Implications of CAD and DNase II in ischemic neuronal necrosis specific for the primate hippocampus

Abstract

The exact molecular mechanism of ischemic neuronal death still remains unclear from rodents to primates. A number of studies using lower species animals have suggested implication of apoptosis cascade, while using monkeys the authors recently claimed necrosis cascade by calpain-induced leakage of lysosomal cathepsins (calpain-cathepsin hypothesis). This paper is to study implications of apoptotic versus necrotic cascades for the development of hippocampal CA1 neuronal death in the primate brain undergoing complete global ischemia. Here, we focused on two terminal cell death effectors; caspase-activated DNase (CAD) and lysosomal enzyme DNase II, in the monkey CA1 sector undergoing 18 min ischemia. The expressions of their mRNA and proteins, and the subcellular localizations as well as ultrastructure and speci®c DNA gel electrophoresis were examined. Expression of CAD was much less in the normal brain, compared with the lymph node or heart tissues. On day 1 after ischemia, however, CAD mRNA and protein were signi®cantly increased in the CA1 sector, and then CAD protein immunohistochemically showed a translocation from the perikarya into the nucleus. Activated DNase II protein was signi®cantly increased on days 2 and 3 after ischemia, and also showed a similar translocation indicating lysosomal leakage. Although the post-ischemic CA1 neurons showed positive terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining on days 3±5, they showed eosinophilic coagulation necrosis on light microscopy, and frank membrane disruption and mild chromatin condensation on electron microscopy. Furthermore, DNA smear pattern typical for necrosis was observed instead of DNA laddering. These data altogether suggest that the post-ischemic CA1 neuronal death of the monkey occurs not by apoptosis but by necrosis with participations of lysosomal enzymes DNase II and cathepsins as well as CAD. The interactions between apoptotic (caspase-3 and CAD) and necrotic (calpain, cathepsin and DNase II) cascades should be studied further.

Activation of caspase-activated deoxyribonuclease and neuroprotective effect of caspase-3 inhibitor after focal cerebral ischemia-reperfusion injury

“…This process is an important pathway to neuronal injuries in rats after cerebral ischemia-reperfusion. The time-dependent relationship between caspase-3 and apoptosis further supports the theory that caspase-3-mediated mechanism play a key role in the final execution of neuronal apoptosis in various forms of central nervous system injuries (Yakovlev et al, 1997;Namura et al, 1998;Springer et al, 1999;Zhang et al, 1999;Citron et al, 2000;Clark et al, 2000;Sharp et al, 2000;Cao et al, 2001;Graham and Chen, 2001;Tsukada et al, 2001). Furthermore, we observed that caspase-3 inhibitor Ac-DEVD-CHO diminished CAD activation and prevented endogenous DNA fragmentation after ischemia.…”

“…Previous studies have suggested that caspase-3 dependent CAD activity play an essential role during neuronal apoptosis after ischemia in the brain (Chen et al, 1998;Tsukada et al, 2001;Luo et al, 2002;Abas et al, 2010;Yan et al, 2010). Hence, antiapoptotic mechanisms through caspase inhibition may play neuroprotective role in the brain after focal ischemic injury.…”

Caspase-independent pathways of hair cell death induced by kanamycin in vivo

“…Specifically, the calpain-cathepsin cascade is a major pathway regulating necrosis or necroticlike cell death. 9,36 One of the targets of a calpain-cathepsin cascade and of caspase-3 is PARP1. Hydrolysis by these two proteases yields different cleavage fragments and results in different forms of cell death.…”

Cooperation between C1q and DNase I in the clearance of necrotic cell–derived chromatin

“…In necrotic cells generated under mild conditions (heat treatment at 56°C for 30 minutes or treatment with low amounts of detergents or alcohol), permeabilization of the cytoplasmic membrane usually precedes the degradation of nuclei by several hours. DNA digestion is performed either by an attack of endogenous lysosomal DNase II (55) or by enzymes that invade the necrotic body from the outside (e.g., DNase I from serum or interstitial fluid). Finally, in necrotic cells generated by harsh conditions, such as repeated freeze-thaw cycles, the DNA is immediately fragmented as a result of the physical stress (data not shown).…”