Mutant Cu,Zn superoxide dismutases and familial amyotrophic lateral sclerosis: evaluation of oxidative hypotheses

Free Radical Biology & Medicine, Vol. 34, No. 11, pp. 1383–1389, 2003 Copyright © 2003 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/03/$–see front matter

doi:10.1016/S0891-5849(03)00153-9

Review Article MUTANT Cu,Zn SUPEROXIDE DISMUTASES AND FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS: EVALUATION OF OXIDATIVE HYPOTHESES STEFAN I. LIOCHEV

and IRWIN

FRIDOVICH

Department of Biochemistry, Duke University Medical Center, Durham, NC, USA (Received 2 January 2003; Revised 7 February 2003; Accepted 28 February 2003)

Abstract—FALS-associated missense mutations of SOD1 exhibit a toxic gain of function that leads to the death of motor neurons. The explanations for this toxicity fall into two broad categories. One involves a gain of some oxidative activity, while the second involves a gain of protein: protein interactions. Among the postulated oxidative activities are the following: (i) peroxidase action; (ii) superoxide reductase action; and, (iii) the enhancement of production of O⫺ 2 by partial reversal of the normal SOD activity, which then leads to increased formation of ONOO⫺. We will herein concentrate on evaluating the relative merits of these oxidative hypotheses and consider whether the experiments with transgenic animals that purport to disprove these oxidative explanations really do so. © 2003 Elsevier Inc. Keywords—Superoxide dismutase, Amyotrophic lateral sclerosis, Superoxide reductase, Superoxide, Free radicals

INTRODUCTION

the aggregates or to the depletion of the mSOD-associated proteins and consequent loss of their normal functions. In this review, we concentrate on the oxidative hypotheses and, after evaluating them, examine the impact of manipulating the levels of Cu,Zn SOD, H2O2scavenging enzymes, or the copper chaperone that delivers Cu to the SOD.

Approximately 20% of cases of familial ALS have been associated with ⬃100 point mutations in the Cu,Zn SOD [1–5]. Transgenic mice expressing several FALS-associated mutant Cu,Zn SOD (mSODs) develop paralysis in early adulthood, while those expressing the wild-type (WT) enzyme remain normal [5]. This fact, together with the genetic dominance of FALS and the observations that Cu,Zn SOD-null mice do not become paralyzed [6] and that most mSODs retain catalytic activity, establishes that a gain, rather than a loss, of function is the basis of the neurotoxicity of mSODs. But what is that gain of function? Two types of hypotheses have been proposed. One is that mSODs exhibit enhanced oxidative activity by acting as peroxidases [7–10], superoxide reductases (SORs) [11], or by actually producing O⫺ 2 rather than scavenging it, leading to ONOO⫺ formation [12,13]. The second is that the mSOD proteins are prone to aggregation, perhaps due to instability or to association with other proteins [3,7,14 –16]. In this case, toxicity could be due to

DISMUTATION OF Oⴚ 2

The established function of the SODs [17] proceeds by two sequential reactions with O⫺ 2 , as shown below: E ⫺ Cu(II) ⫹ O2⫺ ^ E ⫺ Cu(I) ⫹ O2

(a)

E ⫺ Cu(I) ⫹ O2⫺ ⫹ 2H⫹ ^ E ⫺ Cu(II) ⫹ H2O2

(b)

with the sum of these reactions being: SOD - 0 H2O2 ⫹ O2 O2⫺ ⫹ O2⫺ ⫹ 2H⫹ | Both reactions a and b proceed with rate constants of ⬃3 ⫻ 109 M⫺1s⫺1, and for both the half reactions of this catalytic cycle reaction to the right is vastly favored over the reverse reactions [18 –20]. In the case of Escherichia

Address correspondence to: Prof. Irwin Fridovich, Duke University Medical Center, Department of Biochemistry, Durham, NC 27710, USA; Tel: (919) 684-5122; Fax: (919) 684-8885; E-Mail: fridovich@ biochem.duke.edu. 1383

