Partial deficiency of manganese superoxide dismutase exacerbates a transgenic mouse model of amyotrophic lateral sclerosis

Partial Deficiency of Manganese Superoxide Dismutase Exacerbates a Transgenic Mouse Model of Amyotrophic Lateral Sclerosis Ole A. Andreassen, MD,* Robert J. Ferrante, PhD,† Peter Klivenyi, MD,* Autumn M. Klein, PhD,† Leslie A. Shinobu, MD, PhD,* Charles J. Epstein, MD,‡ and M. Flint Beal, MD*§

The pathogenesis of neuronal cell death as a consequence of mutations in copper/zinc superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis may involve oxidative damage and mitochondrial dysfunction. We examined whether crossing transgenic mice with the G93A SOD1 mutation with transgenic mice with a partial depletion of manganese superoxide dismutase (SOD2) would affect the disease phenotype. Compared with G93A mice alone, the mice with partial deficiency of SOD2 and the G93A SOD1 mutation showed a significant decrease in survival and an exacerbation of motor deficits detected by rotorod testing. There was a significant exacerbation of loss of motor neurons and substantia nigra dopaminergic neurons in the G93A mice with a partial deficiency of SOD2 compared with G93A mice at 110 days. Microvesiculation of large motor neurons was more prominent in the G93A mice with a partial deficiency of SOD2 compared with G93A mice at 90 days. These findings provide further evidence that both oxidative damage and mitochondrial dysfunction may play a role in the pathogenesis of motor neuron death associated with mutations in SOD1. Andreassen OA, Ferrante RJ, Klivenyi P, Klein AM, Shinobu LA, Epstein CJ, Beal MF. Partial deficiency of manganese superoxide dismutase exacerbates a transgenic mouse model of amyotrophic lateral sclerosis. Ann Neurol 2000;47:447– 455

The finding that point mutations in copper/zinc superoxide dismutase (SOD1) are associated with approximately 20% of the cases of familial amyotrophic lateral sclerosis (FALS) has focused interest on the possible involvement of oxidative damage in disease pathogenesis.1,2 Substantial evidence suggests that the mutations in SOD1 cause disease not from a loss of function of the enzyme, but from gain of an adverse property of the mutant SOD1 protein. Evidence supporting this includes the lack of a disease phenotype in mice deficient in SOD1, the dominant inheritance pattern in FALS, the lack of correlation between enzyme activity and disease severity, and the finding that overexpression of the mutant enzyme in transgenic mice leads to motor neuron degeneration.3– 6 All of the mutant enzymes, including those with mutations effecting the active site of the enzyme bind copper in vivo.7 The mutations, however, reduce the affinity of the enzyme for zinc, which may then further destabilize the protein backbone of the enzyme.8,9

Changes in the protein backbone of the enzyme may relax the conformation of the active channel and increase the ability of the mutant enzyme to react with hydrogen peroxide and to generate hydroxyl radicals.10 –12 The mutant enzyme may also be more efficient in using peroxynitrite as an enzyme substrate, and thereby catalyze the formation of 3-nitrotyrosine.13 SOD1 with ALS-associated mutations binds zinc less efficiently and, when depleted of zinc, shows an increased efficiency to nitrate tyrosines.8 A consequence of increased oxidative damage may be mitochondrial damage. Mitochondria are known to be particularly susceptible to oxidative damage. The major free radical scavenging enzyme in mitochondria is manganese superoxide dismutase (SOD2). Studies of homozygous knockout mice deficient in SOD2 show that they develop a cardiomyopathy and central nervous system damage resulting in death within a few weeks of birth.14,15 Hemizygote SOD2 knockout mice have a 50% reduction in SOD2 in liver mitochondria and

From the *Neurochemistry Laboratory, Neurology Service, Massachusetts General Hospital and Harvard Medical School; and †Departments of Neurology, Pathology, and Psychiatry, Boston University School of Medicine, Boston, and Department of Veterans Affairs, Bedford, MA; ‡Departments of Neurology, Neurosurgery, Pediatrics, Biochemistry, and Biophysics, University of California, San Francisco, CA; and §Neurology Department, New York Hospital-Cornell Medical Center, New York, NY.

Received Jul 8, 1999, and in revised form Nov 16. Accepted for publication Nov 16, 1999. Address correspondence to Dr Beal, Neurology Department, New York Hospital-Cornell Medical Center, 525 East 68th Street, New York, NY 10021.

