. The decrease in enzyme activity was not attributed to a decrease in the alpha or beta kinase subunit protein expression level. Thus, the significant inhibition of the enzyme occurred without changes in kinase protein expression, suggesting a long-lasting, post-translational modification of the enzyme. This is the first published report of a persistent, post-translational alteration of CaM kinase II activity in a model of epilepsy characterized by spontaneous recurrent seizure activity.

Global forebrain ischemia results in decreased immunoreactivity of calcium/calmodulin-dependent protein kinase II

Previous studies utilizing crude brain homogenates have shown that forebrain ischemia results in inhibition of calcium/calmodulin-dependent protein kinase II (CaM kinase II) activity without large-scale proteolysis of the enzyme. In this report, a monoclonal antibody (1C3-3D6) directed against the β- (60-kDa) subunit of CaM kinase II that does not recognize ischemically altered enzyme was utilized to further investigate the ischemia-induced inhibition of CaM kinase II. Immunohistochemical investigations showed that the ischemia-induced decreased immunoreactivity of CaM kinase II occurred immediately following ischemic insult in ischemia-sensitive cells such as pyramidal cells of the hippocampus. No decrease in CaM kinase II immunoreactivity was observed in ischemia-resistant cells such as granule cells of the dentate gyrus. The decreased immunoreactivity was observed for CaM kinase II balanced for protein staining and calmodulin binding in vitro. In addition, autophosphorylation of CaM kinase II in the presence of low (7 μM) or high (500 μM) ATP did not alter immunoreactivity of the enzyme with 1C3-3D6. The data demonstrate the production of a monoclonal antibody that recognizes the β-subunit of CaM kinase II in a highly specific manner, but does not recognize ischemic enzyme. Together with previous studies, the data support the hypothesis that rapid, ischemia-induced inhibition of CaM kinase II activity may be involved in the cascade of events that lead to selective neuronal cell loss in stroke.

A cellular mechanism for dendritic spine loss in the pilocarpine model of status epilepticus

Purpose:  Previous studies have documented a synaptic translocation of calcineurin (CaN) and increased CaN activity following status epilepticus (SE); however, the cellular effect of these changes in CaN in the pathology of SE remains to be elucidated. This study examined a CaN‐dependent modification of the dendritic cytoskeleton. CaN has been shown to induce dephosphorylation of cofilin, an actin depolymerization factor. The ensuing actin depolymerization can lead to a number of physiological changes that are of interest in SE.

Pilocarpine‐Induced Status Epilepticus Causes N‐Methyl‐D‐Aspartate Receptor‐Dependent Inhibition of Microsomal Mg2+/Ca2+ ATPase‐Mediated Ca2+ Uptake

Abstract: Status epilepticus is associated with sustained and elevated levels of cytosolic Ca2+. To elucidate the mechanisms associated with changes of cytosolic Ca2+ after status epilepticus, this study was initiated to evaluate the effect of pilocarpine‐induced status epilepticus on Mg2+/Ca2+ ATPase‐mediated Ca2+ uptake in microsomes isolated from rat cortex, because the Ca2+ uptake mechanism plays a major role in regulating intracellular Ca2+ levels. The data demonstrated that the initial rate and overall Ca2+ uptake in microsomes from pilocarpinetreated animals were significantly inhibited compared with those in microsomes from saline‐treated control animals. It was also shown that the inhibition of Ca2+ uptake caused by status epilepticus was not an artifact of increased Ca2+ release from microsomes, selective isolation of damaged microsomes from the homogenate, or decreased Mg2+/Ca2+ ATPase protein in the microsomes. Pretreatment with the NMDA antagonist dizocilpine maleate blocked status epilepticus‐induced inhibition of the initial rate and overall Ca2+ uptake. The data suggest that inhibition of microsomal Mg2+/Ca2+ ATPase Ca2+ uptake is involved in NMDA‐dependent deregulation of cytosolic Ca2+ homeostasis associated with status epilepticus.

Long-lasting decrease in neuronal Ca2+/calmodulin-dependent protein kinase II activity in a hippocampal neuronal culture model of spontaneous recurrent seizures.

Ca2+/calmodulin-dependent protein kinase II (CaM Kinase II) activity was evaluated in a well-characterized in vitro model of epileptiform activity. Long-lasting spontaneous recurrent seizure (SRS) activity was induced in hippocampal neuronal cultures by exposure to low Mg2+ media for 3 h. Analysis of endogenous Ca2+/calmodulin-dependent phosphorylation revealed a significant long-lasting decrease in 32P incorporation into the alpha (50 kDa) and beta (60 kDa) subunits of CaM kinase II in association with the induction of SRS activity in this preparation. Ca2+/calmodulin-dependent substrate phosphorylation of the synthetic peptides, Autocamtide-2 and Syntide II, was also significantly reduced following the induction of SRSs and persisted for the life of the neurons in culture. The decrement in CaM kinase II activity associated with low Mg2+ treatment remained significantly decreased when values were corrected for changes in levels of alpha subunit immunoreactivity and neuronal cell loss. Addition of the protein phosphatase inhibitors, okadaic acid and cyclosporin A, to the phosphorylation reaction did not block the SRS-associated decrease in substrate phosphorylation, indicating that enhanced phosphatase activity was not a contributing factor to the observed decrease in phosphate incorporation. The findings of this study demonstrate that CaM kinase II activity is decreased in association with epileptogenesis observed in these hippocampal cultures and may contribute to the production and maintenance of SRSs in this model.