Iron porphyrin treatment extends survival in a transgenic animal model of amyotrophic lateral sclerosis

Authors


Address correspondence and reprint requests to M. Flint Beal, Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, NY 10021, USA. E-mail: fbeal@med.cornell.edu

Abstract

Oxidative damage, produced by mutant Cu/Zn superoxide dismutase (SOD1), may play a role in the pathogenesis of amyotrophic lateral sclerosis (ALS), a devastating motor neuron degenerative disease. A novel approach to antioxidant therapy is the use of metalloporphyrins that catalytically scavenge a wide range of reactive oxygen and reactive nitrogen species. In this study, we examined the therapeutic potential of iron porphyrin (FeTCPP) in the G93A mutant SOD1 transgenic mouse model of ALS. We found that intraperitoneal injection of FeTCPP significantly improved motor function and extended survival in G93A mice. Similar results were seen with a second group of mice wherein treatment with FeTCPP was initiated at the onset of hindlimb weakness—roughly equivalent to the time at which treatment would begin in human patients. FeTCPP-treated mice also showed a significant reduction in levels of malondialdehyde (a marker of lipid peroxidation), in total content of protein carbonyls (a marker of protein oxidation), and increased neuronal survival in the spinal cord. These results therefore provide further evidence of oxidative damage in a mouse model of ALS, and suggest that FeTCPP could be beneficial for the treatment of ALS patients.

Abbreviations used
ALS

amyotrophic lateral sclerosis

FeTCPP

iron 5,10,15,20-tetrakis-4-carboxyphenyl porphyrin

SOD1

Cu/Zn superoxide dismutase

Amyotrophic lateral sclerosis (ALS) is an adult-onset, rapidly progressive, neurodegenerative disorder with an unknown etiology. It is characterized by the degeneration of motor neurons in the spinal cord, brainstem and motor cortex. The characteristic physical findings of ALS are the combination of upper motor neuron and lower motor neuron signs that include muscle weakness, atrophy and spasticity. With its inexorable progression, patients usually die within 3–5 years of symptom onset due to secondary aspiration pneumonia or respiratory failure. ALS is most commonly sporadic, with about 10% of cases being inherited as an autosomal dominant familial form.

Since the discovery in 1993 that a mutated form of Cu/Zn superoxide dismutase (SOD1) associates with approximately 20% of cases of familial ALS (Rosen et al. 1993), various theories have been postulated about what the gain-of-function caused by the mutant SOD1 could be. One hypothesis centers around the loss of substrate specificity and subsequent aberrant redox activity of the active site copper, resulting in oxidative stress (Cookson and Shaw 1999; Cleveland and Liu 2000). It was proposed that mutations to SOD1 lead to a more open or relaxed active site (Deng et al. 1993) that would increase the generation of hydroxyl radicals from hydrogen peroxide (Wiedau-Pazos et al. 1996) or peroxynitrite from nitric oxide (Beckman et al. 1993). Furthermore, it was shown that several SOD1 mutants have decreased zinc binding affinity (Crow et al. 1997) and that, in the absence of zinc, cellular reductants such as ascorbic acid rapidly reduce Cu(II) to the Cu(I) form in the enzyme's active site, which would lead to a net production of superoxide and, in the presence of nitric oxide, production of peroxynitrite (Estevez et al. 1999). These possibilities, however, have been questioned by the finding that crossing mutant SOD1 transgenic mice with mice deficient in the copper chaperone protein for SOD (CCS), required for most of the copper loading in SOD, does not alter the disease phenotype (Subramaniam et al. 2002). This finding, however, does not rule out aberrant copper incorporation into SOD1 in the absence of the chaperone. Indeed, Subramaniam et al. reported that 20–30% of SOD1 activity remained even in the absence of CCS. Moreover, Goto et al. (2000) have reported that in the absence of CCS, copper binds abnormally to SOD1, which increases the possibility that it would possess abnormal redox activity.