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there are increases in oxidative damage in liver mitochondria at 2 to 4 months of age.16 Hemizygote SOD2 show no evidence of central nervous system damage, but they show increased vulnerability to focal ischemia.17 If oxidative damage and mitochondrial dysfunction play a role in the pathogenesis of motor neuron death in transgenic mouse models of ALS, then a partial deficiency of SOD2 may exacerbate the disease phenotype. In the present experiments, we therefore examined the effects of crossing transgenic mice with the G93A SOD1 mutation associated with familial ALS with SOD2 hemizygote mice. Materials and Methods Animals Transgenic mice with the G93A human SOD1 (G1H/⫹) mutation [B6SJL-TgN (SOD1-G93A)1 Gur; Jackson Laboratories, ME] were first bred with the CD-1 strain, and then back-crossed with negative female littermates for five generations. The offspring were typed by using a polymerase chain reaction assay on tail DNA. Heterozygous SOD2 knockout mice15 were bred into the background strain (CD-1). Female SOD2 mice were crossed with male G93A transgenic mice, and the offspring were genotyped by using polymerase chain reaction assay on tail DNA. This breeding resulted in the following four genotypes: hemizygous SOD1-G93A (FALS), hemizygous SOD2 knockout (SOD2), SOD2/FALS, and wild type (wt), with nearly 25% of each type. The first generation was used for behavioral and survival studies, and the next generations were used for histology.

thetized and then transcardially perfused with phosphatebuffered 4% paraformaldehyde. The brain and the spinal cord were postfixed for 24 hours, then cryoprotected in a graded series of 10% and 20% glycerol/2% dimethyl sulfoxide solution. Tissue specimens were subsequently serially cut on a cryostat at 50 ␮m, stored in six separate collection wells, and stained for Nissl by using cresyl violet as previously described.18 Cut tissue sections of the midbrain were immunostained for tyrosine hydroxylase (TH) (TH antisera, 1⬊1,000 dilution; Eugene Tech International, Paramus, NJ) and dopamine transporter (DAT) (DAT antisera, 1⬊500 dilution; Chemicon International, Temecala, CA). DAT labels neurons of the substantia nigra pars compacta.19 Midbrain sections through both the left and right substantia nigra from the bregma levels at 3.08 to 3.16 mm and intra-aural levels at 0.72 to 0.64 mm20 in the four groups of mice were analyzed by microscopic-videocapture. Stereologic counts of Nissl-, TH-, and DAT-positive neurons within the substantia nigra pars compact were computed by using Neurolucida (Microbrightfield, Colchester, VT) image analysis software. Size discriminated neuronal counts at 250⫻ magnification were made in both ventral horn areas from serially cut tissue sections of the lumbar spinal cord in each of the four groups of mice by using the Neurolucida system. The diameter classes were more than 25 ␮m (L, large), less than 25 ␮m but more than 15 ␮m (M, medium), and less than 15 microns (S, small). All cells were counted from within the ventral horn below an imaginary line drawn laterally across the spinal cord from the central canal. The dissector counting technique was used in which all neurons were counted in an unbiased selection of serial sections in a defined volume of the spinal cord and substantia nigra.21,22

Behavior At 70 days of age, mice were given 7 days to become acquainted with the rotorod apparatus (Columbus Instruments, Columbus, OH) before testing started. During testing, the animals were placed on the rod, which rotated for 20 rpm, and the speed was increased by 1 rpm every 10 seconds until the animal fell off, and the speed of rotation at which the mouse fell off was used as the measure of competence on this task. Each mouse had three trials. When the performance of the mice deteriorated, and they were unable to run at the initial speed of 20 rpm, the start speed was set at 10 rpm, and when they fell off at this speed, the start speed was set at 0 rpm. Animals were tested every other day until they could no longer perform the task.

Survival The initial sign of the disease phenotype was a resting tremor that progressed to gait abnormalities, paralysis of the hindlimbs, progressing to paralysis of the forelimbs, and, at the end stage, a nearly complete paralysis was present. The animals were killed when they were no longer able to groom or were unable to roll over within 10 seconds after being pushed to their side. This time was taken as the time of death.

Histology At 90 and 110 days of age, 8 animals in each group (FALS, SOD2, SOD2/FALS, and wt littermates) were deeply anes-

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Statistical Analysis Statistical comparisons were by one-way analysis of variance, by repeated measures analysis of variance, or by Mantel-Cox test for survival analysis.

