Method of modulating radical formation by mutant cuznsod enzymes

Familial amyotrophic lateral sclerosis (FALS)-associated mutant CuZnSODs, A4V and G93A, have been discovered to catalyze the generation of hydroxyl radical from hydrogen peroxide at higher rates than that of wild type CuZnSOD. The copper chelator diethyldithiocarbamate (DDC) has been found to inhibit both radical generation and SOD activity of mutant CuZnSODs A4V and G93A at DDC concentrations significantly lower than those required to inhibit wild type CuZnSOD enzyme. In a neural cell culture model of FALS, DDC reverses the effect of four FALS-associated mutants, but does not alter the survival of cells expressing only wild type CuZnSOD. Thus, radical formation may be modulated and ALS treated in subjects with a mutant CuZnSOD enzyme by the administration of copper chelating agents. Treatment can also be affected by the administration of radical scavenging agents, or the administration of expression inhibitors specific for the mutant genes.

FIELD OF THE INVENTION 
The present invention relates to methods for modulating radical formation 
and treating a subject with amyotrophic lateral sclerosis (ALS; Lou 
Gehrig's disease). In a particular aspect, the invention relates to 
methods of treating subjects with a mutant copper-zinc superoxide 
dismutase (CuZnSOD) protein. Invention treatment involves administration 
of a copper chelating agent to prevent or reduce the ability of the mutant 
CuZnSOD protein to generate hydroxyl radicals, or to decrease the 
peroxidase activity of the mutant CuZnSOD protein. Alternatively, 
invention treatment can be accomplished by administration of a radical 
scavenging agent to scavenge the hydroxyl radicals formed by the mutant 
CuZnSOD protein. In a further aspect, the invention relates to methods of 
treating a subject having DNA encoding a mutant CuZnSOD protein by 
administration of an agent to inhibit the expression of such DNA. 
BACKGROUND OF THE INVENTION 
ALS is a motor neuron degenerative disease that affects approximately one 
person in ten thousand. About 10-15% of cases are familial (R. H. Brown, 
Jr., Cell 80:687 (1995)), and 20-25% of familial ALS (FALS) cases are 
associated with dominantly inherited mutations in sod1, the gene that 
encodes human CuZnSOD (D. R. Rosen, et al., Nature 362:59 (1993)). Initial 
studies of the FALS-associated CuZnSOD mutants demonstrated reduced 
enzymatic activity (H. X. Deng, et al., Science 261:1047 (1993)). However, 
subsequent studies in a transgenic mouse model (M. E. Gurney, et al., 
Science 264:1772 (1994); M. E. Ripps, et al., Proc. Natl. Acad. Sci. 
U.S.A. 92:689 (1995); C. A. Pardo, et al., Proc. Natl. Acad. Sci. U.S.A. 
92:954 (1995)) and a cell culture model (S. Rabizadeh, et al., Proc. Natl. 
Acad. Sci. U.S.A. 92:3024 (1995)) of FALS have pointed to a dominant, 
gain-of-function effect of the FALS-associated CuZnSOD mutants. Neural 
cell death is observed in these model systems despite the fact that mutant 
CuZnSODs are expressed in addition to the normal expression of human wild 
type CuZnSOD. Furthermore, yeast sod 1 null mutants are rescued by 
FALS-associated mutant human CuZnSOD as efficiently as by wild type 
CuZnSOD, indicating a high level of enzymatic activity by the mutant 
proteins (Rabizadeh (1995), supra). Although results from these studies 
support the existence of a dominant, gain-of-function effect of the 
mutants, the nature of the function gained remains undetermined (Brown, 
(1995); J. D. Rothstein, et al., New Engl. J. Med. 326:1464 (1992); S. H. 
Appel, et al, Eur. J. Neurol. Sci. 118:169 (1993); J. S. Beckman, et al., 
Nature 364:584 (1993); and J. F. Collard, et al., Nature 375:61 (1995)). 
CuZnSOD is a homodimeric enzyme containing one copper and one zinc ion per 
subunit. It is a major antioxidant enzyme, found in virtually all 
eukaryotic cells. CuZnSOD catalyzes the disproportionation of superoxide 
to O.sub.2 and H.sub.2 O.sub.2 (J. M. McCord and I. Fridovich, J. Biol. 
Chem. 244:6049 (1969); and I. Fridovich, Ann. Rev. Biochem. 64:97 (1995)). 
In 1975, Hodgson and Fridovich reported that CuZnSOD could be inactivated 
by H.sub.2 O.sub.2, and suggested that the mechanism for this inactivation 
was oxidative inactivation of active site histidine residues (Biochem. 
14:5294 (1975); Biochem. 14:5299 (1975)). They observed further that some 
but not all hydroxyl radical trapping reagents could protect the enzyme 
from inactivation by H.sub.2 O.sub.2 (Id.). In 1989, Cabelli et al., (See, 
J. Biol. Chem. 264:9967 (1989)) concluded that the reaction of H.sub.2 
O.sub.2 with CuZnSOD proceeds by a series of steps, starting with 
reduction of the oxidized (Cu.sup.II) form of the enzyme (step 1, below). 
