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(The FASEB Journal. 1999;13:2318-2328.)
© 1999 FASEB

The roles of free radicals in amyotrophic lateral sclerosis: reactive oxygen species and elevated oxidation of protein, DNA, and membrane phospholipids

DANXIA LIU*,{dagger}1, JING WEN*, JING LIU* and LIPING LI*

Departments of
* Neurology and
{dagger} Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-0653, USA

1Correspondence: Gail Borden Bldg., Route 0653, Department of Neurology, University of Texas Medical Branch, Galveston, TX 77555-0653, USA. E-mail: DLiu{at}utmb.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUMMARY
 REFERENCES
 
To explore whether reactive oxygen species (ROS) play a role in the pathogenesis of amyotrophic lateral sclerosis (ALS), a unique microdialysis or microcannula sampling technique was used in mice transfected with a mutant Cu,Zn-superoxide dismutase (SOD1) gene from humans with familial ALS, mice transfected with the normal human SOD1 gene, and normal mice. We demonstrate for the first time that the levels of hydrogen peroxide (H2O2) and the hydroxyl radical (·OH) are significantly higher, and the level of the superoxide anion (O2·-) is significantly lower in ALS mutant mice than in controls, supporting by in vivo evidence the hypothesis that the mutant enzyme catalyzes ·OH formation by the sequence: O2·- -> H2O2 -> ·OH. This removes doubts regarding the relevance of elevated ROS in FALS raised by in vitro experiments. The levels of oxidation products are also significantly higher in the mutant mice than in controls, consistent with some previous reports. Only the superoxide concentration differs between two controls among all the measurements. Our findings correlate in vivo a gene mutation to both elevated H2O2 and ·OH and increased oxidation of cellular constituents. The elevated H2O2 in mutant mice indicates impairment of its detoxification pathways, perhaps by changed interactions between SOD1 and H2O2 detoxification enzymes.—Liu, D., Wen, J., Liu, J., Li, L. The roles of free radicals in amyotrophic lateral sclerosis: reactive oxygen species and elevated oxidation of protein, DNA, and membrane phospholipids.


Key Words: mutation of Cu,Zn-superoxide dismutase gene • transgenic mouse • hydrogen peroxide • hydroxyl radical • superoxide anion


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUMMARY
 REFERENCES
 
AMYOTROPHIC LATERAL SCLEROSIS (ALS) is an untreatable, age-related, fatal motor neuron degenerative disease whose cause is unknown (1) . The finding of a single-site mutation in the Cu,Zn-superoxide dismutase (SOD1) gene in familial ALS (FALS) patients (2 , 3) linked this disease to free radicals (4) . The goal of this study was to reveal with sound in vivo experiments whether reactive oxygen species (ROS), including superoxide anion (O2·-), hydrogen peroxide (H2O2), and hydroxyl radical (·OH), play a role in neuronal degeneration in ALS. To explore how mutant SOD1 (mSOD1) causes ALS, Gurney and colleagues (5) produced a transgenic mouse model by introducing a human (with ALS disease) mutant SOD1 (mSOD1) gene (Gly 93 -> Ala, G93A) into mice; these transfected mice developed symptoms resembling human ALS. Since then over 50 different SOD1 mutants have been identified in FALS families (6) and a number of transgenic mouse models with different mutation sites have been developed.

There has been a debate between advocates of loss-of-function and those for gain-of-function hypotheses as to whether free radicals play a role in the pathogenesis of ALS. Screening revealed SOD1 mutations with reduced SOD1 activity in 16 of 73 (22%) ALS families (7) , suggesting that a loss of SOD1 function sometimes occurs in ALS. Mutations of the SOD1 gene reduce superoxide dismutase activity (2 , 3 , 8 9 10 11) ; this should elevate levels of O2·-. Elevated O2·- may produce very destructive ·OH by the metal-catalyzed Haber-Weiss reaction through the sequence O2·- -> H2O2 -> ·OH (12) . However, using their model, Gurney and colleagues found that transgenic mice expressing high levels of mutant human SOD1 protein became paralyzed even though the animal’s own normal SOD1 gene remained intact, while similar overexpression of normal human SOD1 did not produce ALS (5) . This finding challenges the loss-of-function hypothesis. This result and the significantly increased expression of SOD1 mRNA in spinal cord motor neurons in sporadic ALS (13) suggest that the mSOD1 protein gains a new function that damages motor neurons (5) —a gain-of-function hypothesis.