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coli, it has been shown that the endogenous level of SOD intercepts at least 95% of the flux of O⫺ 2 , in competition with all other targets of O⫺ [21]. Although H2O2 is one 2 product of this dismutation reaction, SOD can decrease or increase H2O2 production from O⫺ 2 depending, in part, on whether the predominant competing reactions involve ⫺ the oxidative action of O⫺ 2 or either the addition of O2 to ⫺ NO or the reductive action of O2 [22]. PEROXIDATIVE ACTIVITIES OF Cu,Zn SOD

The ability of Cu,Zn SOD to catalyze the oxidation of diverse substrates by H2O2 was first observed in a carbonate-buffered medium [23]. The role of bicarbonate in the peroxidations observed was not suspected until reported by Sankarapandi and Zweier [24] and others [25]. Subsequent work [19,20,26 –28] has explained the role of bicarbonate as follows: E ⫺ Cu(II) ⫹ H2O2 ^ E ⫺ Cu(I) ⫹ O2⫺ ⫹ 2H⫹

(b-)

E ⫺ Cu(I) ⫹ H2O2 ^ E ⫺ Cu(II) ⫺ OH ⫹ OH⫺

(c)

E ⫺ Cu(II) ⫺ OH ⫹ HCO3⫺ ^ E ⫺ Cu(II) ⫹ H2O ⫹ CO3⫺• (d) with the sum of these reactions being: SOD 2H2O2 ⫹ HCO3⫺ | - 0 CO3⫺• ⫹ O2⫺ ⫹ 2H2O ⫹ H⫹ The CO⫺• 3 so produced is a strong oxidant [27–29] and after leaving the active site can oxidize NAD(P)H and urate [19]. As written (reactions b- ⫹ c ⫹ d), this process produces O⫺ 2 . The rate constants for reactions b and c have ⫺1 ⫺1 been estimated to be only ⬃50 M s and ⬃13 M⫺1s⫺1, respectively [19]. Hence, O⫺ 2 production by these reactions is not kinetically significant. Furthermore, in the absence of scavengers of E ⫺ Cu(II) ⫺ OH, such as HCO⫺ 3 , the equilibrium of reaction c lies far to the left [19]. Km for HCO⫺ 3 in this overall process is high, i.e., ⬃100 mM [19]. This HCO⫺ 3 -dependent peroxidase activity is unlikely to have significance in vivo in the case of the WT enzyme because in vivo H2O2 is low, i.e., ⬃1 ⫻ 10⫺8 M and in vivo HCO⫺ 3 is only one-fourth of the Km for this reactant. ⫺ An O⫺ 2 -dependent and HCO3 -independent peroxidase activity was seen with one mSOD (H48Q), but not with several other mSODs [10]. This activity is explained by the following reactions: E ⫺ Cu(II) ⫹ O2⫺ ^ E ⫺ Cu(I) ⫹ O2

(a)

E ⫺ Cu(I) ⫹ H2O2 ^ E ⫺ Cu(II) ⫺ OH ⫹ OH⫺

(c)

E ⫺ Cu(II) ⫺ OH ⫹ RH2 ^ E ⫺ Cu(II) ⫹ H2O ⫹ RH•

(d⬘)