This is followed by the reaction of HO.sub.2.sup.- with the reduced 
(Cu.sup.I) form, in a Fenton-like reaction, to produce hydroxyl radical 
(step 2, below). Hydroxyl radical produced in step 2 can be scavenged by 
small anionic scavengers such as formate (step 3 below, where X.sup.- is a 
Scavenger). 
EQU Enz-Cu.sup.II +H.sub.2 O.sub.2 .fwdarw.Enz-Cu.sup.I +O.sub.2.sup.- 
+2H.sup.+( 1) 
EQU Enz-Cu.sup.I +HO.sub.2.sup.- +H.sup.+ .fwdarw.Enz-Cu.sup.II (.OH)+OH.sup.-( 
2) 
EQU Enz-Cu.sup.II (.OH)+X.sup.- .fwdarw.Enz-Cu.sup.II +X.+OH.sup.-( 3) 
In addition to its activity as a SOD, CuZnSOD catalyzes oxidation of 
substrates by H.sub.2 O.sub.2 at rates competitive with its own oxidative 
inactivation by the same reagent (Hodgson and Fridovich, (1975), supra 
(both articles), and Cabelli, (1989), supra). A convenient substrate used 
to study this type of reaction is the spin trapping agent 
5,5'-dimethyl-1-pyrroline N-oxide (DMPO), which reacts with H.sub.2 
O.sub.2 to give its electron paramagnetic resonance (EPR)-detectable 
hydroxyl adduct, DMPO-OH. 
In 1990, it was demonstrated that hydroxyl radicals are generated in the 
reaction of H.sub.2 O.sub.2 with CuZnSOD (M. B. Yim, et al., Proc. Natl. 
Acad. Sci. U.S.A. 87:5006 (1990)). Three distinct fates for the hydroxyl 
radicals were described (Hodgson and Fridovich, (1975), supra (both 
articles); Yim, et al., (1990), supra; and M. B. Yim, et al., J. Biol. 
Chem. 268:4099 (1993)). First, the hydroxyl radicals could react directly 
with CuZnSOD, irreversibly inactivating the enzyme. Second, the hydroxyl 
radicals could exit from the active site channel, causing oxidative damage 
to other molecules within the short range of hydroxyl radical diffusion. 
Third, the radicals could be scavenged by small anionic molecules (e.g., 
formate or glutamate) capable of entering the active site channel. 
Although such a reaction would prevent damage to CuZnSOD, thereby 
prolonging the enzyme's half-life, the resulting scavenger radicals (e.g., 
formyl or glutamyl radicals) could potentially diffuse away from the 
enzyme and interact with targets beyond the range of hydroxyl radical 
diffusion (Yim, et al., (1993), supra). Based upon this evidence, it has 
not been possible to establish whether the function gained in 
FALS-associated CuZnSOD mutants is best described as an increase in the 
generation of hydroxyl radicals, or an increase in peroxidase activity. 
Therefore, reference to either activity, throughout this specification, is 
meant to encompass either possibility. 
Thus, it remains to be determined what is the cause of hydroxyl radical 
production by CuZnSOD, and what effect it has on cellular biochemistry. It 
also remains to be determined whether there is any connection between ALS 
and hydroxyl radical production by (or increased peroxidase activity of) 
CuZnSOD. Therefore, it remains to be discovered what can be done to 
modulate hydroxyl radical production by CuZnSOD, and thereby treat ALS. 
BRIEF DESCRIPTION OF THE INVENTION 
FALS-associated mutant CuZnSODs, A4V and G93A, have been discovered to 
catalyze the oxidation of substrates using hydrogen peroxide (peroxidase 
activity) at higher rates than that of wild type CuZnSOD. The copper 
chelator diethyldithiocarbamate (DDC) has been found to inhibit this 
activity in the mutant CuZnSODs A4V and G93A at concentrations 
significantly lower than required for DDC to inhibit wild type CuZnSOD. In 
a neural cell culture model of FALS, DDC reverses the effect of four 
FALS-associated mutants, but does not alter the survival of cells 
expressing only wild type CuZnSOD. 
This invention demonstrates that ALS caused by mutant CuZnSODs is 
treatable. Thus, copper chelating agents are shown to be capable of 
inhibiting the hydroxyl radical forming activities of mutant CuZnSOD 
enzymes at levels that do not have a significant effect on the SOD 
activity of the mutant or wild type enzyme. Thus the beneficial actions of 
the enzyme may be preserved while treating the harmful, disease-causing 
actions.

DETAILED DESCRIPTION OF THE INVENTION 
In accordance with the present invention, there are provided methods of 
treating a subject with ALS (or a subject susceptible to developing ALS) 
by administering to said subject an amount of a copper chelating agent 
effective to ameliorate the symptoms of ALS. 