It has been demonstrated in vitro that SOD1 can catalyze dissociation of H2O2 to ·OH (14 , 15) and that the ·OH-generating function of mSOD1 (G93A and A4V) is enhanced relative to that of the normal SOD1 enzyme (16 17 18) . X-ray crystallographic studies show that the active channel of the mSOD1 containing copper and zinc is slightly larger than that of the normal SOD1 enzyme (3) ; thus, the metal atoms are more accessible to H2O2. The mutant SOD1 might catalyze more ·OH formation because its Cu2+ is more exposed. However, there is strong disagreement regarding the possibility that the mutant SOD1 gains a new function to catalyze ·OH formation from H2O2 (19) , as there is no strong in vivo evidence supporting this in vitro observation because of a lack of appropriate techniques to analyze O2·- and H2O2 in vivo. Only the levels of ·OH have been measured, but results are somewhat conflicting. For example, it was reported that ·OH levels are not significantly different between mutant (G37R) SOD1 transgenic mice and controls (20) . In contrast, we found that the level of ·OH is significantly higher in the cerebrospinal fluid (CSF) in G93A transgenic mice than in normal and SOD1 transgenic mice (21) , and an elevated level of ·OH was found in the extracellular space in the brains of G93A transgenic mice by other investigators (22) . It was also reported recently that there is no difference in catalytic capability in vitro between mutant SOD1 and normal SOD1 in producing ·OH from H2O2, questioning the previous conclusion (23) . To resolve these apparent inconsistencies, the present study examined in vivo the in vitro finding that on mutation SOD1 gains a new function, catalyzing ·OH formation from H2O2. We determined the levels of ROS in the G93A transgenic mice (mSOD1 mice), normal SOD1 transgenic mice (SOD1 mice), and normal mice (or the littermates of G93A mice, Nc mice) using a microdialysis or microcannula sampling technique. Our results indicate that the in vitro finding of ·OH formation by mSOD1 using H2O2 as a substrate reported previously (14 15 16 17 18) is a realistic pathway in vivo, demonstrating a role of ROS in ALS.

Attack of elevated free radicals on proteins, DNA, and membrane phospholipids initiate oxidative damage to these molecules, eventually destroying cells (24 25 26 27 28) . It has been hypothesized that oxidative damage to these major cell components may be a final common pathway to cell destruction and death in a variety of neuronal degenerative disorders. However, there is also strong disagreement concerning the role of oxidative stress in the pathogenesis of ALS (19 , 29) , and the experimental results are contradictory. The levels of malondialdehyde (MDA)—an end product of membrane lipid peroxidation—were increased in the spinal cord sections of FALS and sporadic ALS (SALS) patients compared to control patients (30) , in the cerebral cortex (31) , and in the spinal cord (32) in mutant (G93A) SOD1 transgenic mice. The antioxidants vitamin E and selenium delay the appearance of ALS symptoms in these mice (33) . However, the MDA level was not increased in G37R transgenic mice (20) . The level of 8-hydroxyl-2-deoxyguanosine (8-OHdG), a marker of DNA oxidation, was increased in the motor cortex in SALS but not in FALS patients (30) ; it was also 10-fold higher in the spinal cord tissue in ALS patients than in controls (34) . Protein carbonyl content—a marker of protein oxidation—was elevated by 85% in postmortem brain tissue from ALS patients (8) . It was also increased in G93A transgenic mice (35) and in the motor cortex of SALS but not FALS patients (30) . Because of these contradictions, the present study reexamined concentrations of these oxidative markers in the G93A transgenic mice and controls. Our results support that the mutation of SOD1 indeed induces oxidative stress and correlate SOD1 mutation to elevated H2O2, ·OH and oxidative stress.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 SUMMARY
 REFERENCES
 
Animal preparation
The procedures on mice were approved by the University of Texas Medical Branch Animal Care and Use Committee and were in accord with the NIH guide for the Care and Use of Laboratory Animals.

Three groups of mice purchased from the Jackson laboratory were used: mice transfected with the mutant SOD1 gene (G93A) from humans with FALS-B6SJL-TgN(SOD1-G93A)1Gur,mice transfected with normal human SOD1 gene B6SJL-TgN(SOD1)2Gur, and normal control mice (B6SJLF1 or littermates of mSOD1 and SOD1 mice) without gene transfection. For measurements of ·OH and the oxidative products (protein carbonyl content, 8-OHdG, and MDA), the onset of the ALS symptoms in the mSOD1 mice we used was delayed due to a small transgenic copy number; they became incapacitated within 8 months. Therefore, mSOD1, SOD1, and Nc mice were used at 6–6.5 months of age in these experiments. The mSOD1 mice used for the measurement of O2·- and H2O2 carried a high copy number of the mutant gene and died by 4–5 months; for those mutants, therefore, 2.5- to 3-month-old mSOD1 mice and matching age SOD1 and Nc mice were used while paralysis was developing in the mutant strains. We tested whether the levels of O2·- were different among the littermates of mSOD1 mice, littermates of SOD1 mice, and the normal control mice (B6SJLF1); no difference was found. Therefore the B6SJLF1 normal control mice are a valid control for mSOD1 mice; our definition of normal control (Nc) mice includes B6SJLF1 mice and littermates of mSOD1 and SOD mice.

A microdialysis or microcannula loop was implanted into the terminal cistern in the intrathecal space of the mouse spinal cord; CSF was sampled from there so as to avoid damage to the spinal cord caused by insertion of the sampling probe to the tissue (Fig. 1C ). The cauda equina in the terminal cistern is surrounded by CSF. There is enough space to implant the loop without injuring the nerves and there is no cord in this area that might be damaged by the loop. The dialysis zone is 2 cm long, so slight differences in the length of the uncoated zones did not affect the accuracy of the results.



