Elsevier

Neurobiology of Disease

Volume 20, Issue 2, November 2005, Pages 608-614
Neurobiology of Disease

Role of caspase-3 in ethanol-induced developmental neurodegeneration

https://doi.org/10.1016/j.nbd.2005.04.014Get rights and content

Abstract

Acute, transient exposure to ethanol causes a widespread pattern of caspase-3 activation and neuroapoptosis in the developing rodent brain. To determine whether caspase-3 activation is an essential step in ethanol-induced developmental neuroapoptosis, we treated homozygous caspase-3 knockout mice or wild-type mice on postnatal day 7 with an apoptosis-inducing dose of ethanol and examined the brains at appropriate survival times for evidence of apoptotic neurodegeneration. In caspase-3 knockout mice, the cell death process evolved more slowly than in wild-type mice, and morphological changes observed were not those typically associated with apoptosis. However, neuronal cell counts performed 2 weeks post-treatment revealed that the extent of neuron loss was similar in wild-type and caspase-3-deficient mice. We conclude that absence of functional caspase-3 alters the time course and morphological characteristics of the neurodegenerative process but does not prevent ethanol-induced neuron death.

Introduction

Kerr, Wyllie, and colleagues (Kerr et al., 1972, Wyllie et al., 1980) coined the term “apoptosis” and specified certain morphological changes that characterize this cell death process. In mammalian cells, apoptosis is considered a gene-regulated phenomenon involving the coordinated action of several groups of protein molecules that trigger cell death and cause the cell to be degraded in an orderly sequence of steps. Two major apoptotic pathways have been described, one designated as “extrinsic” and the other “intrinsic”. Caspases, a family of cysteine-containing, aspartate-specific proteases, characteristically are key players in both pathways. In the extrinsic pathway, the cell death signal activates caspase-8, which can directly or indirectly activate effector caspases, including caspase-3. In the intrinsic pathway, proapoptotic members of the Bcl-2 family, such as Bax, translocate to mitochondrial membranes, causing increased membrane permeability and leakage of cytochrome c into the cytoplasm. Cytochrome c binds to APAF-1 and caspase-9, causing activation of caspase-9, which in turn cleaves and activates effector caspases, such as caspases-3, -6, and -7. Activated effector caspases target many structural and functional proteins and lead to the proteolytic degradative morphological changes that typify apoptosis (Adams and Cory, 1998). Mammalian developing brains express high levels of caspase-3 zymogen, which is cleaved and activated under various circumstances involving neuronal apoptotic cell death (Roth, 2001, Roth and D'sa, 2001).

It has been shown in several recent studies that transient exposure of infant rats (Ikonomidou et al., 2000) or mice (Olney et al., 2002a, Olney et al., 2002b) to ethanol during the developmental period of synaptogenesis triggers widespread apoptotic degeneration of neurons in many brain regions. The neurodegenerative reaction transpires rapidly over a period of hours to end-stage cell death, meets ultrastructural criteria for apoptosis (Dikranian et al., 2001), is Bax-dependent (Young et al., 2003), and involves ultrastructurally detectable dissolution of mitochondrial membranes (Dikranian et al., 2001), redistribution of cytochrome c, and a robust display of immunohistochemically detectable caspase-3 activation (Olney et al., 2002b). Caspases-6, -7, and -8 are not involved in this neuroapoptosis phenomenon (Olney et al., 2002a, Olney et al., 2002b, Young et al., 2003), which appears to preferentially use the intrinsic mitochondrial pathway and caspase-3 as the primary protease effector molecule.

Early evidence identifying caspase-3 as a key proapoptotic effector molecule, and a growing belief that apoptosis may play a role in neurodegenerative diseases, stimulated interest in caspase-3 inhibitors as neuroprotective drugs. There is evidence that inhibition of caspase-3 can suppress DNA fragmentation and possibly reduce tissue damage in animal models of cerebral ischemia (Endres et al., 1998, Han et al., 2002, Hara et al., 1997). It has also been reported that caspase-3 knockout mice are less sensitive to ischemic neurodegeneration compared to wild-type controls (Le et al., 2002). These findings have been interpreted as evidence that apoptosis induced by ischemic stress can be inhibited by blocking caspase-3 activation. Oppenheim et al. (2001) studied neurons in the brain stem and spinal cord of caspase-3 knockout mouse embryos (E16.5), and reported that absence of caspase-3 delayed but did not prevent apoptotic (programmed) cell death that occurs naturally in the developing CNS. This finding suggests that programmed cell death may be a form of apoptosis that does not require caspase-3 activation and, therefore, could presumably occur even in the presence of a caspase-3 inhibitor.

Since neuroapoptosis can be induced by ethanol in many regions of the in vivo developing mouse brain during the first week of neonatal life, this provides an opportunity to study the role of caspase-3 activation in apoptotic cell death induced by an environmental stressor in various CNS neurons during an important stage in development (synaptogenesis). Accordingly, in the present study, we have administered an apoptogenic dose of ethanol to homozygous caspase-3 knockout and wild-type mice on P7 (peak of the synaptogenesis period) and evaluated the brains for evidence of neuroapoptosis. For quantitative observations and a detailed ultrastructural evaluation, we restricted our focus to the anterodorsal thalamic (ADT) nucleus, a brain region that is particularly sensitive to ethanol-induced neuroapoptosis.

Section snippets

Animals and treatment

The generation of caspase-3−/− mice and the detection of endogenous and disrupted caspase-3 gene have been described previously (Kuida et al., 1996). Caspase-3-deficient C57BL6J mice are able to grow to adulthood and have no obvious neuropathological abnormalities (Leonard et al., 2002). The knockout mice used in this manuscript were backcrossed to C57BL6J mice for more than 10 generations and are considered to be of pure C57BL6J background. Seven-day-old wild-type (WT) and caspase-3−/− C57BL/6

Activated caspase-3 is detectable in wild-type but not caspase-3−/− mice after ethanol

In WT mice, ethanol elicited a robust caspase-3 activation response (Fig. 1) distributed in a widespread pattern identical to that we have previously described in ethanol-treated infant mice (Olney et al., 2002a, Olney et al., 2002b). In some neuronal groups, this reaction was demonstrable by immunohistochemistry as early as 4 h after ethanol administration, and in others, it became detectable at about 8 h. In the majority of brain regions of WT mice, caspase-3 activation peaked at about 8 to

Discussion

Here, we show that the neuroapoptotic cell death process induced by ethanol in the infant mouse brain during synaptogenesis does not require caspase-3 activation. Absence of the caspase-3 gene altered the morphological appearance of the dying neurons and caused them to undergo a more protracted death process, but it did not prevent them from dying. Our findings are consistent with previous in vitro findings of D'Mello et al. (2000) and in vivo findings of Oppenheim et al. (2001). D'Mello et al.

Acknowledgments

The authors would like to thank Dr. Richard Flavell of Yale University for caspase-3 mutant mice. This study was supported in part by NIH grant NS35107 to KAR and AG 11355, DA 05072, HD 37100, and NARSAD 2000 Toulmin Distinguished Investigator Award to JWO.

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