Results As shown in Figure 1, the survival of SOD2/FALS mice was significantly reduced compared with FALS mice. A log rank (Mantel-Cox) test showed a significant ( p ⫽ 0.0081) decrease in survival. The mean decrease in survival was 11 days. As shown in Figure 2, the performance of the SOD2/FALS mice was significantly worse than that of the FALS mice at time points of 106 days of age and greater. At 90 days, there was a significant loss of the total number of neurons (29.4% reduction) observed within the ventral horns of the lumbar cord from SOD2/ FALS transgenic mice compared with SOD2 and nontransgenic littermate controls (Table 1 and Fig 3). Large ventral horn neurons from SOD2/FALS transgenic mice were most severely involved (53% loss), with medium and small neurons less affected (35.5% and 26.8% loss, respectively). There was a small loss of neurons in the G93A group; however, it did not reach significance in comparison with SOD2 mice or non-

Fig 1. Effects of crossing G93A mice with mice partially deficient in SOD2 on survival. The graph on the left shows the cumulative probability of survival while that on the left shows the mean survival. Survival was significantly decreased in the SOD2/FALS mice. *p ⬍ 0.05. FALS ⫽ familial amyotrophic lateral sclerosis; Sod2 ⫽ SOD2 or manganese superoxide dismutase.

Fig 2. Effects of crossing G93A mice partially deficient in SOD2 on rotorod performance from 80 to 145 days of age. The SOD2/FALS mice showed significantly worse motor performance than the FALS mice alone. FALS ⫽ familial amytrophic lateral sclerosis; Sod2 ⫽ SOD2 or manganese superoxide dismutase.

transgenic littermate controls. Light microscopic examination showed microvesiculation within large, ventral horn motor neurons. While present in the G93A mice, these vacuoles were most prominent with the SOD2/ FALS transgenic mice (Fig 4). No differences were found between SOD2 mice and nontransgenic littermate controls. In contrast to the changes observed in the lumbar spinal cord, neuronal loss within the sub-

stantia nigra was not observed at 90 days as assessed by Nissl, TH, and DAT immunostaining within any of the groups of mice in comparison with littermate nontransgenic mice. At 110 days, there was marked atrophy of the spinal cord in SOD2/FALS transgenic mice in comparison with FALS, SOD2, and littermate nontransgenic controls. A greater neuronal loss of the total number of

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Table 1. Neuronal Counts from the Ventral Horn of the Lumbar Spinal Cord in 90-Day-Old ALS Transgenic Mice with the G93A Human SOD1 Mutation, SOD2/FALS Mice, SOD2 Mice, and Littermate Control Mice

SOD2/FALS mice G93A mice SOD2 mice Littermate control mice

Small

Medium

Large

Total

167 ⫾ 14.6a 198 ⫾ 15.3 219 ⫾ 11.2 228 ⫾ 9.8

20 ⫾ 5.7a 24 ⫾ 5.3 28 ⫾ 4.8 31 ⫾ 2.3

8 ⫾ 3.9b 12 ⫾ 3.1 15 ⫾ 2.1 17 ⫾ 1.8

195 ⫾ 13.9b 234 ⫾ 14.1 262 ⫾ 8.9 276 ⫾ 8.7

In 8 SOD2/FALS, 7 G93A, 8 SOD2, and 8 littermate control mice, the total number of Nissl-stained neurons was counted within the ventral horn of serial cut lumbar cord tissue sections by videomicroscopic capture and Neurolucida image analysis software. Neurons were segregated into diameter classes, small (⬍15 ␮m), medium (⬍25 but ⬎15 ␮m), and large (⬎25 ␮m). Data are mean ⫾ SEM values. p ⬍ 0.01; bp ⬍ 0.001, compared with both littermate control and SOD2-deficient mice.

a

ALS ⫽ amyotrophic lateral sclerosis; SOD1 ⫽ copper,zinc superoxide dismutase; SOD2 ⫽ manganese superoxide dismutase; FALS ⫽ familial ALS.