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  		Figure 1. Preparation and implantation of sampling loops. The loop was made from a coated microdialysis catheter (as described in Materials and Methods) with a Nichrome-Formvar wire inserted through the catheter. Then the catheter was bent to form a U-shaped loop. A) The microdialysis loop. One end of the loop was glued to a PE 20 tube that was attached to a syringe pump; the other end of the loop was inserted into a vial for collecting dialysates. B) The cannula loop. After coating, four pairs of holes were made within a 2 cm zone through the wall of the catheter at 0.5 cm intervals. Both ends of the loop were then attached to a push-pull syringe pump with two lengths of PE 20 tubing. C) Implantation of the loop. After a laminectomy on the top of vertebra L5, a small hole was made in the exposed dura and the loop was implanted through the hole caudal to the terminal cistern in the mouse intrathecal space. The loop was fixed on the vertebra L5 by super glue and the incision was closed.

Microdialysis sampling
The microdialysis catheter (200 µM ID and 300 µM OD, 11 kDa molecular weight cutoff; Filtral, AN 69-HF) was coated with a thin layer of epoxy (Devcon, Danvers, Mass.) except for a 2 cm long dialysis zone. A 1 mm wide ink mark extending away from the trailing edge of the dialysis zone was made on the catheter before coating, as described in our previous publications (36 , 37) . A Nichrome-Formvar wire (0.0026'', A-M systems, Everett, Wash.) was inserted through the catheter (38) . Then the catheter was bent to form a U-shaped loop. One end of the loop was glued to a PE 20 tube (Fig. 1A ). The mouse was anesthetized with 3% halothane and maintained with 0.5–1% halothane in an oxygen (3 L/min) and air mixture (1:1). After a laminectomy performed at vertebra L5 by removing half of the vertebra, a small hole was made in the exposed dura and care was taken not to damage the nerve. The mouse was then clamped in a frame and the dialysis loop was implanted through the hole caudal to the terminal cistern in the mouse intrathecal space until the ink mark just entered the dura (Fig. 1C ). Then the loop was fixed and the incision was closed. The PE 20 tube was then attached to a syringe pump for sampling. The free end of the loop was inserted into a collecting vial in ice for collecting dialysates. Artificial cerebrospinal fluid (ACSF) was pumped through the loop at a rate of 2.5 µl/min. The composition of the ACSF (in mM) was 151 Na+, 2.6 K+, 0.9 Mg2+, 1.3 Ca2+, 132 Cl-, 21.0 HCO3-, 2.5 HPO42-, and 3.5 glucose. The ACSF was bubbled with 95% O2/5% CO2 prior to each experiment to adjust the pH to 7.2 (36 , 37 , 39) . Sampling from cerebrospinal fluid was begun 60 min after implantation of the loop in order to allow the release of substances due to the insertion of the dialysis loop to subside to a stable baseline. Body temperature was maintained throughout the experiment by using a heating blanket.

Microcannula sampling
The cannula was prepared as above from the same catheter. After coating, four pairs of holes were made within a 2 cm zone through the wall of the catheter at 0.5 cm intervals, as we reported previously (40) . Cannula insertion is the same as for a dialysis loop, except both ends of the loop were attached to a push-pull syringe pump with two lengths of PE 20 tubing (Fig. 1B ). ACSF was pumped through the cannula at a rate of 15 µl/min.

Analysis of ·OH in microdialysates
The level of ·OH was determined by measuring the hydroxylation products of salicylate, 2,3- and 2,5- dihydroxybenzoic acids (2,3- and 2,5-DHBA), as reported (41 , 42) and slightly revised by us. Salicylate (5 mM) in ACSF (pH 7.2±0.2) was pumped through the microdialysis loop implanted in the terminal cistern in the intrathecal space of the mouse spinal cord as a ·OH trapping agent and the dialysates collected were measured by high-performance liquid chromatography (HPLC ) with electrochemical detection (ECD). A Shimadzu HPLC with an LC-10AD pump, a SIL-10AXL autoinjector, and a Waters Spherisorb S3 ODS2 (3 µM particle, 15 cm x 4.6 mm) analytical cartridge column were used to separate these two isomers and an ESA Coulochem II ECD was used for detection. The voltages used were 250 mV to detect 2,3- and 2,5-DHBA, 750 mV to detect salicylate, and 775 mV for the guard cell. The mobile phase was 0.03 M potassium citrate and 0.03 M sodium acetate, and the elution flow rate was 1 ml/min. 2,5-, and 2,3-DHBA (Sigma, St. Louis, Mo.) were injected as external standards for quantitation.

