Familial amyotrophic lateral sclerosis (FALS) has been linked in some families to dominant mutations of the SOD1 gene encoding Cu,Zn superoxide dismutase (Cu,ZnSOD). We have used a transgenic model of FALS based on expression of mutant human Cu,ZnSOD to explore the etiology and therapy of the genetic disease. Expression of mutant, but not wild-type, human Cu,ZnSOD in mice places the brain and spinal cord under oxidative stress. This causes depletion of vitamin E, rather than the typical age-dependent increase in vitamin E content as occurs in nontransgenic mice and in mice expressing wild-type human Cu,ZnSOD. Dietary supplementation with vitamin E delays onset of clinical disease and slows progression in the transgenic model but does not prolong survival. In contrast, two putative inhibitors of the glutamatergic system, riluzole and gabapentin, prolong survival. However, riluzole did not delay disease onset. Thus, there was clear separation of effects on onset, progression, and survival by the three therapeutics tested. This suggests the hypothesis that oxidative damage produced by the expression of mutant Cu,ZnSOD causes slow or weak excitotoxicity that can be inhibited in part by alerting glutamate release or biosynthesis presynaptically.

The G1y 93-*AIa mutation in the Cu,Zn superoxide dismutase (Cu,Zn-SOD) gene (SOD 1) found in some familial amyotrophic lateral sclerosis (FALS) patients has been shown to result in an aberrant increase in hydroxyl radical production by the mutant enzyme that may cause oxidative injury to spinal motor neurons. In the present study, we analyzed the extent of oxidative injury to lumbar and cervical spinal cord proteins in transgenic FALS mice that overexpress the SOD1 mutation [TgN(SOD1-G93A)G1H] in comparison with nontransgenic mice. Total protein oxidation was examined by spectrophotometnc measurement of tissue protein carbonyl content by the dinitrophenylhydrazine (DNPH) assay. Four ages were investigated: 30 (pre-motor neuron pathology and clinical disease), 60 (after initiation of pathology, but predisease), 100 (~50%loss of motor neurons and function), and 120 (near complete hindlimb paralysis) days. Protein carbonyl content in 30-day-old TgN(SOD1-G93A)G1 H mice was twice as high as the level found in age-matched nontransgenic mice. However, at 60 and 100 days of age, the levels were the same. Then, between 100 and 120 days of age, the levels in the TgN (SOD1 -G93A) Gi H mice increased dramatically (557%) compared with either the nontransgenic mice or transgenic animals that overexpress the wild-type human Cu,Zn-SOD [TgN(SOD1)N29]. The 100-120-day increase in spinal cord protein carbonyl levels was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation and western blot immunoassay, which enabled the identification of heavily oxidized individual proteins using a monoclonal antibody against DNPH-derivatized proteins. One of the more heavily oxidized protein bands (14 kDa) was identified by immunoprecipitation as largely Cu,Zn-SOD. Western blot comparison of the extent of Cu,Zn-SOD protein carbonylation revealed that the level in spinal cord samples from 120day-old TgN (SOD1 -G93A) Gi H mice was significantly higher than that found in age-matched nontransgenic or TgN(SOD1)N29 mice. These results suggest that the increased hydroxyl radical production associated with the G93A SOD1 mutation and/or lipid peroxidation-derived radical species (peroxyl or alkoxyl) causes extensive protein oxidative injury and that the Cu,Zn-SOD itself is a key target, which may compromise its antioxidant function. Key Words: Transgenic mouse-Familial amyotrophic lateral sclerosis-Protein oxidation-Car-bonyl content-Dinitrophenylhydrazine-Western immunoblot.

Abstract:The time course and intensity of brain hydroxyl radical ('OH) generation were examined in male CF-I mice during the first hour after moderate or severe concussive head injury. Hydroxyl radical production was measured using the salicylate trapping method in which the production of 2.3-and/or 2.5-dihydroxybenzoic acid (DHBA) in brain 15 min after salicylate administration was used as an index of 'OH formation. In mice injured with a concussion of moderate severity asdefined by the I-h posttraumatic neurologic recovery (grip score), a 60% increase in 2,5-DHBA formation was observed by 1 min after injury compared with that observed in uninjured mice. The peak in DHBA formation occurred at 15 min after injury (+67.5%; p < 0.02, compared with uninjured). At 30 min, the increase in DHBA lost significance, indicating that the posttraumatic increase in brain 'OH formation is a transient phenomenon. In severely injured mice, the peak increase in DHBA (both 2,3-and 2,5-) was observed at 30 min after injury, but also fell off thereafter as with the moderate injury severity. Preinjury dosing of the mice with SKF-525A (50 mg/kg i.p.), an inhibitor of microsomal drug oxidations, did not blunt the posttraumatic increase in salicylate-derived 2,5-DHBA, thus showing that it is not due to increased metabolic hydroxylation. Neither injury nor SKF-525A administration affected the DHBA plasma levels. However, saline perfusion of the injured mice to remove the intravascular blood before brain removal eliminated the injury-induced increase in 2,5-DHBA, but did not affect the baseline levels seen in uninjured mice. This implies that the source of the increased DHBA in the injured mice is the microvasculature, probably the endothelium. The administration of the 2 1 -aminosteroid lipid antioxidant, tirilazad mesylate, which possesses 'OH scavenging properties, also attenuated the posttraumatic increase in DHBA, further supporting that it reflects an increase in 'OH radical formation. These results are the first direct demonstration of the occurrence and time course of increased 'OH production in injured brain. Key Words: Hydroxyl radical-Concussion-Salicylate trapping-Brain. Hall E. D. et al. Brain hydroxyl radical generation in acute experimental head injury. J. Neurochem. 60, 588-594 (1 993).

