Role of caspase-3 in ethanol-induced developmental neurodegeneration Chainllie Young, a Kevin A. Roth, b Barbara J. Klocke, b Tim West, c,d David M. Holtzman, c,d,e Joann Labruyere, a Yue-Qin Qin, a Krikor Dikranian, a and John W. Olney a, * a Department of Psychiatry, Campus box 8134, Washington University School of Medicine, 660 South Euclid, St. Louis, MO 63110, USA b Department of Pathology, University of Alabama School of Medicine, Birmingham, AL 35294, USA c Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110, USA d Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO 63110, USA e Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110, USA Received 1 December 2004; revised 13 April 2005; accepted 19 April 2005 Available online 31 May 2005 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 homozy- gous 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 neurodegenera- tion. 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. D 2005 Elsevier Inc. All rights reserved. Keywords: Caspase-3 knockout; Alcohol; Apoptosis; Development Introduction Kerr, Wyllie, and colleagues (Kerr et al., 1972; Wyllie et al., 1980) coined the term ‘‘apoptosis’’ and specified certain morpho- logical 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 perme- ability and leakage of cytochrome c into the cytoplasm. Cyto- chrome 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,b) to ethanol during the developmental period of synapto- genesis 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,b; Young et al., 2003), which appears to preferentially use the intrinsic mitochon- drial 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 0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2005.04.014 * Corresponding author. Fax: +1 314 747 0346. E-mail address: olneyj@psychiatry.wustl.edu (J.W. Olney). Available online on ScienceDirect (www.sciencedirect.com). www.elsevier.com/locate/ynbdi Neurobiology of Disease 20 (2005) 608 – 614