Analysis of H2O2 in microdialysates
The levels of H2O2 in the intrathecal space were determined using a unique method developed in our laboratory (43) . FeCl2 (0.2 mM) and salicylate (1 mM) in water (pH 3.0) were pre-added to the collecting vial (1:1, perfusate:reacting solution). The Fe2+ pre-added in the vial will catalyze dissociation of H2O2 sampled by the microdialysis loop and collected into the vial to ·OH by the Fenton reaction. The produced ·OH rapidly attacks salicylate to produce 2,3- and 2,5-DHBA, which were measured by HPLC with EC detection as described above. The 2,3-DHBA used as a specific marker (44) was calibrated to amount of H2O2 using a standard curve. The calibration curve was prepared as follows: 100 µl H2O2 in water at concentrations of 0.5, 1.0, 1.5, 2.0, and 4.0 were added to test tubes containing 100 µl FeCl2 (0.2 mM) and salicylate (1 mM) in water (pH 3.0); the DHBAs produced were measured by HPLC. The calibration curve was presented as 2,3-DHBA (µM) vs. H2O2 (µM). A linear calibration curve was obtained in this concentration region.

Analysis of O2·- in perfusates and spinal cord tissue
The levels of O2·- in the terminal cistern were sampled by a microcannula and determined by measuring reduced cytochrome c in the perfusates using our unique method (40) . After the initial ACSF perfusion, the perfusing fluid was changed to cytochrome c (50 µM in ACSF) solution and equilibrated for 30 min. Three samples were collected at 20 min intervals. The collected perfusates were centrifuged at 12,500 x g for 15 min to precipitate blood and tissue fragments. Reduced cytochrome c in the supernatant was measured by a spectrophotometer at a wavelength of 550 nm. To determine the level of O2·- in the spinal cord tissue, 500 µM cytochrome c was infused into the intrathecal space by the loop through the holes made on the wall at a flow rate of 1 µl/min for 30 min. Then a laminectomy was performed from the L5-T10 vertebrae, the spinal cord was frozen in situ by liquid nitrogen (-196°C), which also killed animals. The tissue was removed and transferred into a vial containing 500 µM cytochrome c in ACSF, homogenized, centrifuged at 13,000 x g for 15 min, and the supernatant was passed through a 30,000 molecular weight ultrafiltration membrane (Micron Separation Inc., Westborough, Mass.). Absorbance of the ultrafiltrate was measured at 550 nm.

Analysis of malondialdehyde in microdialysates
The MDA concentration was measured in the microdialysates sampled from the CSF in the terminal cistern of the mouse intrathecal space by the dialysis loop. The microdialysates were reacted directly with the TBA reagent (45 , 46) and the products of the TBA reaction were analyzed by HPLC (Beckman, Fullerton, Calif.) with a fluorescence detector (Shimadzu RF10A), using a method developed in our laboratory (47) .

Analysis of protein carbonyl content in spinal cord tissue
Some amino acid residues in protein can be converted to carbonyl derivatives by metal-catalyzed oxidation (48) ; therefore, protein carbonyl content was used as a marker of protein oxidation. After a laminectomy at vertebrae L5-T10, the spinal cord was frozen in situ by liquid nitrogen (-196°C), which also killed the animal. The tissue was then removed and stored at -80°C for analysis. To analyze protein carbonyl content, protein was first separated from DNA according to the method of Beland and co-workers (49) . Briefly the frozen tissue was homogenized in 8 M urea-0.24 M sodium phosphate-1% sodium dodecyl sulfate-10 mM EDTA, pH 6.8, using a 20:1 solution to tissue ratio (v:w). The solution was then extracted with an equal volume of chloroform-isoamyl alcohol-phenol (24:1:25) saturated with the homogenizing buffer. The resulting emulsion was separated into two phases by centrifugation at 4000 rpm. The aqueous phase on the top of the organic phase was removed for analysis of damaged DNA. Protein in and above the organic phase was precipitated by addition of an equal volume of acetone. Protein carbonyl content was determined by characterizing protein oxidation spectrophotometrically after labeling with 2,4-dinitrophenylhydrazine (DNPH) by slightly modifying the procedure of Levine and co-workers (50) . After the final ethyl acetate/ethanol wash, the protein pellets were dissolved in 6 M guanidine containing 20 mM potassium phosphate (pH 2.3) and the 370/280 nm absorbance ratio was determined. Protein concentrations were calculated using an absorption coefficient of 1 mg/ml at 280 nm. The carbonyl contents were calculated from the 370 nm/280 nm absorbance ratio of each sample minus the respective control, using a molar absorption coefficient of 22,000 M-1 cm-1 at 370 nm.