In another embodiment, there are provided methods of treating a subject 
having a mutant sod1 gene by inhibiting peroxidase activity of said mutant 
sod1 gene. 
In yet another embodiment, there are provided methods of treating a subject 
having a mutant CuZnSOD protein by administering to said subject a radical 
scavenging agent in an amount effective to scavenge radicals formed by 
said mutant CuZnSOD protein. 
In still another embodiment, there are provided methods of modulating 
radical formation in a subject having a mutant CuZnSOD protein by 
administering to said subject a copper chelating agent in an amount 
effective to modulate radical formation by said mutant CuZnSOD protein. 
In a further embodiment, there are provided methods of modulating radical 
formation in cells having a mutant CuZnSOD protein by administering to 
said cells a copper chelating agent in an amount effective to modulate 
radical formation by said mutant CuZnSOD protein. 
In a still further embodiment, there are provided methods of treating a 
subject having DNA encoding a mutant sod1 gene by administering to said 
subject an inhibitor in an amount effective to inhibit expression of said 
DNA encoding a mutant sod 1 gene. 
The present invention is based on the hypothesis that the FALS-associated 
mutant CuZnSODs might enhance the catalytic generation of radicals from 
H.sub.2 O.sub.2 or the peroxidase activity of the enzyme. First, in a cell 
culture model of FALS, mutant human CuZnSODs (A4V, G37R) increase 
apoptosis (programmed cell death), whereas the wild type CuZnSOD inhibits 
apoptosis (Rabizadeh (1995); and L. J. S. Greenlund, et al., Neuron 14:303 
(1995)). Apoptosis in many paradigms is mediated by reactive oxygen 
species (T. A. Sarafian and D. E. Bredesen, Free Radic. Res. 21:1 (1994)). 
Second, analysis of the locations of the FALS-associated CuZnSOD mutations 
(Deng (1993), supra) suggests the possibility of more open CuZnSOD 
structures, which could conceivably allow greater access of 
radical-forming reactants to the active site copper. Third, it has been 
observed in cell culture that ALS mutant CuZnSODs have reduced half-lives 
in comparison to wild type CuZnSOD (D. R. Borchelt, et al., Proc. Natl. 
Acad. Sci. U.S.A. 270:3234 (1994)). This finding is compatible with 
enhanced enzyme damage due to hydroxyl radical generation (although it is 
equally compatible with enhanced proteolysis due to an alteration in 
enzyme structure). 
In order to test the hypothesis that FALS-associated mutations augment 
peroxidase activity, recombinant wild type CuZnSOD and mutant CuZnSOD A4V 
(indicates that the valine at amino acid residue 4 in the enzyme has been 
replaced with an alanine) and G93A (alanine at position 93 was replaced 
with glycine) DNA constructs were expressed in Saccharomyces cerevisiae, 
and the expressed proteins purified to homogeneity (McCord and Fridovich 
(1969), supra) (for details, see Example 6). Copper and zinc were removed 
by repeated dialysis, as described previously (C. R. Nishid, et al., Proc. 
Natl. Acad. Sci. U.S.A. 91:9906 (1994)), and the apoenzymes were then 
remetallated by the gradual addition of CuSO.sub.4 (Y. Lu, et al., J. Am. 
Chem. Soc. 115:5907 (1993)). The degree of metallation was confirmed using 
atomic absorption. EPR studies were then performed, using DMPO (Yim 
(1993), supra). 
In the presence of H.sub.2 O.sub.2 and DMPO, the fully metallated mutant 
and wild type CuZnSOD enzymes all produced the quadruplet signal 
characteristic of the hydroxyl radical adduct DMPO-OH (FIG. 1(A)-(D)). 
Double integration of the signals from the adduct demonstrated that higher 
concentrations of hydroxyl radical were trapped in the case of the 
FALS-associated mutant enzymes. For example, the signal from CuZnSOD 
mutant A4V was 3.0.+-.1.1 times the wild type CuZnSOD signal (n=10; 
p&lt;0.0005 by unpaired two-tailed t-test; range=2.1-5.7 times the wild type 
CuZnSOD signal); and from CuZnSOD mutant G93A, 2.1.+-.0.3 times the wild 
type CuZnSOD signal (n=5; p&lt;0.025 by unpaired two-tailed t-test; 
range=1.8-2.6 times the wild type CuZnSOD signal). The amplitude of the 
signal generated by the CuZnSOD mutant A4V was reproducibly greater than 
that from the CuZnSOD mutant G93A, which was, in turn reproducibly greater 
than that from wild type CuZnSOD. 
The apoenzymes, by contrast, did not produce hydroxyl radicals detectable 
by EPR (FIG. 2(A)). With increasing degrees of metallation, the enzymes 
gave increasing DMPO-OH specific EPR signals due to hydroxyl radical 
production (FIG. 2(B)-(E)). Differences between wild type and mutant 
enzymes were seen at all degrees of metallation. The increase in the EPR 
signals was not simply due to free copper in solution, since free 
Cu.sup.2+ without any enzyme does not give a significant EPR signal (FIG. 
