Inhibition of interleukin 1beta converting enzyme family proteases reduces ischemic and excitotoxic neuronal damage

Abstract

The interleukin 1beta converting enzyme (ICE) family plays a pivotal role in programmed cell death and has been implicated in stroke and neurodegenerative diseases. During reperfusion after filamentous middle cerebral artery occlusion, ICE-like cleavage products and tissue immunoreactive interleukin 1beta (IL-1beta) levels increased in ischemic mouse brain. Ischemic injury decreased after intracerebroventricular injections of ICE-like protease inhibitors, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.FMK), acetyl-Tyr-Val-Ala-Asp-chloromethylketone, or a relatively selective inhibitor of CPP32-like caspases, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone, but not a cathepsin B inhibitor, N-benzyloxycarbonyl-Phe-Ala-fluoromethylketone. z-VAD.FMK decreased ICE-like cleavage products and tissue immunoreactive IL-1beta levels in ischemic mouse brain and reduced tissue damage when administered to rats as well. Only z-VAD.FMK and acetyl-Tyr-Val-Ala-Asp-chloromethylketone reduced brain swelling, and N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone did not attenuate the ischemia-induced increase in tissue IL-1beta levels. The three cysteine protease inhibitors significantly improved behavioral deficits, thereby showing that functional recovery of ischemic neuronal tissue can follow blockade of enzymes associated with apoptotic cell death. Finally, we examined the effect of z-VAD.FMK on excitotoxicity and found that it protected against alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-induced or to a lesser extent N-methyl-D-aspartate-induced excitotoxic brain damage. Thus, ICE-like and CPP32-like caspases contribute to mechanisms of cell death in ischemic and excitotoxic brain injury and provide therapeutic targets for stroke and neurodegenerative brain damage.

Figure 1

Effects of z-VAD.FMK and cathepsin B inhibitor (z-FA.FMK) on infarct volume (A) and area (B), and neurological deficits (C) after transient focal ischemia in SV-129 mice. Animals were subjected to MCA filament occlusion for 2 hr and reperfused for 18 hr as described. Vehicle (○), z-VAD.FMK (27 ng; •), z-VAD.FMK (80 ng; ▵), z-VAD.FMK (240 ng; ▴), and z-FA.FMK (240 ng; □) were injected i.c.v. 15 min before ischemia and immediately after reperfusion. Infarct area was determined in each of five coronal sections (2 mm) from anterior (2 mm from anterior pole) to posterior (10 mm; B). z-VAD.FMK (80 and 240 ng) decreased infarct volume and neurological deficits, whereas z-FA.FMK (240 ng) did not. After treatment with z-FA.FMK and z-VAD.FMK (27 ng), infarct areas did not differ from vehicle in the 5 coronal sections (data not shown). Data are presented as means ± SE (n = 5–12). ∗, P < 0.05; ∗∗, P < 0.01 vs. vehicle.

Figure 2

Effects of z-VAD.FMK on infarct size after transient focal ischemia in rats. Animals were subjected to filament MCA occlusion for 2 hr and reperfused for 22 hr. z-VAD.FMK was injected i.c.v. 15 min before ischemia and 10 min after reperfusion. Open bar, vehicle; hatched bar, z-VAD.FMK (16 ng); shaded bar, z-VAD.FMK (54 ng); solid bar, z-VAD.FMK (160 ng). Data are represented as means ± SE (n = 7–9). ∗, P < 0.05; ∗∗, P < 0.01 vs. vehicle.

Figure 3

Effects of z-VAD.FMK on ICE-like protease expression in brain after left MCA filament occlusion and reperfusion. Immunoreactive products p35 and p20 increased in the ipsilateral hemisphere at 3 hr after reperfusion. z-VAD.FMK (80 ng) administered 15 min before ischemia and immediately upon reperfusion decreased immunoreactive products p35 and p20 in the ipsilateral hemisphere at 3 and 18 hr after reperfusion. There was no significant difference between normal brain (sham) and the nonischemic contralateral hemisphere (data not shown). L, Left (ischemic); R, right (nonischemic) hemisphere.

Figure 4

Effect of z-VAD.FMK and z-DEVD.FMK on ischemia-induced immunoreactive IL-1β levels in brain. Treatment with z-VAD.FMK (120 ng × 2) but not z-DEVD.FMK (120 ng × 2) 15 min before ischemia and at reperfusion reduced the expected augmentation of IL-1β ipsilaterally after 30 min of reperfusion after 2 hr of MCA occlusion. Control animals were treated with the same volume of vehicle. Data are presented as the mean ± SEM (n = 6 or 7). ∗∗, P < 0.01 vs. vehicle.