Analysis of 8-hydroxy-2-deoxyguanosine in DNA in spinal cord tissue
Hydroxyl radical reacts with DNA to form several products, including 8-OHdG, which can be quantitated at extremely low levels (<20 fmol) by the use of HPLC with EC detection (51 , 52) . The frozen tissue was homogenized and DNA was separated from protein as described above. The DNA in the aqueous phase was purified, digested, and 8-OHdG was analyzed using a reported procedure modified by us. The aqueous phase was dialyzed first and the dialysate dried on a centrifugal evaporator. The sample then was extracted and purified. The RNA in the DNA solution was enzymatically digested according to reported methods (53 , 54) . The DNA in the solution was extracted, purified, digested (55) , and measured with a UV spectrophotometer at 254 nm. The pH of the digest was adjusted to 5.2 for analyzing 8-OHdG by HPLC-EC detection. The procedure for HPLC analysis was according to the standard method of ESA, Inc. A Shimadzu HPLC with a Waters ODS2 column and an ESA ECD was used. Voltages of the guard cell, oxidative electrode, and analytical electrode were 450, 125, and 400 mV respectively. The mobile phase used for isocratic elution was 5% methanol in 100 mM sodium acetate (pH adjusted to 5.2 with H3PO4) at a 1 ml/min flow rate (ESA, Inc., standard method).

Statistical test
An unpaired t test in Sigma plot was used in all measurements to determine whether the mean results obtained from mSOD1, SOD1, and Nc mice were significantly different.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 SUMMARY
 REFERENCES
 
The mutation of SOD1 significantly increases the levels of ·OH
Figure 2 illustrates the concentrations of 2,3- and 2,5-DHBA measured in the dialysates sampled from CSF. The average level of 2,5-DHBA (mean±SD) is 980 ± 240 nM in mSOD1 mice (n=8), 300 ± 120 nM in SOD1 mice (n=5), and 400 ± 190 nM in Nc mice (n=6): ~3.3-fold higher in mSOD1 mice than in SOD1 mice (P=0.0001) and 2.5-fold higher than in Nc mice (P=0.0005). The average level of 2,3-DHBA (mean±SD) is 650 ± 130 nM in mSOD1 mice, 330 ± 90 nM in SOD1 mice, and 350 ± 190 nM in Nc mice: ~2.0-fold higher in mSOD1 mice than in SOD1 mice (P=0.0006) and 1.9-fold higher than in Nc mice (P=0.005). Both 2,5- and 2,3-DHBA levels are significantly higher in mSOD1 mice than in SOD1 and Nc mice, but there is no significant difference between the SOD1 and Nc mice (P=0.3 for 2,5-DHBA and 0.8 for 2,3-DHBA), direct in vivo evidence that SOD1 mutation elevates the levels of ·OH.



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  		Figure 2. Upper panel: concentrations of 2,3- and 2,5-DHBA in microdialysates. ACSF was first pumped through the system for 1 h after placement of the dialysis loop, then salicylate was administered through the loop. After a 30 min equilibration, three samples were collected at 20 min intervals. The levels of 2,3- and 2,5-DHBA (hydroxyl radical markers) were measured by HPLC with EC detection in the three groups of mice (8 mSOD1, 5 SOD1, and 6 Nc mice). Concentrations for each animal are the averages of three dialysis samples. Each bar represents the average data for that group of experiments (mean±SD). Lower panel: typical chromatograms. a) Standards of 2,3- (5 pmol) and 2,5-DHBA (20 pmol). b) Chromatogram of 10 µl dialysate obtained from a mSOD1 mouse. The peaks contain 14.9 pmol 2,5-DHBA and 7.0 pmol 2,3-DHBA. c) Chromatogram of 10 µl dialysate obtained from a SOD1 mouse. The peaks contain 4.8 pmol 2,5-DHBA and 3.6 pmol 2,3-DHBA. d) Chromatogram of 10 µl dialysate obtained from a normal control mouse. The peaks contain 6.6 pmol 2,5-DHBA and 3.7 pmol 2,3-DHBA.

The mutation of SOD1 significantly increases the levels of H2O2
Figure 3A, B illustrates the concentrations of 2,3- and 2,5-DHBA measured in the dialysates, and Fig. 3C illustrates the concentration of H2O2 after calibration. The average level of H2O2 (mean±SD) is 2.8 ± 0.3 µM in mSOD1 mice (n=4), 1.9 ± 0.2 µM in SOD1 mice (n=3), and 2.1 ± 0.4 µM in Nc mice (n=9). The levels of H2O2 are significantly higher in mSOD1 mice than in SOD1 (P=0.005) and Nc mice (P=0.007), but there is no significant difference between the SOD1 and Nc mice (P=0.6), indicating that mutant SOD1in the mSOD1 mice also elevates H2O2 levels.



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  		Figure 3. The concentrations of H2O2 in microdialysates. ACSF was first pumped through the system for 1 h after placement of the dialysis loop, then three samples were collected in vials containing FeCl2 and salicylate at 20 min intervals. The reaction products, 2,3- and 2,5-DHBA, were measured by HPLC with EC detection in the three groups of mice. The concentration of H2O2 was obtained using a calibration curve as described in Materials and Methods. Concentrations for each animal are the averages of the three dialysis samples. Each bar represents the average data for that group of experiments (mean±SD). A, B) Concentration of 2,3- and 2,5-DHBA measured in the dialysates. C) Concentration of H2O2 calibrated from 2,3-DHBA by the calibration curve.