1(D)). Neither could the spectra be explained simply by the preferential 
loss of zinc from the mutant CuZnSODs, since the zinc-free derivatives of 
wild type CuZnSODs gave results similar to the fully metallated wild type 
enzyme. 
Previous studies of the reaction of wild type CuZnSOD with H.sub.2 O.sub.2 
suggested strongly that the reaction occurs at the active site of the 
enzyme, i.e., at the copper ion bound to each subunit. It was predicted, 
therefore, that the reaction would be inhibited by a copper-ion chelating 
agent that would either bind to or remove the copper from the active site 
of the protein. Diethyldithiocarbamate (DDC) is a chelator capable of 
first binding to and then removing copper ions from CuZnSOD at 
physiological pH, and it has been shown to inhibit the SOD activity of 
CuZnSOD both in vitro and in vivo (H. P. Misra, J. Biol. Chem. 264:11623 
(1979)) 
Therefore, the effect of adding DDC to wild type CuZnSOD, mutant CuZnSOD 
A4V, and mutant CuZnSOD G93A was studied. A fundamental difference in the 
copper reactivity of the mutant enzymes in comparison to the wild type 
enzyme was demonstrated by their disparate responses. Whereas the wild 
type CuZnSOD showed a concentration-dependent increase in peroxidase 
activity following the addition of DDC, both mutant CuZnSOD A4V and mutant 
CuZnSOD G93A showed a progressive decrease in peroxidase activity with 
increasing concentrations of DDC (FIG. 3(A)-(C) and FIG. 4(A)-(B)). 
Furthermore, it was found that equimolar DDC (relative to enzyme subunit 
copper) had no effect on wild type CuZnSOD enzymatic activity, as reported 
previously (Misra (1979), supra), while the activity of both mutant 
CuZnSODs was inhibited even when only 0.25 equivalents of DDC per copper 
were added (FIG. 5). 
It was reasonable to postulate that if the peroxidase activity was 
important in SOD-associated FALS and was inhibited by DDC, then a low 
concentration of DDC would inhibit the process of neural degeneration 
associated with the expression of mutant CuZnSODs. The effect of adding 
DDC to a neural cell culture model of FALS, in which overexpression of the 
wild type CuZnSOD inhibits apoptosis while similar levels of expression of 
FALS-associated mutant CuZnSODs enhance apoptosis (Rabizadeh (1995), 
supra), was therefore investigated. The conditionally-immortalized rat 
nigral neural cell line CSM 14.1 (M. Durand, et al., Soc. Neurosci. Abs. 
16:40 (1990); and L. T. Zhong, et al., Proc. Natl. Acad. Sci. U.S.A. 
90:4533 (1993)) was transfected with vectors carrying wild type CuZnSOD or 
the mutant CuZnSODs A4V, G37R, G41D, or G85R. As had been observed 
previously (Rabizadeh (1995), supra), overexpression of the wild type 
CuZnSOD inhibited apoptosis induced in the cells by serum withdrawal, 
whereas all four mutant CuZnSODs tested were found to enhance apoptosis 
over the control. DDC (25-100 .mu.M) added to the cultures inhibited 
apoptosis induced by all four mutant CuZnSODs from 30-70%, but had no 
effect on cells overexpressing wild type CuZnSOD (FIGS. 6 and 7). DDC at a 
concentration of 500 .mu.M decreased the viability of the cells expressing 
wild type CuZnSOD but continued to rescue the cells expressing the mutant 
CuZnSODs. DDC at a concentration of 1 mM or greater was toxic in all 
groups. 
Thus, one aspect of the invention is the modulation of radical formation 
and the treatment of ALS by administration to a subject of a copper 
chelating agent in a pharmaceutically acceptable vehicle. Classes of 
copper chelating agents contemplated for use in the practice of the 
present invention include in particular those with access to the active 
site channel of mutant CuZnSOD. Particular copper chelating agents 
contemplated for use in the practice of the present invention include DDC, 
penicillamine, catechol, diethylenetriaminepentaacetic acid, 
diisopropylsalicylate, dithizone, ethylenediaminetetraacetic acid, 
tetraethylenepentamine, triethylenetetramine, tetrakis-2-(pyridylmethyl) 
ethylenediamine, and the like. 