where RH2 is urate or another cellular reductant and with the sum of these reactions being: SOD - 0 RH• ⫹ O2 ⫹ OH⫺ ⫹ H2O RH2 ⫹ H2O2 ⫹ O2⫺ | The sum of reactions a ⫹ c ⫹ d⬘ represents the oxidation of RH2 by an mSOD-catalyzed interaction of O⫺ 2 and H2O2. The sum of reactions a and c are analogous to what has been called the Haber-Weiss reaction. This activity of H48Q could be demonstrated using low [H2O2]. The H48Q enzyme must have exhibited a greater kc than the WT enzyme and must have had a widened solvent access channel to the active site that allowed urate to approach the bound oxidant (E ⫺ Cu(II) ⫺ OH). The SOD activity of H48Q was only ⬃1%, as great as that of the WT SOD. The poor dismutation activity of H48Q could be due to a low ka or to a low kb. If the former, then the WT enzyme would strongly inhibit its peroxidase activity, especially if va and not vc is rate-limiting; if the latter, that would not significantly be the case. It has been proposed that the mSODs produce the bound oxidant by reaction with H2O2 as in reaction c more rapidly than does the WT enzyme [7–9]. Were that the case one should expect more rapid inactivation of the mSODs by H2O2 due to reaction e. E ⫺ Cu共II兲 ⫺ OH ⫹ his 3 E ⫺ Cu共II兲 ⫹ OH⫺ ⫹ his⫹ (e) where his is one of the histidines in the ligand field of the Cu(II). Three mSODs were examined and they were not more sensitive to H2O2 than was the WT [30]. In full accord with this was the observation that mSODs were not more active than the WT enzyme in catalyzing HCO⫺ 3 -dependent peroxidations [31]. There have been some reports of modestly greater peroxidase activity of mSODs [7–9], but these could have been due to Cu(II) bound at other than the active site, as discussed by Valentine [7]. The in vivo peroxidative action of mSOD has been explored by the deletion of glutathione peroxidase (GSHPx) [32]. The GSHPx knockout was not more adversely affected by expression of mSOD [32], which argues against a peroxidase activity for the toxic gain of function of the mSOD. However, this result is equivocal since there are more than one GSHPx gene and elevation of [H2O2] in the GSHPx knockout was not demonstrated. In a contrasting study [33], overexpression of the

Superoxide dismutase and Lou Gehrig’s Disease

mitochondrial GSHPx-4 significantly protected against the toxicity of G93A mSOD. Thus, studies such as these do not decide the issue of the importance of the peroxidase activity of the FALS-associated mSODs. The most serious weakness with this peroxidase hypothesis is that, excluding the case of H48Q, there are no data showing that kc is greatly increased in the mSODs. Nevertheless, that enhanced peroxidative activity of some mutant SOD1s contribute to the toxic gain of function of these mSODs remains a possibility. WEAKENED Zn(II) BINDING AND DECREASED STABILITY

It has been shown that FALS-associated mSODs are less stable in the apo, but not in the holo, state [34]. Moreover, the loss of stability of the apo mSODs correlated with the severity of the disease imposed on patients. This result is in accord with the reports that mSODs exhibit lower affinity for Zn(II) [12,13,35,36]. It follows that a greater fraction of mSODs than of WT SOD will be free of Zn(II) (Cu,E SOD) in vivo, and it has been shown that the Cu,E SOD is toxic in the cases of both WT and mutant enzymes [12,13,36]. Additionally, a Zn-deficient diet has been reported to hasten the development of paralysis caused by G93A in transgenic mice [37]. What is the basis of the toxicity of the Cu,E SOD? We consider two mechanisms: (i) the Cu,E SOD exhibits increased SOR activity; and, (ii) the Cu,E SOD actually ⫺ produces O⫺ 2 , which leads to ONOO formation. SOR ACTIVITY OF mSODs

The SOR activity of Cu,Zn SOD [11] is attributed to the reduction of the active site Cu(II) by some cellular reductant other than O⫺ 2 , followed by its reoxidation by O⫺ as in reactions f and b. 2 E ⫺ Cu共II兲 ⫹ RH2 ^ E ⫺ Cu共I兲 ⫹ RH• ⫹ H⫹ ⫺ 2

⫹

E ⫺ Cu共I兲 ⫹ O ⫹ 2H ^ E ⫺ Cu共II兲 ⫹ H2O2

(f) (b)

with the sum of these reactions being:

