Aberrant DPKC activation in the spinal cord of Wobbler mouse: a model of motor neuron disease Kunjan R. Dave, a Ami P. Raval, a Jesu ´s Purroy, a Ilias G. Kirkinezos, a Carlos T. Moraes, a Walter G. Bradley, a,b and Miguel A. Pe ´rez-Pinzo ´n a,b, * a Department of Neurology, University of Miami School of Medicine, Miami, FL 33101, USA b Department of Neuroscience, University of Miami School of Medicine, Miami, FL 33101, USA Received 9 October 2003; revised 12 July 2004; accepted 24 August 2004 Available online 23 November 2004 Protein kinase C (PKC) was suggested to play a role in the pathology of amyotrophic lateral sclerosis (ALS) patients. Activation of PKC delta (DPKC) modulates mitochondrially induced apoptosis. The goal of the present study was to define whether DPKC activation occurs in Wobbler mouse spinal cord (a model of motor neuron disease). The level of DPKC in the soluble fraction was significantly decreased in the spinal cord of Wobbler mice, which was associated with a significant increase in DPKC cleavage. Since caspase-3 is known to cleave DPKC, we determined caspase-3 activation in the Wobbler mice spinal cord, immunohistochemically. The results demonstrated intense immunor- eactivity for activated caspase-3 in corticospinal tract motor neurons of Wobbler mice spinal cord. We hypothesize from these results that caspase-3 activation cleaves DPKC, which in turn promotes an aberrant signal transduction pathway in the Wobbler spinal cord. D 2004 Elsevier Inc. All rights reserved. Keywords: Amyotrophic lateral sclerosis; Neuronal death; Apoptosis; Cytochrome c ; Neurological disorders; Immunohistochemistry; SOD1 mice Introduction Amyotrophic lateral sclerosis (ALS) is a devastating disease that results in degeneration of both upper and lower motor neurons of the brain, brain stem, and spinal cord (Brownell et al., 1970). The cause of most types of ALS remains uncertain, and the disease is incurable. There is a wide range of theories of the etiology of ALS (Bradley, 1995; Bradley and Krasin, 1982; Lange et al., 1983). The two most currently accepted theories are glutamate excitotoxicity (Bristol and Rothstein, 1996; Rothstein, 1995; Rothstein et al., 1992) and oxidative damage (Browne and Beal, 1994). It is possible that oxidative damage occurs via mitochondrial dysfunc- tion. In fact, the involvement of mitochondrial dysfunction in ALS was suggested by histopathological and biochemical mitochondrial abnormalities reported in both sporadic and familial ALS patients (Ferrante et al., 1997). We recently reported mitochondrial respiration dysfunction in two animal models of motor neuron disease: in complex I, III, or IV in the motor cortex and or spinal cord of Wobbler mice (Dave et al., 2003a; Xu et al., 2001) and in complex IV in the brain and spinal cord of the SOD1 transgenic mouse (Kirkinezos et al., 2001). We further demonstrated that mitochondrial dysfunction develops at an early stage of the disease in spinal cord and is more pronounced in the motor cortex later in the disease (Dave et al., 2003a). This type of mitochondrial dysfunction will generate reactive oxygen species, which can promote oxidative stress. Oxidative stress can affect many cellular systems. One such system is the neuronal signal transduction pathway (Droge, 2002; Finkel, 2001, 2003; Martindale and Holbrook, 2002; Owuor and Kong, 2002). In fact, an abnormal activation of a key signal transduction molecule, protein kinase C (PKC), was suggested as one of the factors involved in the etiology of ALS (Krieger et al., 1996). PKC is a protein kinase that in normal conditions phosphorylates serine and threonine residues, thus changing the kinetics of specific proteins. However, defining how PKC is involved in ALS is complicated, as there are many PKC isozymes that play multiple and sometimes opposite cellular roles. Among the PKC isozymes, PKC delta (yPKC) is a good candidate to play a role in the pathology of ALS, since it is known to modulate mitochondrial-induced apoptosis (Anantharam et al., 2002; Li et al., 1999; Majumder et al., 2000). Thus, the goal of the present study was to define whether yPKC activation occurs at the onset of disease in Wobbler mouse spinal cord, a model of motor neuron degeneration (Mitsumoto and Bradley, 1982). 0969-9961/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2004.08.017 * Corresponding author. Department of Neurology (D4-5), PO Box 016960, University of Miami School of Medicine, Miami, FL 33101, USA. Fax: +1 305 243 5830. E-mail address: perezpinzon@miami.edu (M.A. Pe ´rez-Pinzo ´n). Available online on ScienceDirect (www.sciencedirect.com). www.elsevier.com/locate/ynbdi Neurobiology of Disease 18 (2005) 126 – 133