εPKC May Contribute to the Protective Effect of Hypothermia in a Rat Focal Cerebral Ischemia Model
Background and Purpose— Protein kinase C epsilon (εPKC) has been implicated as a neuroprotectant in vitro. We studied εPKC activation (by its localization and proteolysis) in a rodent stroke model and correlated the effects of hypothermia with εPKC activity after cerebral ischemia.
Methods— Rats were subjected to permanent distal middle cerebral artery occlusion plus 1 hour of bilateral common carotid artery occlusion. Body temperatures were maintained at 37°C or 30°C during common carotid artery occlusion. Brains were harvested at 10 minutes, 4 hours, and 24 hours after common carotid artery release, and the cortex corresponding to the ischemic core and penumbra was dissected. εPKC localization after stroke was assessed by Western blot and immunofluorescence microscopy. A caspase-3 inhibitor was used to test whether εPKC cleavage is caspase dependent.
Results— εPKC in the membrane fraction and whole-protein homogenates decreased moderately in the penumbra but decreased markedly in the ischemic core. Hypothermia blocked this decrease in both the ischemic core and penumbra. Confocal microscopy confirmed that neuronal εPKC expression decreased in the ischemic core at 4 hours after reperfusion, and this loss was prevented by hypothermia. Two carboxyl-terminal cleavage products of εPKC with molecular masses of 43 and 35 kDa were detected. Although the protein band of 43 kDa decreased after stroke, the 35-kDa band increased. Such changes were not dependent on caspase-3. However, hypothermia blocked changes in the cleavage form of 35 kDa but not 43 kDa after stroke.
Conclusions— Moderate hypothermia preserves εPKC activity after stroke.
Protein kinase C epsilon (εPKC) belongs to the novel subgroup of the PKC family and has been associated with antiapoptotic functions in a variety of cellular systems.1–3 Several studies have shown that εPKC is activated via subcellular translocation from the cytosol to the particulate membrane fraction.4–6 In addition, generation of a 43-kDa carboxyl-terminal catalytic fragment by caspase activity also results in the activation of εPKC7–9 (Figure 1A).
εPKC is an important component of the signal transduction pathways in ischemic preconditioning–induced neuroprotection.10 A recent study demonstrated that ischemic preconditioning neuroprotection could be emulated in vitro with an agonist of εPKC or blocked with its antagonist.6 Despite its importance, the spatial kinetics of εPKC activation after focal cerebral ischemia are unknown.
The neuroprotective effects of hypothermia are not understood completely. Recently, we have shown that proapoptotic δPKC activation via subcellular translocation and proteolytic cleavage is suppressed by hypothermia in a rat focal cerebral ischemia model.11 In this report we study εPKC activation in response to stroke and the effects of hypothermia on the kinetics of its activity.
Focal Cerebral Ischemia and Hypothermia
Experimental protocols were approved by the Stanford University Administrative Panel on Laboratory Animal Care. Focal ischemia was generated as described12 in male Sprague-Dawley rats (weight, 350 to 450 g). The distal middle cerebral artery was permanently cauterized, and the bilateral common carotid arteries (CCAs) were transiently occluded for 1 hour. Core temperature was maintained at 37°C throughout the surgery for normothermic animals; for hypothermia, the temperature was maintained at 30°C during ischemia by spraying 70% methanol onto the rat’s body (Figure 1B).12
Whole-Cell Homogenization, Subcellular Fractionation, and Western Blots
At 10 minutes, 4 hours, and 24 hours after CCA release, rat brains were harvested, and tissue corresponding to the ischemic core and penumbra was dissected as described12 (Figure 1C). Samples from sham-operated rats were also prepared. For whole-cell homogenates, tissues were homogenized in cell lysis buffer (Cell Signaling Technology) and centrifuged, and the supernatant was collected.12 Subcellular cytosolic (soluble) and membrane (particulate) fractions were also prepared as described.12 Proteins (10 μg) were loaded and separated by SDS-PAGE.12 A primary εPKC antibody (C-15, 1:10 000, Santa Cruz Biotechnologies) was probed, and signals were detected by enhanced chemiluminescence (Amersham). Densities of protein bands were analyzed with the use of ImageJ (NIH).
Caspase-3 Inhibitor Injection and εPKC Cleavage Assay
A cell-permeable caspase-3–specific inhibitor, Z-DQMD-FMK (Calbiochem), was dissolved in dimethyl sulfoxide and PBS (Z-DQMD-FMK, 0.3 μg/μL in 1% dimethyl sulfoxide in PBS; vehicle, 1% dimethyl sulfoxide in PBS). Rats were anesthetized and placed in stereotactic frames. The drug solution (1.5 μg) or the vehicle was injected into the ventricular space ipsilateral to the ischemia (5 μL; from bregma: 0.9 mm posterior, 1.5 mm lateral, 3.5 mm deep) at 2 hours after CCA release. At 24 hours after CCA release, brains were removed, and whole-cell extracts were prepared for full-length or cleaved εPKC detection by Western blots.