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Thus SOR activity depends on the competition between reactions a and f. The ratio of the rates of those reactions is given by: Va ka[O2⫺] ⫽ Vf kf[RH2] ⫺10 If we take in vivo [O⫺ M [41,42] 2 ] to be 1 ⫻ 10 ⫺3 and [ascorbate] to be 1 ⫻ 10 M and apply the rate constants ka ⫽ 3 ⫻ 109 M⫺1s⫺1 and kf ⫽ 0.3 M⫺1s⫺1 [13] for the Cu,Zn SOD, we get va/vf ⫽ 1000. Hence, with the holo enzyme the SOD activity exceeds the SOR activity by a factor of 103. But the rate of reduction of the Cu,E SOD by ascorbate [13,35] has been reported to be 3000 times faster than the reduction of the holo enzyme [13]. This changes the ratio va/vf for the Cu,E SOD to 0.33, assuming that va is not much changed by the removal of Zn(II), which appears to be the case [43]. Therefore, for the Cu,E SOD, SOR activity will be comparable to the SOD activity under physiological conditions. So why might SOR activity be a problem? One possibility is that SOR action depletes the SOR reductant, ascorbate in our example. Another is that SOR action involves univalent oxidation of the SOR reductant to the corresponding free radical and further reactions of that radical might be injurious. Since FALS is most often genetically dominant, the mutant protein is toxic in the presence of a like amount of the WT enzyme. As such, we must consider how the level of WT SOD influences the SOR activity of the mSOD. The three reactions involved in the SOD plus SOR activities are (a), (f), and (b), and their rates can be written as follows:

va ⫽ ka关O2⫺兴 关E ⫺ Cu共II兲兴 vf ⫽ kf关RH2兴 关E ⫺ Cu共II兲兴 vb ⫽ kb关O2⫺兴 关E ⫺ Cu共I兲兴 In the steady state, the rates of the E ⫺ Cu(I)-producing reactions (a ⫹ f) must equal the rate of the E ⫺ Cu(I)-consuming reaction (b). Hence: ka关O2⫺兴 关E ⫺ Cu共II兲兴 ⫹ kf关RH2兴 关E ⫺ Cu共II兲兴 ⫽ kb关O2⫺兴 关E ⫺ Cu共I兲兴 共1兲

SOD RH2 ⫹ O ⫹ H | - 0 RH• ⫹ H2O2 ⫺ 2

⫹

dividing by [E ⫺ Cu(II)], we get The SOR action of Cu,Zn SOD has been demonstrated using HNO [38,39], 3-hydroxy anthranilic acid [40], or ferrocyanide [11] as the reductants. For SOR activity to be significant vis a vis SOD activity, the rate of reaction f must approach that of reaction a.

ka关O2⫺兴 ⫹ kf关RH2兴 ⫽

kb关O2⫺兴 关E ⫺ Cu共I兲兴 关E ⫺ Cu共II兲兴

since ka is known to be equal to kb, we can simplify this to:

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关E ⫺ Cu共I兲兴 kf关RH2兴 ⫽1⫹ 关E ⫺ Cu共II兲兴 kb关O2⫺兴

(2)