A presently preferred copper chelating agent contemplated for use in the 
practice of the present invention is DDC. DDC is preferably used to treat 
a subject by administration of about 0.001 to about 1 g of DDC per kg body 
weight of the subject. Of course, The required dosage will vary with the 
severity of the condition and with the duration of desired treatment, and 
this should be determined by and administered under the guidance of a 
physician. In a presently preferred embodiment, 0.1 g of DDC per kg body 
weight is administered to the subject. More important than the amount 
administered to a subject is the concentration of the DDC that is achieved 
in the subject's neural cells. The optimal intracellular concentration of 
DDC is relatively the same as the intracellular concentration of the 
CuZnSOD enzyme. Thus, the preferred intracellular concentration is in the 
range of about 0.01 to about 1 .mu.M. Preferably, a sufficient amount of 
DDC is administered so as to achieve an extracellular DDC concentration in 
the range of about 10 to about 500 .mu.M. In a presently preferred 
embodiment, the extracellular DDC concentration will be about 100 .mu.M. 
When penicillamine is used, it is preferably administered in an amount of 
about 0.001 to about 1 g of penicillamine per kg body weight of the 
subject. In a presently preferred embodiment, 0.1 g of penicillamine per 
kg body weight is administered to the subject. More important than the 
amount administered to a subject is the concentration of the penicillamine 
that is achieved in the subject's neural cells. The optimal intracellular 
concentration of penicillamine is relatively the same as the intracellular 
concentration of the CuZnSOD enzyme. Thus, the preferred intracellular 
concentration is in the range of about 0.01 to about 1 .mu.M 
penicillamine. Preferably, a sufficient amount of penicillamine is 
administered so as to achieve an extracellular penicillamine concentration 
in the range of about 10 to about 500 .mu.M. In a presently preferred 
embodiment, the extracellular penicillamine concentration will be about 
100 .mu.M. 
Another aspect of the invention is the modulation of radical formation and 
the treatment of ALS by administration to a subject of a radical 
scavenging agent in a pharmaceutically acceptable vehicle. Classes of 
radical scavenging agents include thiol reagents, lipid-soluble 
antioxidants, water-soluble antioxidants, spin-trapping agents, and the 
like. Particular radical scavenging agents contemplated for use in the 
invention include DMPO, tocopherol, ascorbate, N-acetylcysteine, 
N-t-.alpha.-phenylnitrone, and the like. 
A presently preferred radical scavenging agent is DMPO. DMPO is preferably 
used to treat a subject by administration of about 0.001 to about 1 g of 
DMPO per kg body weight of the subject. In a presently preferred 
embodiment, 0.01 g of DMPO per kg body weight is administered to the 
subject. More important than the amount administered to a subject is the 
concentration of the DMPO that is achieved in the subject's neural cells. 
Preferably, a sufficient amount of DMPO is administered so as to achieve a 
cellular DMPO concentration in the range of about 10 to about 1000 .mu.M. 
In a presently preferred embodiment, the cellular DMPO concentration will 
be about 45 .mu.M. 
It has been shown that ALS caused by mutant sod1 genes may be inherited in 
a dominant manner. Thus, afflicted subjects may have both a wild type and 
a mutant version of the gene within their genome. In these subjects, if 
the expression of the mutant gene could be inhibited, the disease state 
would be reduced or abated, while the wild type gene could express a 
sufficient amount of the active, wild type enzyme to provide the SOD 
activity required by the patient. 
Therefore, another aspect of the invention is the modulation of radical 
formation and the treatment of ALS by administration to a subject of an 
expression inhibitor in an amount effective to inhibit expression of the 
DNA encoding the mutant sod 1 protein. A variety of methods to achieve 
this aim will be apparent to those of ordinary skill in the art, including 
the use of antisense DNA or RNA, the use of retroviral constructs that 
will integrate specifically into the mutant gene to prevent expression 
thereof, adenoviral vectors, and the like. 
Many mutant sod1 genes have been elucidated, including A4V, G93A, G37R, 
G41D, G85R, I112T, I113T, D90A, E100, L106, V148, H43, H46R, L38V, L144, 
and the like. Of these, A4V is known to be involved in a particularly 
severe form of ALS, and therefore may be the most suited to treatment by 
the invention methods. 
The results of the work done in developing the invention demonstrate a 
qualitatively different chemistry for the active site copper of the 
CuZnSOD mutants A4V and G93A as compared to the wild type enzyme. The 
alteration in reactivity of the CuZnSOD mutants A4V and G93A relative to 
the wild type CuZnSOD enzyme is demonstrated by their responses to the 
chelating agent DDC. Whereas hydroxyl radical generation by the wild type 
CuZnSOD is slightly enhanced by stoichiometric amounts of DDC, similar 
amounts markedly reduce hydroxyl radical generation by the mutants (FIGS. 
3 and 4). Additionally, wild type CuZnSOD shows no loss of SOD activity at 
concentrations of DDC that cause substantial reduction of the SOD activity 
of CuZnSOD mutants A4V and G93A (FIG. 5). Results both from previous 
studies (Yim (1990), supra) and the present invention show that hydroxyl 
radical generation requires an active CuZnSOD; neither apoenzyme nor 
boiled, inactivated enzyme demonstrates the effect. Furthermore, the 
enhancement of hydroxyl radical generation by the CuZnSOD mutants A4V and 
G93A is not mimicked by zinc-free derivatives of wild type CuZnSOD nor by 
adding free copper to the solution. Thus, the enhancement of hydroxyl 
radical generation by the CuZnSOD mutants A4V and G93A is due to the 
reactivity of copper within the active site of CuZnSOD. 