Significantly lower levels of O2·- in the mutant mice than in controls
Figure 4A illustrates levels of O2·- in the perfusates collected from the mSOD1, SOD1, and Nc mice. The average absorbance of reduced cytochrome c (mean±SD) is 0.060 ± 0.011 in mSOD1 mice (n=6), 0.037 ± 0.004 in SOD1 mice (n=3), and 0.130 ± 0.030 in Nc mice (n=6). In contrast to the results of H2O2 and ·OH, the levels of O2·- are significantly higher in normal control mice than in mSOD1 (P=0.0007) and SOD1 mice (P=0.002). The level of O2·- is also significantly higher in mSOD1 than in SOD1 mice (P=0.01). Figure 4B shows the levels of O2·- in the spinal cord tissue of the mSOD1, SOD1, and Nc mice. The average absorbance of reduced cytochrome c (mean±SD, absorbance/g wet weight tissue) is 158 ± 10 in mSOD1 mice (n=3), 138 ± 10 in SOD1 mice (n=3), and 186 ± 12 in Nc mice (n=4). Consistent with the levels measured in perfusates, the level of O2·- in the spinal cord tissue of normal control mice is significantly higher than in mSOD1 (P=0.02) and SOD1 mice (P=0.003), and is also higher in mSOD1 than in SOD1 mice. This is the first in vivo measurement of the levels of O2·- in the CSF and spinal cord.



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  		Figure 4. The levels of superoxide in perfusates and spinal cord tissue. ACSF was first pumped through the system at 15 µl/min for 1 h after placement of the perfusing loop, then cytochrome c (50 µM in ACSF) was administered through the loop. After 30 min equilibration, three samples were collected at 20 min intervals. The perfusates were centrifuged, and reduced cytochrome c in the supernatant was measured by a spectrophotometer at a wavelength of 550 nm. To determine the levels of O2·- in the spinal cord tissue, 500 µM cytochrome c was infused into the intrathecal space by the loop through the holes made in the wall at a flow rate of 1 µl/min for 30 min. Then a laminectomy was performed and the cord was removed after freezing in situ by liquid nitrogen. The tissue was dropped into a vial containing 500 µM cytochrome c in ACSF, then homogenized and centrifuged; the supernatant was passed through a 30,000 molecular weight ultrafiltration membrane. The absorbance of ultrafiltrate was measured at 550 nm. Each bar represents the average data for that group of experiments (mean±SD). A) The absorbance of reduced cytochrome c measured in the perfusates collected from mSOD1 (n=6), SOD1 (n=3), and Nc mice (n=6). Concentrations for each animal are the averages for the three perfusates. B) The absorbance of reduced cytochrome c measured in spinal cord tissue in mSOD1 (n=3), SOD1 (n=3), and Nc mice (n=4).

Mutation of SOD1 significantly increases membrane peroxidation
Figure 5 indicates the concentrations of MDA in the dialysates collected from the CSF in mSOD1, SOD1, and Nc mice. The average level of MDA (mean±SD) is 70 ± 15 nM in mSOD1 mice (n=8), 40 ± 5 nM in SOD1 mice (n=7), and 30 ± 10 nM in Nc mice (n=9). The MDA level in dialysates in mSOD1 mice is 1.8-fold that in SOD1 mice (P=0.0005) and 2.3-fold higher than in Nc mice (P=0.00005). There was no significant difference between SOD1 and Nc mice (P=0.1), suggesting that overexpression of normal SOD1 does not increase peroxidation of membrane lipids. The significantly higher levels of MDA in mSOD1 mice than in SOD1 and Nc mice demonstrate that oxidative damage to membrane phospholipids indeed occurs after the induction of the mutant SOD1 gene.



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  		Figure 5. The concentrations of MDA in microdialysates. ACSF was pumped through the system for 1 h after the placement of the dialysis loop to establish the basal levels of MDA. Three samples were then collected at 20 min intervals. The MDA in microdialysates was measured by HPLC and fluorescence detection in mSOD1 (n=8), SOD1 (n=7), and Nc (n=9) mice. Three samples from each animal were analyzed and the results averaged. Each bar represents the average data for that group of experiments (mean±SD).

The mutation of SOD1 significantly increases protein oxidation
Figure 6 shows the protein carbonyl content measured in the spinal cord tissue of the three groups of mice (n=7 for each group). The average protein carbonyl content (mean±SD) is 2.31 ± 0.16 nmol/mg protein in mSOD1 mice, 1.95 ± 0.09 nmol/mg protein in SOD1 mice, and 1.94 ± 0.13 nmol/mg in Nc mice. The protein carbonyl content in spinal cord tissue is 1.2-fold higher in mSOD1 mice than in SOD1 (P=0.0002) and Nc (P=0.0004) mice—a significant increase; there is no difference between the two controls (P=0.9), indicating that overexpression of normal SOD1 does not induce protein oxidation, and only mutant SOD1 in mSOD1 mice causes significantly more protein oxidation.



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  		Figure 6. Protein carbonyl content in the spinal cord tissue. Protein carbonyl content in the mouse spinal cord tissue of mSOD1, SOD1, and Nc mice was measured spectrophotometrically after labeling with DNPH as described in Materials and Methods. Each animal generated one datum, and the average protein carbonyl content in each group was obtained from seven animals (mean±SD).

