Evidence for chronic mitochondrial impairment in the cervical spinal cord of a murine model of motor neuron disease Barbara Santoro, a,1 Paolo Bigini, b,1 Giovanna Levandis, a Vincenzo Nobile, a Marco Biggiogera, c Francesca Botti, a Tiziana Mennini, b and Daniela Curti a, * a Department of Cellular and Molecular Physiological and Pharmacological Sciences, University of Pavia, PV, Italy b Laboratory of Receptor Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy c Department of Animal Biology and CNR Institute for Molecular Genetics, University of Pavia, Pavia, Italy Received 18 February 2004; revised 9 July 2004; accepted 9 July 2004 Profound alteration of the oxygen consumption rate (QO 2 ) is present in the cervical spinal cord (CS) of the wobbler mice aged 12 weeks (wr12). Early symptomatic mice at 4 weeks (wr4) show less pronounced changes with decreases of basal QO 2 (P b 0.03) and of QO 2 through complex I (P b 0.04). Mitochondrial respiratory enzyme activities, measured spectrophotometrically in the CS homogenate, show no difference between wr12 and controls, whereas complex I is reduced in the wr4 CS (P b 0.0003). Complex I activity is lower than normal both in wr12 and wr4 CS when measured in motor neurons by mean of a histochemical technique. Electron microscopy (EM) reveals a mixture of normal and morphologically altered mitochondria in wr4 motor neurons. The wobbler lumbar spinal cord is spared even at 12 weeks. Our results demonstrate the presence of mitochondrial abnormalities in the wobbler CS since the first manifestations of the disease. Thus, chronic mitochondrial dysfunction has a contributory role in motor neuron degeneration in the wobbler disease. D 2004 Elsevier Inc. All rights reserved. Keywords: Motor neurons; Wobbler; Mitochondria; Complex I; Oxygen consumption; Neurodegeneration Introduction The wobbler mice are originated by a spontaneous mutation, arisen in the C57Bl/Fa strain, that seems to be transmitted by an autosomal recessive gene wr and maps to chromosome 11 (Kaupmann et al., 1991). However, after sequencing the wobbler critical region, all candidate genes have been excluded and the mutation might affect a conserved noncoding sequence with a cis - acting effect on genes that might be located outside the critical region (Fuchs et al., 2002). The wobbler disease is a multisystemic disease and the affected mice can be easily recognized as early as at 21 days postnatal. The brighting reflexQ has been used to identify homozygous mice even at 3–7 days (Bose et al., 1998). However, the neurological symptoms progressively develop from the 4th to the 8th–9th week of age. Thereafter, a stabilization of the clinical signs takes place (Duchen and Strich, 1968). Histologically, the wobbler phenotype is paralleled by perikaryal vacuolar degener- ation of anterior horn cells with swelling of a and g motor neurons, severe astrogliosis, and strong microglia activation (Ait-Ikhlef et al., 2000; Bigini et al., 2001; Boille ´e et al., 2001, 2003; Hantaz- Ambroise et al., 1994; Mitsumoto and Bradley, 1982; Rathke- Hartlieb et al., 1999). Forelimbs and motor neurons in the cervical spinal cord are primarily affected by the disease (Andrews and Andrews, 1976; Duchen and Strich, 1968; Papapetropoulos and Bradley, 1972). Proton magnetic resonance spectroscopy and immunoreactivity for ubiquitin also show the presence of cerebral pathology (Pioro et al., 1998) supporting the use of the wobbler mouse as a model of amyotrophic lateral sclerosis (ALS). In the last few years, mitochondrial abnormalities have been found in patients affected by chronic neurodegenerative diseases among which ALS patients (Beal, 2000; Bowling and Beal, 1995; Cassarino and Bennet, 1999; Curti et al., 1996; Simonian and Coyle, 1996). Chronic systemic inhibition of mitochondria respiratory complexes leads to selective motor neuron vulnerability (Kaal et al., 2000; Sriram et al., 1998). Besides, drugs acting primarily on energy metabolism delay muscle atrophy and reduce degeneration of the spinal motor neurons in animal models of motor neuron diseases (Ikeda et al., 2000; Klivenyi et al., 1999). Mitochondria participate directly in the signaling pathways of apoptotic and necrotic cell death but the temporal and causal role of mitochondria in motor neuron diseases have not been clarified. Oxygen consumption rate (QO 2 ) was decreased in mitochondria isolated from the brain and spinal cord of motor neuron disease 0969-9961/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2004.07.003 * Corresponding author. Department of Cellular and Molecular Physiological and Pharmacological Sciences, University of Pavia, Piazza Botta 11, 27100 Pavia, Italy. Fax: +39 0382 506385. E-mail address: curtid@unipv.it (D. Curti). 1 These authors contributed to the same extent in this work. Available online on ScienceDirect (www.sciencedirect.com.) www.elsevier.com/locate/ynbdi Neurobiology of Disease 17 (2004) 349 – 357