Any model that explains mutant CuZnSOD-associated FALS satisfactorily must 
account for the dominant inheritance pattern, the fact that different 
CuZnSOD mutations cause the same disease, and the differing degrees of 
severity of the disease caused by different mutations. The enhanced 
production of hydroxyl radicals by FALS-associated CuZnSOD mutants A4V and 
G93A is an effect that is compatible with the required characteristics of 
the mutants for the initiation of motor neuron loss in FALS. First, the 
effect is a gain-of-function effect, and could account for the dominant 
inheritance pattern. Second, multiple FALS-associated mutants demonstrate 
the effect. Third, it is compatible with the finding that FALS-associated 
CuZnSOD mutants induce apoptosis in cultured neural cells, whereas the 
wild type CuZnSOD has an anti-apoptotic effect (Rabizadeh (1995); 
Greenlund et al. (1995); and D. E. Bredesen, Apoptosis II: The Molecular 
Basis of Apoptosis in Disease (Cold Spring Harbor Laboratory Press, 1994) 
pp. 397-421). Fourth, the effect is compatible with the finding that 
copper chelation inhibits the pro-apoptotic effect of the mutants A4V, 
G37R, G41D, and G85R, yet does not affect apoptosis in cells 
overexpressing wild type CuZnSOD. Finally, the effect is more pronounced 
for the CuZnSOD mutant A4V than for the CuZnSOD mutant G93A, and A4V is 
associated with a particularly severe form of FALS (D. R. Rosen, et al., 
Hum. Mol. Genet. 3:981 (1994)). 
Despite the demonstration that peroxidase activity is enhanced by the 
FALS-associated mutations, it is unlikely that free hydroxyl radical is 
itself the predominant damaging species produced (Yim (1993), supra). Free 
hydroxyl radicals react at diffusion-controlled rates and are therefore 
likely to be scavenged by small-molecule antioxidants immediately after 
they are produced in vivo. It is more likely that a small, anionic 
substrate(s), such as formate or glutamate, scavenges the hydroxyl radical 
within the active site channel, forming free radical products such as 
formyl or glutamyl radicals, which in turn may participate in more long 
range oxidative reactions. In fact, the ability of glutamate to prevent 
the inhibition of wild type CuZnSOD by reaction with H.sub.2 O.sub.2 has 
been attributed to such a reaction (Id.). The present data indicate that 
this mode of free radical production is substantially enhanced by the 
FALS-associated CuZnSOD mutants A4V and G93A. 
Motor neurons have been reported to have particularly high levels of 
CuZnSOD, implying that this cell type may require a high rate of 
superoxide dismutation. Indeed, this notion has been supported by the 
finding of relatively restricted motor neuron apoptosis in response to 
CuZnSOD inhibition in organotypic cultures (J. D. Rothstein, et al., Proc. 
Natl. Acad. Sci. U.S.A. 91:4155 (1994)). Although many of the 
FALS-associated CuZnSOD mutants retain the ability to dismutate superoxide 
enzymatically, at least CuZnSOD mutants A4V and G93A also demonstrate a 
high rate of hydroxyl radical generation. Thus, the requirement for 
high-level expression of CuZnSOD may place motor neurons at high risk for 
damage by these mutants. The observation that there are other neurons that 
express sod1 at high levels but do not degenerate in FALS (C. A. Pardo, et 
al., Proc. Natl. Acad. Sci. U.S.A. 92:954 (1995)) might conceivably be 
explained by a difference in the availability of substrate (H.sub.2 
O.sub.2), scavengers (e.g., glutamate), or targets (e.g., glutamate 
transporters). 
The results of the DDC experiments described herein do not discriminate 
between the possibility that DDC causes removal of copper from the active 
site of the mutant enzymes, and the alternate possibility that Cu-DDC 
remains in the active site. Nonetheless, results from Misra (1979, supra) 
indicate that binding of DDC to wild type bovine CuZnSOD does not cause an 
inhibition of SOD activity until sufficient DDC is present to cause 
removal of copper from the active site. It is likely therefore that the 
inhibition of the SOD activity of A4V and G93A by DDC is due to copper ion 
removal from the protein. Thus CuZnSOD mutants A4V and G93A may have a 
lower affinity than wild type CuZnSOD for copper ions in the active site, 
in addition to their enhanced ability to catalyze free radical production 
by hydrogen peroxide. 
The finding that copper chelation with DDC, at a concentration that does 
not decrease wild type SOD enzymatic activity, reduces both hydroxyl 
radical generation and the pro-apoptotic effects of CuZnSOD mutants, 
indicates that the use of copper chelators will prove beneficial in animal 
models of FALS (Gurney (1994), supra; Ripps (1995), supra; and Pardo 
(1995), supra), and in the treatment of patients with FALS-associated sod1 
mutations. 