Fig 3. Lumbar spinal cord sections from 90-day-old wild-type littermate control (A and E), manganese superoxide dismutase (SOD2) (B and F), familial amyotrophic lateral sclerosis (FALS) (C and G), and SOD2/FALS (D and H) mice. There was a significant loss of ventral horn neurons and marked gross atrophy within the SOD2/FALS spinal cord sections in comparison with the littermate control, SOD2, and FALS specimens. Although there was a small loss of ventral hom neurons in the FALS animals, it did not reach statistical significance. Magnification bar in H equals 100 ␮m.

neurons (47.3% reduction) was observed within the ventral horns of SOD2/FALS transgenic mice than that found at 90 days (Table 2 and Fig 5). Large ventral horn neurons were almost entirely absent (92.3% loss), with medium and small neurons less affected (48.3% and 43.3% loss, respectively). At 110 days of age, there was a significant (33.5%) reduction of lumbar ventral horns in the FALS mice (see Table 2 and

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Fig 4). The neuronal loss found in the SOD2/FALS mice was significantly greater than that detected in the FALS mice ( p ⱕ 0.05). SOD2 mice did not differ significantly from littermate nontransgenic controls (see Table 2 and Fig 5). In the substantia nigra within the 110-day-old groups, a significant loss of neurons was found in both the SOD2/FALS and the G93A transgenic mice com-

Fig 4. Photomicrographs of ventral horn neurons in lumbar spinal cord sections from G93A (A) and SOD2/FALS (B) mice at 90 days. Although microvesiculation of neurons is present in the G93A mice, vacuoles are much larger and found in greater numbers in neurons from SOD2/FALS mice. Bar in B equals 20 ␮m. SOD2 ⫽ manganese superoxide dismutase; FALS ⫽ familial amyotrophic lateral sclerosis. Table 2. Neuronal Counts from the Ventral Horn of the Lumbar Spinal Cord in 110-Day-Old ALS Transgenic Mice with the G93A Human SOD1 Mutation, SOD2/FALS Mice, SOD2 Mice, and Littermate Control Mice

SOD2/FALS mice G93A mice SOD2 mice Littermate control mice

Small

Medium

Large

Total

131 ⫾ 13.8b,c 162 ⫾ 13.2b 238 ⫾ 10.9 231 ⫾ 10.6

15 ⫾ 3.5b,c 21 ⫾ 3.7a 26 ⫾ 3.2 29 ⫾ 2.1

1.6 ⫾ 1.1b,c 4.0 ⫾ 0.9b 18 ⫾ 1.2 22 ⫾ 1.9

148 ⫾ 13.3b,c 187 ⫾ 12.8b 282 ⫾ 11.7 281 ⫾ 10.8

In 8 SOD2/FALS, 8 G93A, 6 SOD2, and 6 littermate control mice, the total number of Nissl-stained neurons was counted within the ventral horn of serial cut lumbar cord tissue sections by videomicroscopic capture and Neurolucida image analysis software. Neurons were segregated into diameter classes, small (⬍15 ␮m), medium (⬍25 but ⬎15 ␮m), and large (⬎25 ␮m). Data are mean ⫾ SEM values. p ⬍ 0.01; bp ⬍ 0.001, compared with both littermate control and SOD2-deficient mice. p ⬍ 0.05, compared with the G93A mice.

a

b

For definitions, see Table 1.

pared with both SOD2 and littermate nontransgenic controls. Although neuronal loss was greater in the SOD2/FALS compared with the G93A transgenic mice, this difference was not statistically significant (Table 3). Discussion Two proposals have been made to suggest a disease mechanism for SOD1 mutations. The first is that the mutant SOD1 has an altered substrate affinity that leads to the generation of toxic reaction products, and the second is that the mutant enzyme may be unstably folded so that it precipitates to form toxic cytoplasmic aggregates. The mutations tend to occur in the protein backbone of the enzyme, thereby leading to altered accessibility of the active site copper to a variety of substrates.23 In particular, it has been proposed that there may be more ready access to hydrogen peroxide or peroxynitrite.13 Peroxynitrite can be converted by the catalytic copper of the mutant enzyme into a highly reactive nitronium intermediate, which can then nitrate tyrosine residues on proteins. The mutant enzymes bind zinc less tightly,8,9 which destabilizes the protein backbone and contributes to increased generation of reactive nitronium ions.8 This can result in nitration of the light chain of neurofilaments, which disrupts the ability of neurofilaments to form the normal

