Caspase-3 dependent proteolytic cleavage of ɛPKC after ischemic preconditioning in the organotypic hippocampal slices

Introduction

In an earlier report we established that the epsilon protein kinase c (ɛPKC) played a key role in ischemic preconditioning (IPC) ¹ . Using an in vivo model of cerebral ischemic tolerance, activation of caspase-3 in preconditioned brain was reported ² . Recent reports also suggest that proteolytic activation of ɛPKC by caspases is associated with its anti-apoptotic functions 3 ³ . The goal of the present study was to determine whether caspase-3 is activated after IPC and whether this activation leads to cleavage of ɛPKC with a consequent neuroprotection in the organotypic hippocampal slices.

Methods

The organotypic hippocampal slices from rat pups (9–11 day old) were used as a model for ischemia and IPC^{1, 4}. The cleavage of ɛPKC was determined at 24 h of reperfusion after IPC by Western blot analysis using ɛPKC specific antibody. The Western blots were quantified by densitometric analysis. The effect of caspase-3 activation on cleavage of ɛPKC was determined by pharmacological blockade of caspase-3 using cell permeable inhibitor DMQD-CHO during and after IPC. Cell death was assessed by the propidium iodide (PI) fluorescence technique^{1, 4}.

Results

PI fluorescence at 24 h after lethal OGD was 35.85 ± 2.57% (n = 3) in the IPC group, as compared to 54.59 ± 3.1 (n = 3) in non-preconditioned slices (p<0.05). Superfusion with 50 μM of the caspase-3 inhibitor during IPC and up to 3 h of reperfusion completely blocked neuroprotection afforded by IPC (p<0.05) (PI fluorescence values: 56.50 ± 5.31%, n=7, after combined IPC and caspase-3 inhibitor treatment). These results show that activation of caspase-3 is required for tolerance afforded by IPC. In the next set of experiments, we found that the ratio of uncleaved to cleaved ɛPKC in control slices was 1.61 ± 0.21 (n = 3). In the slices from the IPC group, because of the high levels of cleaved ɛPKC, the ratio of uncleaved to cleaved ɛPKC decreased 61% (p<0.05) (0.63 ± 0.08, n = 3) as compared to the control group (1.61 ± 0.21, n = 3). The ratio of uncleaved to cleaved ɛPKC in the IPC group where the caspase-3 inhibitor was administered during IPC and for an additional 3 h of reperfusion was higher by 62% (1.02 ± 0.03, n = 2) as compared with the IPC alone group.

Conclusion

Our study shows that mild activation of caspase-3 following IPC is neuroprotective, perhaps in part due to cleavage of ɛPKC. This is supported by the anti-apoptotic role of cleaved ɛPKC in other models of apoptosis. We suggest that a better understanding of the mechanisms by which cleaved ɛPKC acts as anti-apoptotic molecule after IPC may shed light into the mechanisms of IPC induced tolerance.