We present data correlating the time courses of hydroxyl radical (.OH) production, lipid peroxidation, and blood-brain barrier (BBB) damage following unilateral head injury in the rat. Using a controlled cortical impact device to inflict head injury, we have directly measured brain .OH levels via the salicylate trapping method, and phosphatidylcholine hydroperoxide (PCOOH) levels via the HPLC-chemiluminescence technique, at 5, 30, and 60 min postinjury. These results were then correlated with the time course of BBB disruption, as measured by the extravasation of Evans blue dye (EB) into the injured cortex, over the same time period. In the present study, .OH levels were 62% higher than sham at 5 min postinjury, 25% higher at 15 min (both p < or = 0.05), and no different from sham at 60 min. PCOOH, on the other hand, increased linearly between 5 and 60 min postinjury. Whereas PCOOH levels were 25% greater than sham at 5 min, they were 35% and 52% higher than sham at 30 and 60 min, respectively (both p < or = 0.05 vs sham). Blood-brain barrier disruption followed a similar time course to PCOOH generation, except that the magnitude of the effect was much greater. Whereas EB extravasation was only slightly elevated in the injured cortex at 5 min postinjury, there was nearly an 8-fold increase at 30 min and an 11-fold increase at 60 min (all p < or = 0.05 vs sham). An additional experiment demonstrated that BBB damage can be attenuated by treatment with the 21-aminosteroid lipid peroxidation inhibitor, tirilazad mesylate (U-74006F). Rats were given a single i.v. injection of 3 or 10 mg/kg of U-74006F 5 min postinjury and killed 30 min postinjury. The 10 mg/kg dose of U-74006F reduced EB extravasation 52% (p < 0.025) in comparison to vehicle-treated controls. This is the first study to correlate the time courses of .OH formation, lipid peroxidation, and BBB disruption in injured brain. The results suggest that there is an immediate, posttraumatic burst in .OH formation, followed by a progressive increase in lipid peroxidation and a similar, although slightly delayed, time-related opening of the BBB. The attenuation of BBB damage by U-74006F suggests that this chain of events can be interrupted by administration of an antioxidant/lipid peroxidation inhibitor.

Transgenic mice that overexpress a mutated human CuZn superoxide dismutase (SOD1) gene (gly93-->ala) found in some patients with familial ALS (FALS) have been shown to develop motor neuron disease, as evidenced by motor neuron loss in the lumbar and cervical spinal regions and a progressive loss of voluntary motor activity. The mutant Cu,Zn SOD exhibits essentially normal dismutase activity, but in addition, generates toxic oxygen radicals as a result of an enhancement of a normally minor peroxidase reaction. In view of the likelihood that the manifestation of motor neuron disease in the FALS transgenic mice involves an oxidative injury mechanism, the present study sought to examine the extent of lipid peroxidative damage in the spinal cords of the TgN(SOD1-G93A)G1H mice over their life span compared to nontransgenic littermates or transgenic mice that overexpress the wild-type human Cu,Zn SOD (TgN(SOD1)N29). Lipid peroxidation was investigated in terms of changes in vitamin E and malondialdehyde (MDA) levels measured by HPLC methods and by MDA-protein adduct immunoreactivity. Four ages were investigated: 30 days (pre-motor neuron pathology and clinical disease); 60 days (after initiation of pathology, but predisease); 100 days (approximately 50% loss of motor neurons and function); and 120 days (near complete hindlimb paralysis). Compared to nontransgenic mice, the TgN(SOD1-G93A)G1H mice showed blunted accumulation of spinal cord vitamin E and higher levels of MDA (P < 0.05 at 30 and 60 days) over the 30-120 day time span. In the TgN(SOD1)N29 mice, levels of MDA at age 120 days were significantly lower than in either the TgN(SOD1-G93A)G1H or nontransgenic mice. MDA-protein adduct immunoreactivity was also significantly increased in the lumbar spinal cord at age 30, 100, and 120 days, and in the cervical cord at 100 and 120 days. The results clearly demonstrate an increase in spinal cord lipid peroxidation in the FALS transgenic model, which precedes the onset of ultrastructural or clinical motor neuron disease. However, the greatest intensity of actual motor neuronal lipid peroxidative injury is associated with the active phase of disease progression. These findings further support a role of oxygen radical-mediated motor neuronal injury in the pathogenesis of FALS and the potential benefits of antioxidant therapy.