Although the precise biochemical mechanism whereby SOD1 mutants are pathologic in ALS remains elusive, there is substantial evidence for increased oxidative damage in a transgenic mouse model associated with mutant G93A SOD1 (Ferrante et al. 1997a). A novel approach to antioxidant therapy is the use of catalytic antioxidants such as low molecular weight metal-containing porphyrins. Originally developed as SOD1 mimetics, both iron(III) and manganese(III) porphyrins have been shown to catalytically decompose several biological oxidants including superoxide, hydrogen peroxide, lipid peroxyl radicals and peroxynitrite (Patel and Day 1999). Metalloporphyrins are effective at blocking oxidant stress both in vitro (Faulkner et al. 1994; Benov and Fridovich 1995; Liochev and Fridovich 1995; Batinic-Haberle et al. 1998) and in vivo (Day and Crapo 1996; Zingarelli et al. 1997; Szabo 1998; Cuzzocrea et al. 1999). Several manganese (Mn[III]) and iron (Fe[III]) porphyrins have been shown to be excellent peroxynitrite scavengers because they react very rapidly with peroxynitrite (second order rate constants of 106−107/m/s) and decompose it catalytically (Crow 2000). Manganese porphyrins require exogenous reductants such as ascorbate to reduce Mn(IV) back to Mn(III) to complete the catalytic cycle (Lee et al. 1997, 1998; Crow 1999; Ferrer-Sueta et al. 1999), whereas iron porphyrins do not require exogenous reductants for the catalytic function. However, because reductants such as ascorbate and glutathione are always present in mammalian tissues, this requirement would not be limiting in vivo.

In this study, we evaluated survival, motor function (rotarod) and weight changes, and carried out histological and oxidative damage marker determinations in iron 5,10,15,20-tetrakis-4-carboxyphenyl porphyrin (FeTCPP) (Fig. 1) and vehicle-treated G93A transgenic mice. We also compared treatment regimens wherein FeTCPP administration commenced prior to the onset of weakness (40 days of age) with a second group wherein FeTCPP treatment was initiated on the first day of hindlimb weakness in each mouse. The latter regimen is comparable with the earliest time at which drug treatment could begin in human patients.

Figure 1.

Structure of FeTCPP.

Materials and methods

Transgenic mice for early (40 days of age) treatment

G93A transgenic familial ALS mice (Gurney et al. 1994) were obtained from Jackson Laboratory (Bar Harbor, ME, USA). We maintained the transgenic G93A hemizygotes by mating transgenic males with B6SJLF1/J hybrid females. Transgenic offspring were genotyped by PCR assay of DNA obtained from tail tissue. Twenty-nine G93A transgenic mice were randomly assigned to a treatment or control (vehicle) group. We also included seven N1029 (wild-type SOD1 transgenics instead of G93A SOD1) mice as a control mouse model for G93A transgenic mice in the histological evaluations. All animal experiments were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Transgenic mice for late (symptom-onset) treatment

G93A overexpressing mice were initially obtained by breeding transgenic males purchased from Jackson Laboratories with c57b6 females also obtained from Jackson Laboratories Offspring were determined to be transgenic by sampling 2 µL of whole blood from the tail and running SOD activity gels (Beauchamp and Fridovich 1971). Transgenic male offspring from the same litter were used as breeders for the second generation of transgenic mice; all mice used for this study were obtained using this litter-mate breeding strategy. Sixteen G93A transgenic mice were randomly assigned to a FeTCPP-treated (n = 9) and untreated control group (n = 7). The mean age of onset in the FeTCPP-treated animals was 101.2 ± 1.4 days and in the controls 104.4 ± 2.9 days, which did not significantly differ.

Drug administration

A 1 mg/mL solution of FeTCPP (Porphyrin Products, Inc., Logan, UT, USA) was prepared by dissolving the compound in aqueous NaOH. The solution was made isotonic by addition of 0.9% NaCl and the pH was adjusted to 7.4 with HCl. The final solution was sterile-filtered and the final concentration of FeTCPP was standardized spectrophotometrically (A409nm = 32 400/m/cm).

Two different treatment regimens were carried out with the transgenic mice: (i) in the early treatment group, 1 mg/kg FeTCPP was administered i.p. once a day beginning at 40 days of age, while 100 µL of PBS (0.15 m potassium phosphate containing 150 mm NaCl, pH 7.4) were administered i.p. to the vehicle group; (ii) in the symptom-onset treatment group, 1 mg/kg of FeTCPP was administered i.p. twice daily following a single 2 mg/kg loading dose beginning on the first day that symptoms appeared in each mouse. Symptom onset was defined as the first day that the mouse displayed abnormal splaying of hindlimbs when lifted by its tail. Hindlimb splaying is a consistent and objective sign that asymmetrical or symmetrical hindlimb weakness and paralysis will follow within 2–3 days.