In the absence of the reductant RH2, 关E ⫺ Cu共I兲兴 ⫽1 关E ⫺ Cu共II兲兴 in accord with the fact that the rate constants for both halves of the catalytic dismutation cycle are ⬃3 ⫻ 109 M⫺1s⫺1. Equation 2 allows us to examine how responsive the state of reduction of mSOD would be to overproduction or elimination of WT SOD. Thus, when: kf关RH2兴 关E ⫺ Cu共I兲兴 ⫽ 1.1 ⫺ ⫽ 0.1 the ratio 关E ⫺ Cu共II兲兴 kb关O2 兴 and 47.6% of the mSOD is in the cupric state. If we decrease [O⫺ 2 ] 5-fold by overproduction of WT SOD, we get: kf关RH2兴 关E ⫺ Cu共I兲兴 ⫽ 1.5 ⫺ ⫽ 0.5 and then 关E ⫺ Cu共II兲兴 kb关O2 兴 and now 40% of the mSOD is cupric. Since the SOR activity is proportional to [E ⫺ Cu(II)], we see that it would not be very responsive to changes in [O⫺ 2 ] and, hence, not very responsive to changes in the level of the WT enzyme. This is especially the case when va ⬎ vf. It follows that any toxicity of an mSOD that was due to its SOR activity would not be much affected by changing the level of WT SOD. Additionally, we must consider that marked overproduction of a usually abundant metallo enzyme such as Cu,Zn SOD may be deleterious, particularly in the presence of mSODs. Firstly, because it diverts cell resources from making other essential proteins; secondly, because it may make the prosthetic metals less available to other metallo enzymes such as Zn(II) for mSOD; and, finally, because the WT enzyme may exert, to a lesser degree, the same toxic action attributed to the mutant enzyme. In this regard, it has been reported that overproduction of WT Cu,Zn SOD is sometimes toxic [44 – 46] and can lead to neuropathology [44]. These comments apply to studies with transgenic murine models of FALS designed to prove that the toxic gain of function of the mSOD is not related to O⫺ 2 [47]. The surprising long-term survival of Cu,Zn SOD knockout mice [6] raises another caution. There may be means of disposing of O⫺ 2 , in addition to the action of SODs, and these may be regulated in response to changes in [O⫺ 2 ]. Hence, changing [Cu,Zn SOD] may not have the anticipated effect on [O⫺ 2 ]. The same caution applies to experiments aimed at changing [H2O2] by ablation of a

GSHPx [32]. The deductions arrived at from such manipulations should be viewed with caution, particularly ⫺ when the levels of O⫺ 2 , H2O2, NO, or ONOO were not actually measured. Pro-oxidative hypotheses of the toxic gain of function of mSOD were challenged by Subramaniam et al. [48], who manipulated the degree of Cu(II) loading of SODs in mSOD-expressing mice by preventing the expression of the copper chaperone for SOD (CCS). There must be alternate means of introducing Cu(II) into the enzyme, since the activity of the Cu,Zn SOD was decreased by only 80 – 85% and not 100%. As noted by Beckman et al. [36], the amount of Cu,Zn SOD was 5-fold greater than in the nontransgenic mice. Hence, the effect of the CCS knockout was to bring the net Cu,Zn SOD activity down to the normal level. The CCS knockout experiment is thus not very informative about the reason for the toxicity of mSODs. An interesting aspect of these experiments is that CCS specifically metallates the Zn-replete enzyme. The non-CCS metallation may not depend on occupancy of the Zn site. In that case, the 15–20% of activity seen in the CCS knockout mice may well have been due to Cu(II)-replete but Zn(II)-depleted enzyme, and this is the form that has been shown to be toxic to neurons and prone to reduction by ascorbate [13]. IS SOD A PLAUSIBLE SOURCE OF Oⴚ 2?

It has been proposed [12,13] that the cuprous form of the mSOD autoxidizes and, thus, produces rather than consumes O⫺ 2 . Consider the reverse of reactions a and b: E ⫺ Cu共II兲 ⫹ H2O2 3 E ⫺ Cu共I兲 ⫹ O2⫺ ⫹ 2H⫹

(b-)

E ⫺ Cu共I兲 ⫹ O2 3 E ⫺ Cu共II兲 ⫹ O2⫺

(a-)

with the net of these reactions being: SOD H2O2 ⫹ O2 O ¡ 2H⫹ ⫹ 2O2⫺ This reversal of the usual SOD reaction has been demonstrated by using excess tetranitromethane (TNM) to trap O⫺ 2 and, thus, to pull these reactions [20,49]. Presumably, NO, which reacts with O⫺ 2 as fast as TNM does, also could pull these reactions. In that case, we could envision the enzyme, or its Zn(II)-depleted form, being reduced by ascorbate, as in reaction f. Could the enzyme then produce O⫺ 2 , and in the presence of NO, produce ONOO⫺ by reaction f followed by reaction a-? Given that the enzyme will be maintained half reduced in the normal dismutation of O⫺ 2 because both reaction a and b proceed at the same fast rate of ⬃3 ⫻ 109 M⫺1s⫺1, the consequence of maintaining the enzyme

