Caspase Independent Programmed Cell Death

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As mentioned previously, apoptosis and PCD have been used interchangeably, and until recently, necrosis was assumed to be a passive process of cell swelling and lysis, based on in vitro evidence. However, there has been for many years in vivo evidence in cerebral ischemia, status epilepticus and hypoglycemia that what was then called "ischemic cell change" and what we now describe as neuronal necrosis involves cell shrinkage and condensation, with nuclear pyknosis (shrinkage), scattered irregular chromatin clumps, and mitochondrial swelling, with disrupted cristae and flocculent densities (Fig. 4.6) (Brown and Brierley 1972, 1973; Griffiths et al. 1984; Auer et al. 1985a, b; Ingvar et al. 1988; Colbourne et al. 1999; Fujikawa et al. 1999, 2000, 2002). As discussed in the section "Morphological Classification of Cell Death," these changes were described independently by Wyllie (1981), and were recognized as being necrotic.

Recently, evidence has accumulated that necrotic cell death can also be programmed, but that it involves caspase-independent mechanisms (Kitanaka and Kuchino 1999; Proskuryakov et al. 2003; Syntichaki and Tavernarakis 2003; Vanlangenakker et al. 2008). For example, in cerebral ischemia and SE activation of both the calcium-dependent cysteine protease calpain I and the DNA repair

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  1. 4.4 (a) shows electron photomicrographs of neurons in the upper blade of the dentate granule cell layer in contralateral (control) hemisphere and ipsilateral hemisphere in rats with left common carotid artery ligation and hypoxia (8% O2) for 60 min in P7 rats 48 h after hypoxia-ischemia. The contralateral hemisphere showed normal neuronal nuclei and cytoplasm. Ipsilaterally, there were two types of degenerating neurons: those with pyknotic nuclei and scattered, irregular chromatin clumps (double black arrows) and those with large, round, chromatin clumps (white arrows in the middle panel and single black arrow in the right panel). These corresponded to necrotic and apop-totic neurons, respectively. (b) shows necrotic neurons in the hippocampal CA1 pyramidal cell layer ipsilaterally 48 h after hypoxia-ischemia in P7, P15, P26 and P60 rats (the P7 and P15 rats had 60 min of hypoxia, the P26 and P60 rats, 30 min of hypoxia). The shrunken neurons are electron-dense, with pyknotic nuclei containing scattered, irregular chromatin clumps, which decreased in size from P7 to P26 and were not visible in P60 rats. The cytoplasm at all ages showed extensive degeneration, with swollen, round mitochondria and disrupted endoplasmic reticulum, with ribo-somal disaggregation. The white arrows in the P7 rat point to round, swollen mitochondria without obvious disruption of cristae, and the arrowheads point to the scattered chromatin clumps. The white arrows in the P60 rat point to the nucleus, with no visible chromatin clumps and with an intact nuclear membrane (from Liu et al. 2004, with permission from Elsevier) -
  2. 4.5 (continued) granule cell layer, showing progressively less active caspase-3 IR with age, and absent caspase-3 IR at P60. Large arrows point to nuclei with active caspase-3 IR, small arrows to large, round chromatin masses without active caspase-3 IR and arrowheads to condensed nuclei without active caspase-3 IR (from Liu et al. 2004, with permission from Elsevier)










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Fig. 4.5 These are confocal images from brain sections of P7 and P60 rats (a), double-labeled with an antibody to active caspase-3 (green) and propidium iodide (PI), which, since brains were perfused with 4% phosphate-buffered paraformaldehyde, penetrates cells and stains nucleic acids of normal and degenerating neurons, essentially serving as a nuclear stain. Rats were subjected to left common carotid ligation and hypoxia, as in Fig. 4.4. Active caspase-3 expression is present in the ipsilateral hippocampal pyramidal cell layer and neocortex of the P7 rat, but there is no expression in the P60 rat. Arrows point to weaker PI staining in the neocortex of the P60 rat. (b) shows left hippocampal PI staining, active caspase-3 immunoreactivity (IR) and a merged image in a P7 rat, with merged images in P15, P26 and P60 rats. There is substantially less active caspase-3 IR in P15 hippocampus, which is only faintly seen in dentate granule cells at P26, and which is absent throughout the hippocampus at P60. (c) shows higher magnification images in CA1 and the upper blade of the dentate