Survival

The initial sign of disease in G93A transgenic mice is a resting tremor that progresses to gait impairment, asymmetrical or symmetrical paralysis of the hindlimbs, followed by complete paralysis at the end stage. For the early treatment group and the corresponding vehicle group, mice were killed when they were unable to roll over within 20 s after being pushed on their sides and this time point was recorded as the time of death. For the symptom-onset treatment group and its corresponding control group, mice were killed when they displayed any signs of labored breathing or when they were no longer able to eat and drink, as evidenced by significant weight loss in a 24-h period. The untreated control group was treated in an identical manner, except that no injections were given. Upon killing, mice were examined for evidence of peritonitis or any obvious abnormalities related to the i.p. injections. Any mice showing signs of peritonitis or pathology related to injections were excluded from the study. Survival was assessed for all four groups.

Motor function testing (rotarod)

For the early treatment group and its corresponding vehicle group, rotarod performances were assessed twice a week in G93A mice starting at 80 days of age. Mice were trained for 2–3 days to become acquainted with the rotarod apparatus (Columbus instruments, Columbus, OH, USA). The testing began by placing the mice on a rod rotating at 12 rev/min, and the time that the mice stayed on the rod (until falling off or staying a maximum of 5 min) was recorded as a measurement of the competence of their motor function. Three trials were performed and the best result of the three was recorded.

Histological evaluation

Mice were anesthetized and transcardially perfused with 0.9% NaCl. Brains and spinal cords were removed and immediately frozen on solid CO2. The spinal cords were fixed in 4% paraformaldehyde in 0.1 m phosphate buffer (pH 7.4) for 48 h, and the tissues were cryoprotected in 30% sucrose overnight. Serial transverse sections (50 µm thick) were cut on a cryostat and collected for Nissl staining and immunohistochemistry. Every ninth section through the lumbar spinal cord was immunostained with a rabbit anti-serum against malondialdehyde-modified protein (provided by Dr Craig Thomas, Hoechst Marion Roussel) using a modified avidin-biotin peroxidase technique (Hsu et al. 1981). Free-floating sections were pre-treated with 3% H2O2 in PBS for 30 min to inactive endogenous peroxidases. All sections were pre-incubated in 1% bovine serum albumin (BSA)/0.2% Triton X-100 in PBS for 30 min on an orbital shaker at room temperature. The sections were incubated in rabbit anti-albumin-modified malondialdehyde (diluted 1 : 1000 in 0.5% BSA/PBS) overnight, followed by biotinylated anti-rabbit IgG (Vector Laboratory, Burlingance, CA, USA) incubation for 1 h (diluted 1 : 200 in 0.5% BSA/PBS). After washing with 0.5% BSA/PBS (3 × 5 min), the sections were incubated in avidin-biotin peroxidase complex (diluted 1 : 200 in PBS) for 1 h. The immunoreaction was visualized using 3,3′-diaminobenzidine tetrahydrochloride dihydrate (DAB) with nickel intensification (Vector) as the chromogen. The sections were mounted onto gelatin-coated slides, dehydrated, cleared in xylene and coverslipped. The specificity of immunostaining was confirmed by pre-incubation of the antibody with antigen and by omission of the primary antibody. The intensity of malondialdehyde immunostaining in the gray and white matter within the lumbar spinal cord was graded blindly using a semi-quantitative scale of weak (+), moderate (+ +) and intense (+ + +).

Sections adjacent to those used for immunocytochemistry were stained with cresyl violet. Every fourth section was analyzed for neuronal volume and number using optical fractionator and nucleator probes of the Stereo Investigator System (Microbrightfield, Colchester, VT, USA). Six tissue sections of the lumbar spinal cord from each mouse were analyzed. All cells were counted from within the ventral horn below a horizontal line across the gray matter through the ventral border of the central canal.

Protein carbonyl measurements

Spinal cords were homogenized in lysis buffer containing 250 mm sucrose, 10 mm MgCl2, 2 mm EDTA pH 6.8, 20 mm HEPES and protease inhibitor (Roche Molecular Biochemicals, Indianapolis, IN, USA). Protein concentration of homogenates was determined using a Bio-Rad (Bio-Rad Laboratories, Hercules, CA, USA) protein detection kit.

Derivatization of the spinal cord homogenates (15 µg of total protein) was performed using the OxyBlot protein oxidation detection kit (Intergen, Purchase, NY, USA) according to the manufacturer's instructions. Then, samples were applied directly to a 4–20% gradient Tris-glycine sodium dodecyl sulfate (SDS) gel (Invitrogen life technolo- gies, Carlsbad, CA, USA) or stored at 4°C for a later use for up to a week.