neurofilament triplet.8 In support of an increase in nitration, we found increased levels of 3-nitrotyrosine in transgenic mice with either the G37R or the G93A mutations24,25 and in the spinal cord in both sporadic ALS patients and in FALS patients with the A4V mutation.26 Another potentially toxic interaction of the mutant SOD1 molecule is with hydrogen peroxide. Hydrogen peroxide can more readily react with the mutant enzyme generating hydroxyl radicals in vitro.11,12,27 Expression of the mutant enzyme but not the wt SOD1 results in increased apoptosis within cultured cells.11,28 Expression of mutant SOD1 in PC12 cells is associated with increased superoxide production and cell death, which is attenuated by copper chelators, Bcl-2, glutathione, vitamin E, and inhibitors of caspases.28 Consistent with increased reactivity with hydrogen peroxide, catalase protects neurally differentiated neuroblastoma cell lines with SOD1 mutations from apoptotic cell death.29 We found evidence of increased “hydroxyl radical-like” activity in G93A mice in vivo by using microdialysis.30 In a similar manner, others found enhanced oxygen radical generation, as assessed with a novel electron spin trap, and increased protein oxidative damage in transgenic mice with the G93A SOD1 mutation.31,32 The formation of intracellular aggregates of SOD1 is

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Fig 5. Lumbar spinal cord sections from 110-day-old wild-type littermate control (A and E), SOD2 (B and F), FALS (C and G), and SOD2/FALS (D and H) mice. Gross atrophy of the spinal cord section and a loss of ventral horn neurons were observed in both the FALS and the SOD2/FALS mice, with a statistically significant greater neuronal loss found in the SOD2/FALS mice. Magnification bar in H equals 100 ␮m. SOD2 ⫽ manganese superoxide dismutase; FALS ⫽ familial amyotrophic lateral sclerosis. Table 3. Nissl, Tyrosine Hydroxylase, and Dopamine Transporter Protein Neuronal Counts in the Substantia Nigra Compacta of 110-Day-Old ALS Transgenic Mice with the G93A Human SOD1 Mutation, SOD2/FALS Mice, SOD2 Mice, and Littermate Control Mice

SOD2/FALS mice G93A mice SOD2 mice Littermate control mice

Nissl

TH

DAT

114 ⫾ 12.5a 123 ⫾ 11.4a 146 ⫾ 7.4 151 ⫾ 6.1

90 ⫾ 6.1a 98 ⫾ 5.2a 118 ⫾ 6.2 120 ⫾ 4.9

71 ⫾ 5.3a 83 ⫾ 5.8a 101 ⫾ 4.8 107 ⫾ 3.9

In 8 SOD2/FALS, G93A, SOD2, and littermate nontransgenic control mice, Nissl, tyrosine hydroxylase (TH), and dopamine transporter (DAT)-positive neurons within the substantia nigra compacta were quantitated by videomicroscopic capture of tissue sections and Neurolucida image analysis software. Data are mean ⫾ SEM values. p ⬍ 0.01, compared with littermate control and SOD2-deficient mice.

a

For other definitions, see Table 1.

another possible toxic mechanism, as has been proposed in diseases with polyglutamine expansions.33,34 The expression of SOD1 cDNAs with mutations, found in association with FALS, led to the formation of cytoplasmic aggregates in cultures of spinal motor neurons.35 The aggregates were not observed after expression of wt SOD1, and the formation of the aggregates was closely linked to apoptotic cell death.35 Upregulation of protein chaperones reduced the number

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of aggregates and cell death in vitro.36 Cytoplasmic inclusions in astrocytes are a prominent pathological feature in G85R transgenic ALS mice.24 Neuronal SOD1-positive inclusions are found in some FALS patients with the A4V mutation.37 Intracytoplasmic inclusions have also been observed in G93A transgenic ALS mice.37,38 A recent study showed that levels of wt SOD1 in ALS mice that express the G85R mutation had no ef-

