Elsevier

Brain Research

Volume 1135, 2 March 2007, Pages 206-218
Brain Research

Research Report
Caspase-dependent programmed cell death pathways are not activated in generalized seizure-induced neuronal death

https://doi.org/10.1016/j.brainres.2006.12.029Get rights and content

Abstract

Activation of the caspase-dependent cell death pathways has been shown in focal seizures, but whether this occurs in prolonged generalized seizures is not known. We investigated whether the initiator caspase in the extrinsic pathway, caspase-8, or the intrinsic pathway, caspase-9, is activated during the first 24 h following lithium–pilocarpine-induced status epilepticus, when neuronal death is maximal and widespread. The thymuses of rats given methamphetamine were used as positive controls for caspase-3-activated cellular apoptosis. Following methamphetamine treatment, caspase-9 but not caspase-8 was activated in thymocytes. However, 6 or 24 h following status epilepticus, none of 26 brain regions studied showed either caspase-8 or -9 activation by immunohistochemistry, western blotting and enzyme activity assays. Our results provide evidence against the activation of the extrinsic and intrinsic caspase pathways in generalized seizures, which produce morphologically necrotic neurons with internucleosomal DNA cleavage (DNA laddering), a programmed process. In contrast, there is increasing evidence that caspase-independent programmed mechanisms play a prominent role in 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.

References (76)

  • K. Heo et al.

    Minocycline inhibits caspase-dependent and -independent cell death pathways and is neuroprotective against hippocampal damage after treatment with kainic acid in mice

    Neurosci. Lett.

    (2006)
  • A. Kondratyev et al.

    Intracerebral injection of caspase-3 inhibitor prevents neuronal apoptosis after kainic acid-evoked status epilepticus

    Mol. Brain Res.

    (2000)
  • A. Kondratyev et al.

    Status epilepticus leads to the degradation of the endogenous inhibitor of caspase-activated DNase in rats

    Neurosci. Lett.

    (2002)
  • S. Lankiewicz et al.

    Activation of calpain I converts excitotoxic neuron death into a caspase-independent cell death

    J. Biol. Chem.

    (2000)
  • T. Li et al.

    Inhibition of caspase-8 attenuates neuronal death induced by limbic seizures in a cytochrome c-dependent and Smac/DIABLO-independent way

    Brain Res.

    (2006)
  • X. Liu et al.

    DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis

    Cell

    (1997)
  • C.L. Liu et al.

    Pathogenesis of hippocampal neuronal death after hypoxia–ischemia changes during brain development

    Neuroscience

    (2004)
  • H. Pollard et al.

    Kainate-induced apoptotic cell death in hippocampal neurons

    Neuroscience

    (1994)
  • B.M. Polster et al.

    Calpain I induces cleavage and release of apoptosis-inducing factor from isolated mitochondria

    J. Biol. Chem.

    (2005)
  • S.Y. Proskuryakov et al.

    Necrosis: a specific form of programmed cell death?

    Exp. Cell Res.

    (2003)
  • B. Puig et al.

    Caspase-3-associated apoptotic cell death in excitotoxic necrosis of the entorhinal cortex following intraperitoneal injection of kainic acid in the rat

    Neurosci. Lett.

    (2002)
  • J.C. Reed

    Mechanisms of apoptosis

    Am. J. Pathol.

    (2000)
  • M. Roy et al.

    Neuronal apoptosis in acute necrotic insults: why is this subject such a mess?

    Trends Neurosci.

    (1999)
  • R.S. Sloviter

    Apoptosis: a guide for the perplexed

    Trends Pharmacol. Sci.

    (2002)
  • J. Takano et al.

    Calpain mediates excitotoxic DNA fragmentation via mitochondrial pathways in adult brains: evidence from calpastatin mutant mice

    J. Biol. Chem.

    (2005)
  • J. Weise et al.

    Expression time course and spatial distribution of activated caspase-3 after experimental status epilepticus: contribution of delayed neuronal cell death to seizure-induced neuronal injury

    Neurobiol. Dis.

    (2005)
  • Y. Wu et al.

    Role of endonuclease G in neuronal excitotoxicity in mice

    Neurosci. Lett.

    (2004)
  • T. Yamashima

    Implication of cysteine proteases calpain, cathepsin and caspase in ischemic neuronal death of primates

    Prog. Neurobiol.

    (2000)
  • J. Zhang et al.

    DNA fragmentation factor 45 mutant mice exhibit resistance to kainic acid-induced neuronal cell death

    Biochem. Biophys. Res. Commun.

    (2001)
  • C. Ananth et al.

    Domoic acid-induced neuronal damage in the rat hippocampus: changes in apoptosis related genes (bcl-2, bax, caspase-3) and microglial response

    J. Neurosci. Res.

    (2001)
  • E. Bonfoco et al.

    Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-d-aspartate or nitric oxide/superoxide in cortical cell cultures

    Proc. Natl. Acad. Sci. U. S. A.

    (1995)
  • E.C. Cheung et al.

    Apoptosis-inducing factor is a key factor in neuronal cell death propagated by BAX-dependent and BAX-independent mechanisms

    J. Neurosci.

    (2005)
  • S. Chua et al.

    Direct cleavage by the calcium-activated protease calpain can lead to inactivation of caspases

    J. Biol. Chem.

    (2000)
  • P.G.H. Clarke

    Developmental cell death: morphological diversity and multiple mechanisms

    Anat. Embryol.

    (1990)
  • D.B. Clifford et al.

    Ketamine, phencyclidine, and MK-801 protect against kainic-acid-induced seizure-related brain damage

    Epilepsia

    (1990)
  • G.M. Cohen

    Caspases: the executioners of apoptosis

    Biochem. J.

    (1997)
  • F. Colbourne et al.

    Electron microscopic evidence against apoptosis as the mechanism of neuronal death in global ischemia

    J. Neurosci.

    (1999)
  • G. Denecker et al.

    Apoptotic and necrotic cell death induced by death domain receptors

    Cell. Mol. Life Sci.

    (2001)
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