Electrophoresis was carried out for 90 min at 125 V, and proteins were transferred to a polyvinylidene difluoride (PVDF) membrane for 60 min at 70 V under cold conditions. The membranes were then blocked with 1% BSA/Tris buffered saline Tween 20 (TBST) for 1 h at room temperature on an orbital shaker. The membranes were then incubated with a primary antibody specific to the dinitrophenyl (DNP) moiety of the protein (dilution of 1 : 150 in 1% BSA/TBST) overnight at 4°C on an orbital shaker. The blot was washed with TBST (5 × 10 min) and incubated with secondary antibody (dilution of 1 : 300 in 1% BSA/TBST) for 2 h. Enhanced chemiluminescence (Supersignal; Pierce, Rockford, IL, USA) was used and exposed to X-ray film for 5 min to detect DNP-conjugated protein. Optical densities were analyzed using the Scion Image software for Windows.

HPLC method for FeTCPP in mouse tissue

Mouse tissues (age of onset-treated group, n = 9) were homogenized in four volumes of water and sonicated. NaOH was added to a final concentration of 50 mm and an equal volume of ethyl acetate was added and vigorously mixed. After centrifugation, the ethyl acetate layer was removed and the aqueous layer acidified with HCl. An equal volume of tert-butyl methyl ether (tBME) was added, mixed vigorously and separated by centrifugation. The tBME layer was removed and taken to dryness in a vacuum centrifuge. Dried extracts were resuspended in a minimum volume of methanol (MeOH) followed by addition of an equal volume of 100 mm KOH. Resuspended samples were clarified by centrifugation and supernatant fluids injected into the HPLC. FeTCPP standards were prepared and suspended in the same MeOH–KOH solution. Separation was achieved using a 4.6 × 150 mm Waters Sperisorb ODS-II column (Waters, Milford, MA, USA) maintained at 48°C in a column oven. FeTCPP was eluted using a gradient consisting of 0.2% heptafluorobutyric acid in water (buffer A), acetonitrile (buffer B) and MeOH (buffer C). FeTCPP in mouse tissues was identified by its retention time (9.96 min) and by its characteristic UV-visible spectrum using a Shimadzu diode-array detector (Shimadzu, Columbia, MD, USA). FeTCPP was quantified via its absorbance at 400 nm relative to standards of pure FeTCPP. Untreated mouse tissues were spiked with known concentrations of FeTCPP (similar to those found in treated mice) and extracted just as with treated mice samples. The extraction efficiency was determined for brain, spinal cord, kidney and liver and used to calculate concentrations of FeTCPP in the original wet tissues. For the purposes of calculating and expressing tissue concentrations of drug, one microgram of wet tissue was considered to be equal to one microliter.

Statistical analysis

Kaplan-Meier survival analysis and the Logrank (Mantel–Cox) test were used for survival comparisons, repeated measures anova for Rotarod comparisons, anova with Newman-Keuls for neuronal cell count and the unpaired t-test for total carbonyl studies. For the symptom-onset study, survival interval data were analyzed by the unpaired t-test.

Results

In this study, G93A mice were treated with FeTCPP using two different regimens: (i) the early treatment group, beginning at 40 days of age and dosed once per day and (ii) the symptom-onset treatment group, with twice-daily dosing beginning with a 2× loading dose on the first day that symptoms appeared in each mouse. Each treatment group had its own corresponding control group. Survival was assessed in all four groups. Motor function was assessed by rotarod performance for the early treatment group and its corresponding vehicle group.

FeTCPP tissue levels

A critical issue is whether FeTCPP penetrates the blood–brain barrier to mediate its therapeutic effects. We therefore measured concentrations of FeTCPP in the brain, spinal cord, kidney and liver of the ‘age of onset’ treatment group of G93A mice (Fig. 2). These results show that FeTCPP does penetrate into the brain and spinal cord and achieves lower µm concentrations. A caveat is that the samples were not perfused. However, we think that blood contamination is unlikely to account for the levels since they were obtained 16–18 h after the last injection, they showed no evidence of blood tingeing and spinal cord contains very little blood.

Figure 2.

Mean FeTCPP concentrations (µm) in the ‘age of onset’ treatment group of G93A mice.

