Research ReportCaspase-dependent programmed cell death pathways are not activated in generalized seizure-induced neuronal death
Introduction
Based upon a developmental classification of cell death (Clarke, 1990), three major morphological subtypes are currently recognized: apoptotic (type I), autophagic (type II) and necrotic (type IIIb). In recent years attention has been focused on the programmed mechanisms contributing to apoptotic cell death—more specifically, the intrinsic (mitochondrial) and extrinsic (Fas death receptor-mediated) caspase-dependent pathways (Cohen, 1997, Earnshaw et al., 1999, Philchenkov, 2004, Reed, 2000, Riedl and Shi, 2004, Stefanis, 2005, Zimmerman et al., 2001). The notion that pathologically induced neuronal death is apoptotic was based initially upon application of two techniques thought at the time to be specific for apoptotic cell death, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), labeling double-stranded DNA fragments (Gavrielli et al., 1992), and agarose gel electrophoresis, showing 180 base pair, internucleosomal DNA cleavage or DNA “laddering” (Wyllie, 1980). However, both TUNEL positivity and DNA laddering may be found in necrotic, as well as apoptotic, cells (Fujikawa et al., 1999, Fujikawa et al., 2000, Fujikawa et al., 2002). This, plus a lack of attention to the morphological similarities and differences between apoptotic and necrotic cells, and controversy regarding the activation of caspase-dependent pathways in excitotoxic neuronal death, has created confusion in the literature (Fujikawa, 2000, Fujikawa, 2002, Roy and Sapolsky, 1999, Sloviter, 2002).
The conventional view that necrotic cell death is a passive process in which cells swell, then lyse, has been called into question by accumulating evidence that necrotic cell death may involve caspase-independent, programmed mechanisms (Denecker et al., 2001, Kitanaka and Kuchino, 1999, Leist and Jäättelä, 2001, Proskuryakov et al., 2003). Seizure-induced neuronal death is morphologically necrotic (Fujikawa et al., 1999, Fujikawa et al., 2000, Fujikawa et al., 2002), but involves programmed processes such as DNA laddering (Filipkowski et al., 1994, Fujikawa et al., 1999, Fujikawa et al., 2000, Fujikawa et al., 2002, Kondratyev and Gale, 2000, Kondratyev et al., 2002, Pollard et al., 1994).
There is conflicting information as to whether the central effector caspase, caspase-3, contributes to neuronal death from prolonged seizures, or status epilepticus (SE) (Ananth et al., 2001, Fujikawa et al., 2002, Henshall et al., 2000, Kondratyev and Gale, 2000, Narkilahti et al., 2003, Puig and Ferrer, 2002, Weise et al., 2005). However, even if caspase-3 does not contribute to SE-induced neuronal death, this does not rule out activation of either caspase-9 or caspase-8, upstream cysteine proteases in the intrinsic mitochondrial and Fas death receptor extrinsic caspase-dependent pathways respectively. For example, it was recently shown in an adult model of hypoxia–ischemia that both caspase-8 and -9 are activated without activation of caspase-3 (Adhami et al., 2006). Both caspase-8 and -9 are activated in a model of focal seizures (Henshall et al., 2001a, Henshall et al., 2001b, Li et al., 2006), but unlike caspase-3, there are no reports of whether either is activated or not in generalized seizure-induced neuronal death. We show for the first time that neither caspase-8 nor -9 is activated during the first 24 h following generalized seizures, when neuronal necrosis is maximal and widespread. This suggests that caspase-independent mechanisms are involved in producing generalized seizure-induced neuronal necrosis with DNA laddering. In fact, there is increasing evidence that caspase-independent mechanisms play a prominent role in seizure-induced neuronal death (see Discussion).
Section snippets
Extent of neuronal damage 6 and 24 h following SE
Six hours following SE, 15 of 26 brain regions examined showed a significant number of acidophilic neurons by H&E stain compared to controls (Table 1), ranging from 10 to 25% (e.g., the ventral subiculum, rhinal and entorhinal cortex, neocortex and septal nuclei) to more than 50% (e.g., the ventral hippocampal dentate hilus). We have shown previously that these acidophilic neurons are necrotic by ultrastructural examination in lithium–pilocarpine-induced SE (LPCSE) (Fujikawa et al., 1999,
Discussion
This study was undertaken in order to determine if LPCSE, which produces morphologically necrotic neurons 6 and 24 h after SE (Fujikawa et al., 2002) and DNA laddering 24 h after SE (Fujikawa et al., 1999, Fujikawa et al., 2002), activates either or both of the two caspase-dependent programmed pathways upstream of caspase-3, about which there is disagreement whether it is activated in seizure-induced neuronal death (Ananth et al., 2001, Fujikawa et al., 2002, Henshall et al., 2000, Kondratyev
Materials
Male Wistar rats (220–350 g) were obtained from Charles River Laboratories (Wilmington, MA, USA). Methylatropine, diazepam, phenobarbital and 0.9% NaCl were obtained from the Pharmacy Service at Sepulveda VA Ambulatory Care Center (North Hills, CA, USA). Lithium chloride, pilocarpine, ethidium bromide, paraformaldehyde, BSA, DNase I, RNase A, Triton X-100, Tween-20, methamphetamine (METH), proteinase K, diaminobenzidine, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), EDTA,
Acknowledgments
This study was funded by the Medical Research Service, Office of Research and Development, Department of Veterans Affairs. Dr. Jeffrey A. Gornbein, a biomathematician at UCLA, provided expert assistance in the statistical analysis of data.
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