Bioenergetic abnormalities in discrete cerebral motor pathways presage spinal cord pathology in the G93A SOD1 mouse model of ALS
Introduction
The rapidly progressing phenotype of muscle weakness and wasting, paralysis, and ultimately death in amyotrophic lateral sclerosis (ALS) is associated with the selective and inexorable loss of CNS motor neurons (Mulder et al., 1986). ALS is distinguished from similar disorders by the involvement of both lower motor neurons in spinal cord and brainstem (innervating skeletal muscle) and of upper motor neurons in cerebral cortex and brainstem constituting the corticospinal and bulbospinal tracts (Tandan and Bradley, 1985). Several different detrimental processes have been identified in the CNS during the disease course in ALS (see Strong, 2003), but the definitive pathogenic mechanism remains unclear. Findings of Cu/Zn superoxide dismutase (SOD1) mutations in 25% of familial ALS (FALS) patients (Ratovitski et al., 1999), and observations that mice expressing mutant SOD1 (mSOD1) recapitulate phenotypic and pathologic features of the disease, suggest that the toxic downstream events induced by mSOD1 may be common to both FALS and sporadic-onset ALS (SALS).
Among the deleterious processes identified in ALS, abnormalities in CNS energy metabolism are well documented. Symptomatic SALS patients display progressive hypometabolism in brain motor regions (Hatazawa et al., 1988). Reports of abnormal structure, number, and localization of mitochondria in ALS motor neurons and skeletal muscle led to suggestions of compromised mitochondrial function (Hirano et al., 1984, Sasaki and Iwata, 1996, Siklos et al., 1996), corroborated by findings of altered respiratory chain enzyme activities in ALS spinal cord and motor cortex (Bowling et al., 1993, Fujita et al., 1996, Browne et al., 1998, Borthwick et al., 1999). Mutant mouse models of ALS have subsequently provided evidence that mitochondrial dysfunction may be critically involved in ALS pathogenesis. Morphological changes in mitochondria are the earliest detected pathologic events in mouse lines overexpressing SOD1 with G37R or G93A mutations (Dal Canto and Gurney, 1995, Wong et al., 1995, Kong and Xu, 1998, Sasaki et al., 2004). In G93A mice, decreased mitochondrial respiration and abnormal enzyme activities have been reported (Browne et al., 1998, Kirkinezos et al., 2005), and agents that enhance energy metabolism extend lifespan and delay symptom onset (Matthews et al., 1998, Klivenyi et al., 1999). In addition, studies of mitochondrial toxins in preparations of mixed cells from spinal cord suggest that motor neurons are especially vulnerable to metabolic compromise (Kaal et al., 2000, Van Westerlaak et al., 2001). There is also evidence that mSOD1, recently shown to enter mitochondria (Higgins et al., 2002, Mattiazzi et al., 2002, Liu et al., 2004, Vijayvergiya et al., 2005), can alter mitochondrial function. Expression of human G37R or G93A mSOD1 in a motor neuron cell line induced morphological abnormalities in mitochondria, reduced mitochondrial complex II and IV activities, and increased cell vulnerability to oxidative stress (Menzies et al., 2002).
Despite these numerous reports, it remains unclear whether energy defects underlie neuronal death in ALS. To address whether mSOD1-induced metabolic defects are key factors in cell death processes, we used fully quantitative in vivo imaging procedures to map functional alterations in CNS glucose metabolism in discrete anatomical regions throughout the brain and spinal cord of conscious G93A mSOD1 mice, at pre- and post-symptomatic ages. We found that CNS glucose metabolism is defective before the first pathological signs of motor neuron disease occur in G93A mice, and that impairments occur primarily in discrete brain motor regions contributing to corticospinal projections. Decreased glucose utilization in the cerebral motor cortex was accompanied by marked reductions in ATP generation. Glucose utilization in the spinal cord, in contrast, was unaltered at this age but becomes impaired as mice age. Results suggest that metabolic defects may be principal components of the neurotoxic mechanism in ALS and provide insight into the temporal involvement of upper and lower motor neurons in this FALS model.
Section snippets
Animals
G93A transgenic mice overexpressing human mutant SOD1 (G93AG1H/B6SJLF1) and N1029 mice overexpressing human wild-type SOD1 (Gurney et al., 1994) were obtained from Jackson Laboratories (Bar Harbor, ME). Lines were maintained by crossing into B6SJLF1 mice (Jackson Laboratories, Bar Harbor, ME). Non-transgenic wild-type littermates served as controls. Mice were housed at room temperature in a 12-h light/dark cycle with free access to food and water. All procedures were reviewed and approved by
Glucose utilization in G93A mouse CNS
Rates of glucose utilization were measured in multiple discrete regions throughout the brains and spinal cords of conscious, freely moving G93AG1H SOD1 mice at ages preceding pathological changes (60 days) and at end-stage of the disorder (120 days), in age-matched wild-type littermate mice and in aged transgenic wild-type (N1029 mice overexpressing human wild-type SOD1, 21 months). Assays used the fully quantitative in vivo [14C]-2-deoxyglucose (2-DG) technique of Sokoloff et al. (1977)
Discussion
Mitochondrial and energetic defects are implicated in the pathogenesis of motor neuron degeneration in ALS, but their precise contribution to the disease mechanism is unclear. Here, we demonstrate that expression of the G93A SOD1 mutation induces energy dysfunction in discrete CNS motor regions long before motor neuron degeneration occurs. The marked reduction in cerebral cortex ATP levels detected in 30-day-old G93A mice, well before pathology and symptom onset, represents the earliest
Acknowledgments
This work was supported by a Department of Defense grant DAMD17-98-1-8620 to SEB, and the ALS Association. We thank Dr. Uwe Reuter for his contributions to pilot experiments, Dr. Nancy Stagliano for surgical expertise, Prof. Michael Moskowitz for surgery space, and Jason Gregorio for genotyping mice.
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