Survival

In the early treatment group, survival was significantly extended by 7 days with FeTCPP. The mean survivals were 135.6 ± 2.4 days (mean ± SE) for FeTCPP-treated mice, and 128.6 ± 1.7 days for vehicle-treated mice (n = 19 per group, p < 0.02 by Mantel–Cox rank test) (Fig. 3a).

Figure 3.

The effect of FeTCPP treatment on survival in G93A transgenic mice. (a) Cumulative probability of survival for mice treated with either FeTCPP (▵) or vehicle (○) beginning at 40 days of age (n = 19 per group). There is a significant increase in survival in FeTCPP-treated mice (p = 0.015, Mantel–Cox rank test). (b) Survival interval (time from symptom onset to death) for mice treated with FeTCPP or nothing beginning at symptom onset. Survival interval increased from 16.7 ± 1.3 days (mean ± SE) in the untreated group (n = 7) to 25.8 ± 2.2 in the FeTCPP-treated group (n = 9) (p = 0.0053, unpaired t-test).

In the symptom-onset treatment group, the mean survival interval from disease onset to euthanasia was also significantly extended by FeTCPP. The mean survival intervals were 25.8 ± 2.2 days (mean ± SE) for FeTCPP-treated mice (n = 9) and 16.7 ± 1.3 days for untreated mice (n = 7). This is an overall increase in survival of 9 days (p < 0.01 by unpaired t-test) (Fig. 3b).

Motor function (rotarod) and weight

Improved rotarod performance was observed in FeTCPP-treated mice between the ages of 112 and 129 days (n = 19/group, p < 0.03 by repeated anova measures) (Fig. 4). There was also a trend of attenuated weight loss in the FeTCPP-treated group from the age of 125 days to 140 days compared with the vehicle group (data not shown).

Figure 4.

The effect of FeTCPP treatment on rotarod performance in G93A transgenic mice from 80 days to 150 days of age. There is a significant improved performance in FeTCPP-treated mice (p = 0.0202 by repeated measures anova). n = 19 per group; □, untreated; ◆, FeTCPP treated.

Immunohistological and stereological evaluation

To evaluate whether FeTCPP treatment is efficacious in preventing oxidative damage and neuronal loss within the spinal cord of treated and vehicle G93A mice, equal numbers of females and males were killed by transcardial perfusion at the age of 113 days, and spinal cords were removed (n = 10 per group). Four spinal cords from each group were used for immunohistochemistry (Table 1 and Fig. 5) and stereological cell count studies (Table 2), while the remaining spinal cords (n = 6 per group) were used to determine indices of oxidative damage (Fig. 6). Immunostaining with malondialdehyde was most robust in neurons of the gray matter, and was more intense in both the gray and white matter of the lumbar spinal cords of the vehicle group, compared with the FeTCPP-treated group (Table 1 and Fig. 5). Malondialdehyde immunostaining from spinal cords of N1029 mice (90 days old, n = 7), as a control mouse model for overexpression of the G93A mutant, showed similar intensities to those in the FeTCPP-treated G93A mice (Table 1). The total cell count of neurons in the G93A vehicle group, the G93A FeTCPP-treated group and the N1029 control group were 225.3 ± 30, 318.3 ± 26 and 350.5 ± 16 (mean ± SE), respectively (n = 4 per group, Table 2).

Table 1.  Relative intensities of malondialdehyde immunoreactivity in the lumbar spinal cord
Animal tag numberMouse typeGroupImmunostaining intensity
4807G93AVehicle+++
4802G93AVehicle+++
4838G93AVehicle++
4859G93AVehicle+++
7444G93AFeTCPP+
4885G93AFeTCPP+
4888G93AFeTCPP+
7438G93AFeTCPP++
11121N1029Control+
11123N1029Control+
11117N1029Control+
11115N1029Control+
11124N1029Control+
11116N1029Control+
Figure 5.

Malondialdehyde immunostained sections through the ventral horn of the lumbar spinal cord in a G93A/vehicle mouse (top), a G93A/FeTCPP mouse (middle) and a N1029 mouse. A marked reduction of malondialdehyde immunoreactivity occurs in both the gray and white matter of the lumbar spinal cord from the FeTCPP-treated mouse. Scale bar = 200 µm.