Rat brains were collected and fixed in 4% paraformaldehyde as described,12 then cut on a vibratome into slices of 50 μm. Sections were stained with anti-εPKC (1:200) and anti-MAP2 (1:500, Sigma) antibodies 4°C overnight, then incubated with FITC or Cy3-conjugated secondary antibodies (1:200; Jackson ImmunoResearch Laboratories) for 2 hours. The slices were mounted with medium containing DAPI (Vector Laboratories) and examined under a laser-scanning confocal microscope (LSM510, Carl Zeiss).
All data are presented as mean±SEM. All statistical analyses were performed with the use of ANOVA followed by the Tukey post hoc test.
In normothermic brains, Western blots indicated that εPKC in whole-cell homogenates decreased significantly at 4 hours after CCA release in the penumbra, and greater decreases were detected in the ischemic core at 4 hours and 24 hours (Figure 1D to 1F). Such decreases in εPKC in both the ischemic core and penumbra at 4 hours (Figure 1D to 1F) were blocked by hypothermia. Carboxyl-terminal cleavage products of εPKC molecular masses of 43 and 35 kDa were detected (Figure 1D and 1E). The 35-kDa fragment increased at 10 minutes and 4 hours, returned to baseline at 24 hours after CCA release in the ischemic core (Figure 1D and 1H), and increased at 4 hours in the penumbra (Figure 1E and 1H). Hypothermia blocked all accumulation of the 35-kDa fragment. In contrast, the larger fragment (43 kDa) decreased in the ischemic core from 4 to 24 hours after stroke but did not change in the penumbra (Figure 1D and 1G). Hypothermia did not block such changes (Figure 1G).
To investigate whether εPKC cleavage after stroke is caused by caspase-3 activation, we examined the effect of a cell-permeable caspase-3–specific inhibitor, Z-DQMD-FMK, on generation of cleaved εPKC fragments (Figure 2). Caspase-3 inhibition did not suppress the decrease in full-length εPKC and the 43-kDa fragment in the ischemic core and penumbra after stroke.
Confocal microscopy indicated that εPKC is distributed in the cytoplasm and axons of neurons in nonischemic brains. Consistent with our results from Western blots, εPKC immunofluorescence decreased in the ischemic core at 4 hours after CCA release, and this decrease was suppressed by hypothermia (Figure 3).
Subcellular distribution of εPKC between the cytosolic and membrane fractions was also studied. In the ischemic core from normothermic brains, εPKC decreased at 4 hours after CCA release in both the cytosolic and membrane fractions compared with controls (Figure 4A and 4C). This decrease in both fractions was blocked by hypothermia. In the penumbra, cytosolic εPKC levels did not change after stroke, and hypothermia did not affect cytosolic εPKC levels. However, membrane-associated εPKC significantly decreased at 4 hours after stroke, and this decrease was blocked by hypothermia (Figure 4B and 4D).
We report here that levels of full-length εPKC in cell lysates and membrane fractions decreased after stroke. This reduction was blocked by hypothermia. The manner by which hypothermia prevents a decrease in εPKC levels after stroke is not understood. It has been demonstrated that εPKC is cleaved by caspases including caspase-3, -7, and -9.9 Our previous study has detected caspase-3 activity in this ischemic model.16 However, caspase-3 inhibition did not blocked decreases in the full-length and 43-kDa εPKC, despite the fact that we have previously shown that δPKC cleavage was blocked via inhibiting caspase-3 activity.11 This suggests that εPKC cleavage is not mediated by caspase-3. Hypothermia may block εPKC cleavage through inhibiting other proteases.
Our characterization of εPKC activation and proteolysis in the normothermic and hypothermic stroke brain is consistent with functional studies showing that εPKC activity can be neuroprotective in an in vitro model.6 The fact that 2 disparate neuroprotective treatments, mild hypothermia and adenosine administration,13 both enhance εPKC activity indicates that this kinase lies at an important node of a neuroprotective cascade.
The present study has some limitations. First, because hypothermia protects ischemic brain through its multiple effects on various detrimental signals, we cannot exclude the possibility that preservation of εPKC levels is secondary to hypothermic protection. However, hypothermia also specifically modifies some gene expression or protein levels to reduce ischemic damage. For example, hypothermia inhibits Bax overexpression 4 hours after 30 minutes of incomplete cerebral ischemia but does not affect Bcl-2, p53, and Mdm-2 expression.14 Yenari et al15 demonstrated that hypothermia transiently attenuated cytochrome c release but did not alter Bcl-2 and Bax expression in a focal ischemia model. Most recently, we found that hypothermia generally maintains the Akt pathway signals but does not attenuate dephosphorylation of GSK-3β.12 In our present study, we found that hypothermia reduces εPKC cleavage and its subcellular translocation, suggesting, but not unequivocally proving, that preservation of εPKC protein level is an important component contributing to the protective effect of hypothermia. Another limitation of this study is that intraischemic hypothermia was employed. The manner by which postischemic hypothermia, a paradigm more relevant to clinical application, blocks εPKC cleavage should be pursued in the future.
The authors thank Dr Bruce Schaar for editing the manuscript and David Kunis for technical assistance.
Sources of Funding
This study was supported by National Institutes of Health, National Institute of Neurological Disorders and Stroke grant R01 NS27292 (Dr Steinberg).
- Received September 12, 2006.
- Accepted October 2, 2006.
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