The invention will now be described in greater detail with reference to the 
following non-limiting examples. 
EXAMPLES 
Example 1 
Hydroxyl radical formation by mutant and wild type CuZnSODs was determined 
by measuring the amount of adduct formed when the radical scavenging agent 
5,5'-dimethyl-1-pyrroline N-oxide (DMPO) was added to the hydroxyl radical 
forming reaction. The amount of the DMPO-OH radical adduct formed was 
determined by measurement of the unique EPR spectra signals of the adduct. 
Reactions were carried out as described (Yim (1990), supra), with minor 
modifications. Reaction mixes consisted of 1.25 .mu.M protein (or 2.5 
.mu.M CuSO.sub.4), 45 mM DMPO (Aldrich) in 23.5 mM NaHCO.sub.3 buffer (pH 
7.4) balanced with 5% CO.sub.2 /95% N.sub.2 gas. 30 mM hydrogen peroxide 
was injected to start the reaction. Spectra developed after 30 to 45 
seconds, were recorded at 5 minutes, and were stable for more than 30 
minutes. EPR spectra were recorded on a Bruker ER 200 D at room 
temperature, operated at 9.5 GHz with a modulation frequency of 100 KHz. 
Conditions were: microwave power 20.7 milliwatts, modulation amplitude 1 
Gauss (G), time constant 10 ms, sweep width 100 G with 2046 point 
resolution. 
The samples were prepared and handled, and all measurements executed, in a 
strictly oxygen-free atmosphere, to prevent radical scavenging by 
molecular oxygen. Buffers were treated with Chelex 100 (Biorad) to 
eliminate the possibility of contamination by trace metals. DMPO was 
purified by filtration with neutral decolorizing charcoal (Aldrich) (G. H. 
Buettner and L. W. Oberley, Biochem. Biophys. Res. Comm. 83:69 (1978)). 
The concentration of DMPO was calibrated spectrophotometrically, using an 
extinction coefficient .SIGMA..sub.226 =7.22.times.10.sup.3 M.sup.-1 
cm.sup.-1 (E. Finkelstein, et al., Arch. Bioch. Biophys. 200:1 (1980)). An 
aqueous flat cell (Wilmad) was used to hold the samples. The 
concentrations of the apoproteins were calculated from the extinction 
coefficient .SIGMA..sub.278 =1.08.times.10.sup.4 M.sup.-1 cm.sup.-1. 
The signals shown in FIG. 1(A)-(D) are representative; measurements were 
repeated 5 to 10 times, always paired with the wild type protein. Double 
integration of these signals using the spin label carbamoyl-proxyl 
(Aldrich) as a standard to quantitate the hydroxyl radical production 
showed that wild type CuZnSOD produced 3.2 .mu.M, CuZnSOD mutant A4V 
produced 15.6 .mu.M, CuZnSOD mutant G93A produced 6.5 .mu.M, and 
CuSO.sub.4 produced 1.9 .mu.M hydroxyl radical in this experiment. 
Example 2 
Hydroxyl radical formation by wild type and mutant A4V CuZnSODs was 
determined with varying degrees of copper metallation of the enzymes. The 
same methodology was used as in Example 1, with the samples containing 
1.25 .mu.M enzyme, 45 mM DMPO and 30 mM H.sub.2 O.sub.2 in 23.5 mM 
NaHC.sub.3 O buffer, pH 7.4. Experiments were repeated three times, and 
representative signals, recorded at 3 minutes post-initiation, are shown 
in FIG. 2(A)-(E). The mutant A4V CuZnSOD produced more adduct at all 
levels of metallation, although neither enzyme produced any measurable 
adduct when no copper was present. 
Example 3 
Hydroxyl radical formation was next measured as varying amounts of DDC were 
added to the reaction. Experiments were performed as described in Example 
1, and repeated 2-3 times. DDC was freshly prepared, and added to the 
CuZnSOD immediately before adding the DMPO and injecting the H.sub.2 
O.sub.2. EPR spectra, shown in FIG. 3(A)-(C), were recorded at 2.5 minutes 
post-initiation. While DDC caused a slight increase in adduct production 
by wild type CuZnSOD enzyme, a sharp decrease in adduct production is seen 
in both mutant enzyme samples. 
FIG. 4 shows the data of FIG. 3(A)-(C) plotted as hydroxyl radical 
formation (i.e., adduct concentration) vs. DDC concentration. The 
calculation of adduct concentrations was as described in Example 1. The 
upper graph shows two separate experiments with wild type CuZnSOD. In the 
lower graph, the diamonds represent the CuZnSOD mutant A4V, and the 
triangles represent the CuZnSOD mutant G93A. 