The mutation of SOD1 significantly increases DNA oxidation
8-OHdG, a marker of DNA oxidation, was measured in the spinal cord tissue of three groups of mice. Each sample containing three or four spinal cords was homogenized and processed as described in Materials and Methods. The average concentration (from three samples) of 8-OHdG in mSOD1 mice is 0.27 ± 0.06 fmol/mg wet tissue or 0.55 ± 0.12 fmol/µg DNA (mean±SD, 10 mSOD1 mouse spinal cords were measured as three samples). 8-OHdG was undetectable in the same amount of tissue from SOD1 or Nc mice. Thus, DNA oxidation occurs in mSOD1 mice.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
SUMMARY
 REFERENCES
 
We demonstrate in vivo for the first time, using G93A transgenic mice, that mutation of SOD1 elevates levels of both H2O2 and ·OH. The unexpected finding that the H2O2 level is increased in the mutant SOD1 transgenic mice but not in the mice overexpressing normal human SOD1 is particularly important, with significant implications about the pathogenesis of ALS. Our results clearly support the gain-of-function hypothesis and indicate that the new function gained by the mSOD1 includes both catalyzing H2O2 conversion to ·OH and disrupting the normal detoxification pathway. A previous study (20) appeared to rule out elevation of ·OH production in mSOD1 transgenic mice, and reports (18 , 23) as to whether mutant SOD1 catalyzes increased production of ·OH from H2O2 in vitro have been contradictory. However, in our in vivo results, mutation of SOD1 clearly raised H2O2 levels and induced more ·OH formation. Using [2,3-DHBA] as an indicator of [·OH], the greater ratio of [·OH]/[H2O2] in mSOD1 mice (0.23) than in SOD1 and Nc mice (0.17 and 0.16 respectively) supports the notion that mSOD1 mice have a higher capability of converting H2O2 to ·OH than SOD1 and Nc mice do. In previous studies, we demonstrated that ·OH (56 , 57) and O2·- (58) generated in vivo in the rat spinal cord blocked electrical conduction, increased levels of extracellular amino acids and prostaglandins (59) , and induced neuronal death. Therefore, the increased levels of H2O2 and ·OH should contribute to neuronal death in the ALS mice and possibly in human with FALS.

Our demonstration that the level of O2·- in the mutant mice is significantly lower than in the control groups, opposite to ·OH and H2O2, indicates that the elevated level of ·OH is produced by the sequence O2·- -> H2O2 -> ·OH. SOD1 mice contain both the SOD1 enzyme from the transfected normal human SOD1 (hSOD1) gene and the mouse’s own SOD1 (endogenous SOD1, eSOD1), mSOD1 mice contain the G93A mutant SOD1 and eSOD1 whereas Nc mice contain only eSOD1. This predicts that the catalytic ability to convert O2·- to H2O2 and thereby reduce the level of O2·- has the order SOD1 mice > mSOD1 mice > Nc mice, and the opposite order for the level of O2·-: Nc mice > mSOD1 mice > SOD1 mice. This is exactly what we found. All together, our results demonstrate that the mutation of SOD1 induced more ·OH formation from elevated H2O2 by the sequence O2·- -> H2O2 -> ·OH, evidence that ROS are involved in the pathogenesis of ALS.

H2O2 produced from O2·- is converted to H2O by catalase and glutathione peroxidase (GSH-Px), the normal detoxification pathway of H2O2, although some H2O2 may be converted to ·OH by SOD1. According to the order of catalytic ability, more H2O2 should be produced in SOD1 mice; however, no accumulation of H2O2 was observed in SOD1 mice compared to Nc mice. This is probably due to efficient H2O2 removal, which is the normal catalytic function of GSH-Px and catalase. mSOD1 mice had significantly higher levels of H2O2 and ·OH than did the SOD1 and Nc mice and significantly lower levels of O2·- than the Nc mice. This suggests that mSOD1 gains a new function, blocking H2O2 conversion to H2O, thereby allowing more ·OH formation from H2O2. In a recent study, Bruijn and co-workers stated that "Although we cannot directly measure the levels of intracellular H2O2, one might predict an increase or lowering of H2O2 caused by increasing or decreasing levels of wild-type SOD1. This in turn would accelerate or slow disease... . ". Because overexpression of hSOD1 or knocking out eSOD1 did not change the onset and progress of the disease, they strongly disagreed with the hypothesis that ROS are involved in the pathogenesis of ALS (19) . However, our measurements on the three groups of mice show that although addition of mutant and normal SOD1 both decrease O2·- concentrations, concentrations of H2O2 increase with the presence of mutated but not normal SOD1, contrary to their assumption. It appears from our results that H2O2 formed by overexpression of hSOD1 is efficiently destroyed, whereas more of that produced by the mutant SOD1 escapes into the tissue where it may produce damaging ·OH. Therefore, another key effect of mutation is blocking the normal H2O2 detoxification pathway, a crucial change whereby mutation of SOD1 leads to ALS. Given that the manipulations performed by Bruijn and co-workers do not have the effects they predicted on H2O2 concentrations, their observations are not evidence against aberrant O2·- destruction being a cause of ALS. It is not possible to discover this problem in vitro by using purified mSOD1. The deleterious SOD1 mutations typically are in the interaction regions crucial to subunit folding and dimer contact (3) , explaining why there is SOD1 aggregation only in the G93A and G85R transgenic mice but not in the mice overexpressing SOD1 (19) . The SOD1 dimer may act in tandem as a superoxide dismutase and a peroxidase. It may work together with the detoxification enzymes such as catalase and GSH-Px to remove the H2O2 produced from O2·-. The mutation-induced aggregation may interrupt by some way the normal relationship among these enzymes, thereby blocking detoxification as evidenced by the accumulation of H2O2 in mSOD1 mice. Increased free radical production in G93A transgenic mice was recently detected by Gurney’s group with a spin trap technique (60) . Although they did not identify specific free radicals, their work supports a relationship between free radicals and SOD1 mutation in FALS. Indeed, the finding that the sensitivity to H2O2 of fibroblasts from FALS patients with an SOD1 mutation is higher than for controls suggests that the mechanism underlying FALS jeopardizes the cell’s defense against free radical stress (61) .