fect on the timing of disease onset or death in the mice.38 The authors concluded that the aggregation of mutant SOD1 and its neurotoxicity are independent of the wild-type molecule and that toxicity was unlikely to arise from superoxide-mediated oxidative stress. It is still possible, however, that peroxidation of H2O2 or catalysis of nitration by peroxynitrite could occur, because both H2O2 and peroxynitrite can be produced independently of SOD1 (for example, by oxidases, flavoproteins, peroxidase and lipoxygenase enzymes, and the cytochrome P450 system). Furthermore, SOD1 does not appear to be critical in free radical defenses because mice deficient in SOD1 show only minor abnormalities.4 We therefore examined whether manipulation of SOD2 could affect the phenotype of transgenic mice with the G93A mutation. SOD2 is the most important scavenger of O2䡠 radicals within mitochondria. SOD2 is critical for protecting neurons from both excitotoxicity and nitric oxide–mediated toxicity.39 Homozygote mice deficient in SOD2 develop both a cardiomyopathy and central nervous system damage and die within a few weeks of birth.14,15 Hemizygote mice show no central nervous system abnormalities, although they show increased sensitivity to focal ischemia.17 We examined the effects of crossing hemizygote SOD2-deficient mice with transgenic mice with the G93A SOD1 mutation. The mice with both SOD2-deficiency and G93A SOD1 mutations show a significant reduction in survival, as well as decreased motor performance on the rotorod test, compared with mice with the G93A SOD1 mutation alone. The mice with both SOD2 deficiency and the G93A mutation also show a significant reduction in numbers of spinal cord motor neurons at 90 days of age, a time at which a significant loss of motor neurons in the mice with the G93A mutation alone could not be detected. At 110 days of age, there was a significant loss of motor neurons in SOD2/FALS mice as well as in mice with the G93A mutation alone. The loss of motor neurons in the SOD2/FALS mice, however, was significantly greater than that in mice with the G93A mutation alone. An interesting observation that links oxidative damage to the pathogenesis of both ALS and Parkinson’s disease is that transgenic mice with the G93A SOD1 mutation have a significant 25% loss of substantia nigra dopaminergic neurons at end-stage illness.40 We recently confirmed this result at 120 days of age and showed that administration of creatine could significantly attenuate the neuronal loss.41 In the present study, we show that there is no significant neuronal loss in the substantia nigra at 90 days of age, but that at 110 days of age, there is a significant approximate 20% loss of dopaminergic neurons in mice with the G93A mutation. The SOD2/FALS mice show a 25% loss of dopaminergic neurons at 110 days of age, which

is significantly greater than the loss seen in the mice with the G93A mutation alone. This observation provides further evidence for a role of oxidative damage in Parkinson’s disease pathogenesis.42 The present results therefore show that manipulation of SOD2 exacerbates the disease phenotype in transgenic mice with the G93A SOD1 mutation. This finding strongly supports a role of oxidative damage in disease pathogenesis. It also supports a role of mitochondrial damage. Mitochondria are particularly vulnerable to oxidative stress, and mitochondrial vacuolization and swelling are among the earliest pathological features in transgenic mice with both the G93A and G37R mutations.3,6,43 Consistent with this, we found that vacuolization was exacerbated in the SOD2/ FALS mice compared with the G93A mice alone. A recent study showed that an increase in mitochondrial vacuolization correlates with a sharp decline in muscle strength in mice with the G93A SOD1 mutation,44 and mitochondria in mice with the G93A mutation accumulate calcium.45 The effects of SOD1 mutations are expected to occur primarily in the cytosol where most SOD1 is localized. Recent studies, however, have shown that an early pathological abnormality in transgenic mice with the G37R mutation is focal accumulations of SOD1 immunoreactivity adjacent to small vacuoles in proximal axons.46 Some evidence also suggests that SOD1 may occur in the intermembrane space of mitochondria as well.47,48 If this were the case, it could help to explain preferential damage to mitochondria, as well as splitting of the inner and outer mitochondrial membranes.6 Preferential damage to mitochondria may occur because of interaction with peroxynitrite. Mitochondria contain a novel isoform of nitric oxide synthase that associates with the inner mitochondrial membrane.49 –51 Mitochondria may therefore be a source of peroxynitrite under pathological conditions, and this would be expected to be exacerbated in mice partially deficient in SOD2, which could lead to nitration and further inactivation of specific enzymes including SOD2 and creatine kinase, both of which are found in mitochondria.52–54 In summary, the present results show that a partial deficiency of SOD2 exacerbates the clinical phenotype and shortens survival in transgenic mice with the G93A SOD1 mutation. These findings further implicate both oxidative damage and mitochondrial dysfunction in the pathogenesis of motor neuron death in these mice.

This study was supported by NIH grants PO1 AG12992 (M.F.B., R.J.F.), NS35255 and NS38180 (M.F.B.), and NS37102 (R.J.F.),

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the Muscular Dystrophy Association and the ALS Association (M.F.B.), NIMH grant MH1-1692 (A.M.K.), and the Veterans Administration (R.J.F.). Dr Andreassen is supported by the Norwegian Research Council. The secretarial assistance of Sharon Melanson is gratefully acknowledged. We thank J. Kubilus for his photographic assistance.

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