Table 2.  Neuronal counts per six 50-μm lumbar spinal cord sections
 SmallMediumLargeTotal
  1. Total number and size distribution of neurons per six sections (50-μm thick) through the lumbar spinal cord are expressed as mean ±SEM. *p < 0.01 compared with N1029 mice, #p < 0.05 compared with G93A/vehicle mice by anovapost-hoc test.

G93A/vehicle mice142.5 ± 2574.8 ± 58 ± 2225.3 ± 30*
G93A/FeTCPP mice205 ± 17103.8 ± 199.5 ± 3318.3 ± 26#
N1029 mice188.5 ± 6136.8 ± 1325.3 ± 3350.5 ± 16
Figure 6.

(a) Western blot immunoassay for protein-bound carbonyl groups in spinal cords from G93A transgenic mice, comparing levels of carbonyl content at the age of 113 days. Total protein (15 µg) from half the spinal cord was treated with dinitrophenylhydrazine (DNPH)-derivatization solution and immunoblotted; proteins were visualized by enhanced chemiluminescence. For each blot, the first three lanes are from the vehicle group and lanes 4, 5 and 6 are from the FeTCPP-treated group. Molecular weights of the proteins are from 220 to 21 kDa, while 21 kDa corresponds to the molecular weight of SOD1. (b) Total protein carbonyl measurements of G93A mice. The optical density of total carbonyl-bound proteins from the vehicle group, indicated by the solid bar, is 163.5 ± 7.8 (mean ± SE). The optical density of total carbonyl-bound proteins from the FeTCPP-treated group, indicated by the shaded bar, is 138.2 ± 7.0 (mean ± SE). There is a significant difference in total carbonyl-bound proteins between the treated mice and the untreated ones (p = 0.0368 by unpaired t-test).

Oxidative damage assessment

Oxidative damage was assessed by measuring total protein carbonyls from spinal cords of both untreated and treated groups (n = 6 per group) at the age of 113 days. Western blot immunoassay (Fig. 6a) for protein-bound carbonyl groups in spinal cords showed bands from 220 to 21 kDa, with 21 kDa corresponding to the molecular weight of SOD1 subunit under these conditions. Quantification of protein carbonyl content by optical density measurement showed a significant decrease in total protein carbonyls in the treated mice, 138.2 ± 7.0 and 163.5 ± 7.8 (mean of arbitrary unit ± SE) in the FeTCPP-treated and vehicle groups, respectively (p < 0.05 by unpaired t-test) (Fig. 6b).

Discussion

These results demonstrate that FeTCPP administration to transgenic G93A ALS mice prior to disease onset increases survival by an average of 7 days. However, it is rarely possible to initiate therapy in humans prior to disease onset because most cases of ALS cannot be predicted. It is therefore encouraging to find that a significant survival effect (9 days) can be obtained even when drug treatment is initiated at the onset of symptoms. FeTCPP was well tolerated at the dose of 1 mg/kg. Some mice showed better improvement than others, which could be caused by different responses to the drug and/or to differences in drug uptake into the CNS. In that regard, we have determined that the pKa for the four ionizable carboxylic acid groups of FeTCPP is 6.42. This means that 9% of an administered dose of FeTCPP remains unionized at physiological pH and is therefore much more likely to cross the blood–brain barrier. The unusually high pKa for FeTCPP may help explain why FeTCPP gained access to the CNS. We found that FeTCPP did cross the blood–brain barrier and achieved low µm concentrations in the brain and spinal cord.

We saw no indication that early administration of FeTCPP delayed the onset of disease as measured by onset of deficits in rotarod performance. However, we observed that mice in the early FeTCPP treatment group maintained their rotarod performances better than the vehicle group. These results suggest that FeTCPP extended survival in our G93A transgenic mice by slowing the progression of the disease. The fact that early administration showed no benefit on survival over treatment at disease onset suggests that FeTCPP may have its effect after disease onset.

Immunostaining for malondialdehyde of the lumbar region of spinal cords showed more intense immunoreactivity in untreated mice than in FeTCPP-treated mice. Protein carbonyl content, an indicator of protein oxidation, was significantly lower in the FeTCPP-treated group. Neuronal counting in the spinal cords showed a neuroprotective effect exerted by FeTCPP treatment.