Salicylate is able to cross the cellular membrane to trap ·OH intracellularly, and the DHBAs produced intracellularly may cross the membrane into the extracellular space because their molecular structure is similar to that of salicylate. Since ·OH cannot cross the cell membrane, the ·OH we measured in the dialysates may actually reflect the elevated intracellular ·OH that causes oxidation of proteins and DNA. Our demonstration that H2O2 levels in the dialysates are also elevated in mSOD1 mice is consistent with this explanation, since H2O2 can cross the cellular membrane. Thus, the higher level of H2O2 measured in CSF may also indicate a higher level of H2O2 in the cells in mSOD1 mice.

The recent discovery that without decomposing to ·OH, peroxynitrate can directly convert salicylate to 2,3- and 2,5-DHBA, questions the specificity of the DHBAs as indicators of ·OH (62 63 64) and challenges our finding. However, there is no evidence that this reaction occurs in vivo. The elevated level of H2O2 in mSOD1 mice supports the notion that the increased levels of ·OH we measured are at least partly formed from H2O2 by the mutant SOD1; however, a contribution of peroxynitrate cannot be ruled out, since we have found that the level of nitric oxide in mSOD1 mice is also significantly higher than in SOD1 and Nc mice (65) .

Based on the observations that overexpression of hSOD1 did not change the onset and progress of the disease, Bruijn and co-workers (19) also denied any possible effect of oxidative stress on the pathogenesis of ALS (19) . It has been suggested that the deleterious effect of mutant SOD1 on motor neurons in ALS is not related to increased oxidant stress caused by increased reactivity of active-site Cu ions (29) . However, our demonstration that all markers of oxidation of protein, DNA, and membrane lipids are significantly higher in mSOD1 mice than in SOD1 and Nc mice, but not between SOD1 and Nc mice, is consistent with some other reports (8 , 32 33 34 35) and strongly supports a correlation between mutation and oxidative stress. The demonstrations of protein, DNA, and membrane lipid oxidation together with elevated level of ·OH in mSOD1 mice support the hypothesis that ·OH-triggered oxidation of major cell components is a pathway for motoneuron damage in FALS. Although the mechanism as to how free radical-initiated oxidation selectively induces motoneuron degeneration has not been revealed, the finding that there are higher levels of SOD1 in motoneurons relative to other neurons (66) implicates a role of free radicals in the selective degeneration of motoneurons in ALS because higher levels of mSOD1 are probably present in those neurons relative to others when mutation occurs. This would produce more .OH than in other cells, thereby increasing oxidative damage.


   SUMMARY
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUMMARY
REFERENCES
 
The present study, using unique microdialysis and microcannula sampling techniques in transgenic mice and well-established biochemical analysis methods, discovered in vivo elevation of H2O2 and ·OH and reduction of O2·- in the presence of mutant SOD1. This supports, by in vivo experiments, the in vitro discovery that mutant SOD1 gains a new function, catalyzing more ·OH production from H2O2 by the sequence O2·- -> H2O2 -> ·OH. Accomplishing this in vivo removes potential doubts that can be raised about the relevance of the in vitro experiments. The data presented also indicate that the new function gained by the mutant SOD1 enzyme is not only catalyzing more ·OH formation, but also blocking the detoxification capability of other defense enzymes. Our verification of increased oxidation of cellular constituents supports that ROS-initiated processes lead to motoneuron degeneration in ALS.


   ACKNOWLEDGMENTS
 
The authors thank D. J. McAdoo for suggested revisions of this study. This work was supported by the grant from the Amyotrophic Lateral Sclerosis Association.


   FOOTNOTES
 
Received for publication April 22, 1999. Accepted for publication June 14, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUMMARY
 REFERENCES
 

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