It is seen that 0.25 equivalent concentrations of DDC (0.62 .mu.M) per 
enzyme subunit copper reduced the hydroxyl radical production by the 
CuZnSOD mutants A4V and G93A to 22% and 32%, respectively. The wild type 
CuZnSOD showed a slight increase in hydroxyl radical generation of 115% 
compared to the hydroxyl radical production without DDC. Even 4.0 
equivalents of DDC per enzyme subunit copper had no negative effect on 
hydroxyl radical production by the wild type enzyme. 
Example 4 
Now that it has been shown that addition of DDC to CuZnSOD mutants reduces 
hydroxyl radical formation, the effect of DDC on the SOD activity of both 
mutant and wild type CuZnSOD enzymes was tested. DDC was freshly prepared, 
added to the proteins, and allowed to incubate for 5 minutes prior to 
measurements. SOD activity was assayed as described previously (Rabizadeh 
(1995); and R. E. Heikkila and F. Cabbat, Anal. Bioch. 75:356 (1976)). 
While high concentrations of DDC (4 equivalents/enzyme copper) had an 
impact on all CuZnSODs, lower concentrations of DDC (less than 1 
equivalent/enzyme copper) had little effect on the SOD activity of wild 
type CuZnSOD (See FIG. 5). 
Example 5 
Next the in vivo efficacy of DDC was tested. Temperature-sensitive nigral 
neural cells (SCM 14.1) were transfected with wild type and A4V, G37R, 
G85R, and G41D mutant CuZnSOD constructs by methods described previously 
(Rabizadeh (1995), supra). Following selection in puromycin (7 .mu.g/ml), 
cells were plated onto 96-well plates. DDC was added at concentrations of 
25 .mu.M-1 mM to cells in serum-free media. Viability was assessed at 60 
hours, by methods described in Rabizadeh (1995), and is shown in FIG. 6. 
There was no significant improvement at any DDC concentration for the 
cells transfected with the wild type CuZnSOD construct. In contrast, all 
mutants showed highly significant improvements in survival (p&lt;0.01 by 
unpaired, two-tailed t-tests; n=4) at 25-500 .mu.M DDC, except G41D (N.S. 
at 25 .mu.M; p 0.25 at 50, 100 .mu.M; p&lt;0.01 at 500 .mu.M DDC) and G85R 
(p&lt;0.05 at 50 .mu.M; p&lt;0.01 at 25, 100, and 500 .mu.M DDC). 
FIG. 7 illustrates the improvement in viability of the various transfected 
cell lines when DDC was added at a concentration of 100 .mu.M, as opposed 
to viability with no added DDC in the growth media. This was calculated as 
follows: 
EQU viability=Vm(x)-Vm(0)!/Vc(0)-Vm(0)! 
where Vm(x) is defined as the viability of mutant transfected cells at x 
concentration of DDC, Vm(0) is defined as viability of mutant transfected 
cells at a DDC concentration of 0, and Vc(0) is defined as viability of 
control transfected cells at a DDC concentration of 0. Standard errors 
were computed by the delta method for computing the asymptotic standard 
error for the estimate of a nonlinear function (D. Colquhoun, Lectures on 
Biostatistics (Clarendon Press, 1971) pp. 39-42). 
While 100 .mu.M DDC increased viability of the various mutant transfected 
cell lines from 30-70%, it had no significant effect on the viability of 
cell lines transfected with the wild type gene. 
Example 6 
CuZnSOD enzyme proteins, used in Examples 1-4, were prepared as follows. 
cDNA clones for wild type CuZnSOD, ALS CuZnSOD mutant A4V, and ALS CuZnSOD 
mutant G93A, under the control of the yeast CuZnSOD promoter, were cloned 
into the yeast shuttle vector YEp351, and transformed into yeast strain 
EG118, which is null for yeast sod1 (Rabizadeh (1995), supra). Cultures of 
10 liters were grown in highly aerated, YEPD medium (5% yeast extract, 10% 
peptone, 2% dextrose) for 24 hours. Cells were harvested by 
centrifugation, and lysed using an equal volume of 0.5 mm glass beads in a 
blender. Protein purification was as described previously (Lu (1993), 
supra) with the addition of a final Sephadex G75 chromatography step. The 
purified proteins were homogeneous, as indicated by SDS-polyacrylamide gel 
electrophoresis. For each batch, 50-200 mg of purified protein was 
obtained. Human CuZnSOD expressed in yeast is properly N-terminal 
acetylated (R. A. Hallewell, et al., Bio/Tech. 5:363 (1987)). The 
identities of the purified mutant proteins were verified by electrospray 
mass spectrometry, which detected the change in mass due to single amino 
acid substitutions. For both wild type and mutant CuZnSOD proteins, the 
observed and predicted masses corresponded, within 2-4 atomic mass units. 
While the invention has been described in detail with reference to certain 
preferred embodiments thereof, it will be understood that modifications 
and variations are within the spirit and scope of that which is described 
and claimed.