Based on previous studies, oxidative damage may play a role in the pathogenesis of ALS (Ferrante et al. 1997a; Andrus et al. 1998; Bogdanov et al. 1998; Hall et al. 1998; Liu et al. 1998). We found significant increases in concentrations of 3-nitrotyrosine, a marker of peroxynitrite-mediated nitration, in the spinal cord and cerebral cortex of G93A transgenic mice (Ferrante et al. 1997b) as well as in the spinal cord of ALS patients (Beal et al. 1997). This supports the possibility that iron porphyrins could prevent oxidative damage by acting as catalytic scavengers of peroxynitrite, although other non-oxidant-related drug mechanisms are certainly possible. The above approach was not possible in the current study due to the large amounts of tissue required. In this study, extension of the life span, improved motor performance, reduced malondialdehyde immunostaining and decreased protein oxidation in G93A transgenic mice provide strong evidence that oxidative stress contributes to disease pathogenesis. Other catalytic antioxidants such as EUK-8 and EUK-134 have also been reported to reduce levels of oxidative stress and prolong survival (Jung et al. 2001). In another recent study, metalloporphyrin catalytic antioxidants exerted neuroprotective effects against focal ischemic insults by decreasing post-ischemic superoxide-dependent oxidative stress (Mackensen et al. 2001).

In addition to the theory that peroxynitrite-mediated nitration may be involved in oxidative damage, another mechanism has been proposed, namely that increased peroxidase activity of mutant SOD1 oxidizes other cellular constituents via copper-mediated production of hydroxyl radical. Polyamine-modified catalase, an antioxidant enzyme that catalyzes the decomposition of hydrogen peroxide, delayed the onset of the disease and increased survival (Poduslo et al. 2000), presumably by eliminating the substrate for peroxidase activity. Other studies support a possible linkage between oxidative injury and excitotoxicity in ALS. For example, riluzole and gabapentin, both putative inhibitors of the glutamatergic system, prolonged survival in G93A transgenic mice (Gurney et al. 1996) while carboxyfullerenes, a class of antioxidants that can also blockn-methyl-d-aspartate (NMDA) receptor-mediated excitotoxicity, delayed both the onset of symptoms and death (Dugan et al. 1997). Minocycline, which has effects both in blocking inflammatory pathways and on release of mitochondrial proteins that trigger apoptosis, is also effective in transgenic mouse models of ALS (Zhu et al. 2002). Non-oxidant mechanisms such as protein aggregation (Cleveland and Liu 2000) have also been implicated in motor neuron disease in transgenic mice. One common pathological hallmark in ALS and motor neuron disease in mice is mitochondrial dysfunction (Menzies et al. 2002). This observation indirectly supports an oxidative hypothesis, given that mitochondria are a primary source of oxygen radicals even under normal physiological conditions. Our previous study of creatine administration, which increases brain phosphocreatine concentration and may inhibit opening of the mitochondrial transition pore, showed improved motor performance and a 17% extension of life span (Klivenyi et al. 1999). Furthermore, it has recently been firmly established that SOD1 is present in mitochondria (Higgins et al. 2002; Mattiazzi et al. 2002), which was unknown when SOD1 mutations were first identified in familial ALS. Potentially important differences between the cytosolic and mitochondrial pools of SOD1, including how the latter obtains copper and zinc, are largely unexplored. Because abnormalities in mitochondrial morphology and function occur as a prelude to motor neuron death, it is intriguing to speculate that SOD1 within the mitochondria may be the critical pool relevant to ALS. It has also been shown in neuroblastoma cells that the entry of SOD1 into mitochondria depends on demetallation, and that heat-shock proteins (Hsp70, Hsp27 or Hsp25) block the uptake of mutant SOD1 (G37R, G41D or G93A) while having no effect on wild-type SOD1. Therefore, it was proposed that binding heat-shock proteins to mutant SOD1 might make them unavailable for their anti-apoptotic functions and ultimately lead to motor neuron death (Okado-Matsumoto and Fridovich 2002).

Due to the involvement of multiple pathways in ALS, it may be possible to produce more effective neuroprotection and survival effects by therapeutic strategies utilizing combinations of two or more therapeutic agents thought to work by different mechanisms, such as FeTCPP with carboxyfullerene or creatine; these combinations, in theory, should yield greater overall survival.

Acknowledgements

This work was supported by ALS Association and NIA grant AG 12992, and the NINDS division of NIH (grants R29 NS35871 and R01 NS40819 [JPC]). We wish to thank Dr Michael Lin for helpful discussion and Dr Feng Li for western blot technical assistance. We also wish to thank Julie Lynch, CVT (Crow Laboratory), for technical support and extraordinary efforts in caring for partially paralyzed mice.

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