Fig. 4.6 Early evidence that necrotic neurons are produced by cerebral ischemia (a), status epilepticus (b) and hypoglycemia (c). (a) shows an ischemic necrotic hippocampal pyramidal neuron in a rat subjected to right common carotid artery ligation and 40 min of hypoxia (Levine preparation) with a short (less than 2 h) recovery period. It is shrunken, electron-dense, with a pyknotic (shrunken) nucleus (N) containing diffuse, irregular chromatin clumps and cytoplasm with numerous vacuoles, some of which are dilated endoplasmic reticulum (arrows point to two) and dilated mitochondria with disrupted cristae. The neuron is surrounded by dilated astrocytic processes and an adjacent astrocytic nucleus (A) (from Brown and Brierley 1972, with permission from Elsevier). (b) shows a seizure-induced necrotic neuron in the hippocampal "stratum polymorph" 60 min after 2-h L-allylglycine-induced status epilepticus. It is also shrunken, electron-dense, with a pyknotic nucleus with scattered, irregular chromatin clumps and cytoplasmic vacuoles, most of which contain Ca2+ pyroantimonate deposits (the arrow points to one such vacuole; arrowheads point to synaptic terminals) (from Griffiths et al. 1984, with permission from Elsevier). (c) shows a hypo-glycemia-induced necrotic neuron in the cerebral cortex of a rat subjected to 60 min of an isoelec-tric EEG, induced by hypoglycemia, and an 8-h recovery period. It is also shrunken, electron-dense, with prominent irregular chromatin clumps in the pyknotic nucleus, large, swollen mitochondria containing flocculent densities and surrounding swollen astrocytic processes. The scale bar is 1.5 mm (from Auer et al. 1985a, with permission from Springer)

enzyme poly(ADP-ribose) polymerase-1 (PARP-1) occur and contribute to neuronal death (Eliasson et al. 1997; Cao et al. 2007). Calpain I translocates to both mitochondrial and lysosomal membranes, causing release from mitochondria of cyt c and apoptosis-inducing factor (AIF) (Gao and Dou 2000; Lankiewicz et al. 2000; Ding et al. 2002; Volbracht et al. 2005) and of lysosomal cathepsins and DNase II from lysosomes (Yamashima et al. 1998, 2003; Tsukada et al. 2001). PARP-1 forms poly(ADP-ribose) (PAR) polymers, which translocate from nuclei to mitochondria, with resultant AIF release (Yu et al. 2002, 2006; Andrabi et al. 2006). AIF translocates to neuronal nuclei, and together with an as yet unknown endonuclease, is responsible for large-scale, 50-kb DNA cleavage (Susin et al. 1999).

The actions of cytosolic cyt c in caspase-independent programmed cell death are largely unknown. Aside from apoptosome formation, cytosolic cyt c translocates to the endoplasmic reticulum, where it binds to inositol (1,4,5) trisphosphate receptors, inducing Ca2+ release that amplifies mitochondrial cyt c release (Boehning et al. 2003). In addition, cyt c translocates to HeLa cell or cerebellar granule cell nuclei following DNA damage, where it induces cytoplasmic translocation of acetylated histone H2A and chromatin condensation (Nur-E-Kamal et al. 2004). We also have evidence that cyt c translocates to neuronal nuclei within 60 min after the onset of generalized SE, together with mitochondrial AIF and endoG and lysosomal cathep-sin B (cath-B) and cathepsin D (cath-D) and DNase II (Zhao et al. Submitted for publication). However, the functional consequences of cyt c, cath-B and cath-D nuclear translocation are not known, and potential large-scale DNA cleavage by AIF and DNA laddering by endoG and DNase II have yet to be shown following SE.

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