Superoxide dismutase and Lou Gehrig’s Disease

fully reduced, by action of an infinite supply of ascorbate, would be to replace SOD action by an equally rapid SOR action. Hence, the enzyme would scavenge O⫺ 2 with equal efficiency in the presence or absence of the reductant. This is inescapable since the ratio of ka/k⫺ a is ⬃3 ⫻ 109 [18,20], and this ratio precludes SOD from acting as a source of O⫺ 2 under any conceivable in vivo conditions. Consequently, the proposal of Estevez and Beckman [12,13] may be discounted. The only way that Zn-deficient enzyme could be a source of O⫺ 2 would be if its k⫺ becomes much larger than that of Cu,Zn SOD, a which is ⬃1 M⫺1s⫺1 [18,20]. But, Beckman et al. [12] have stated that the holo enzyme can produce ONOO⫺ about as well as the Zn(II)-deficient enzyme. This indicates that the Zn-deficient enzyme exhibits a k⫺ a not very different than that characteristic of the holo enzyme. Moreover, there are multiple sources of O⫺ 2 formation in the cell whose magnitude greatly exceeds that from reaction a- of the Zn(II)-deficient enzyme. Perhaps 0.1% of total intracellular O⫺ 2 could derive from the autoxidation of reduced SOD, which at the same time scavenges 1000 times more O⫺ 2 than it produces. Given the availability of O⫺ produced by other sources, SOD-dependent 2 O⫺ production would make a trivial contribution to 2 ONOO⫺ formation by the reaction of O⫺ 2 with NO. This practical irreversibility of the SOD reaction has been discussed in more detail elsewhere [20,50].

ADDITIONAL PRO-OXIDATIVE ACTIVITIES OF Cu,Zn SOD

Winterbourn et al. [51] have reported recently that Cu,Zn SOD exerts a low level thiol oxidase activity. The mechanism presented involves the binding of the thiols to the SOD followed by their oxidation by O2 with the generation of H2O2. Possibly some mSODs and/or their Zn(II)-depleted forms could exhibit this thiol oxidase activity to a greater degree than that seen with the WT enzyme. This possibility is not excluded by the transgenic experiments involving the manipulation of GSHPx or CCS for reasons already discussed. Another activity involves the oxidation of hydrogen sulfide (HS⫺) by Cu,Zn SOD [52]. In this case, the oxidant was O⫺ 2 and, thus, the activity seems related to SOR activity. However, unlike true SOR activity, reduction and reoxidation of the Cu(II) center does not appear to be required.

FURTHER CONSIDERATIONS

To aid appreciation of the possible significance and some of the complexities of our presentation to this point, we offer the following thoughts. We have assumed

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that the rate constants for both half reactions of the catalytic cycle of SOD are equal, i.e., that ka ⫽ kb. This is the case for the WT Cu,Zn SOD but may not be true for the Zn-depleted or for some of the FALS-associated mutant Cu,Zn SODs. Thus, for the Zn-depleted WT enzyme, kb is slower than ka and decreases with increasing pH [43]. The H48Q mutant mSOD has only trivial activity [10], probably due to the limitation of ka [53], and indeed WT Cu,Zn SOD effectively inhibits the O⫺ 2dependent peroxidase activity of this mSOD [10]. The ratio of reduced to oxidized enzyme in the presence of O⫺ 2 plus a SOR substrate (RH2) may be expressed as: 关E ⫺ Cu共I兲兴 ka kf关RH2兴 ⫽ ⫹ 关E ⫺ Cu共II兲兴 kb kb关O2⫺兴 In the event that ka is not equal to kb, the ratio of reduced to oxidized enzyme will not be 1.0 even in the absence of RH2, and this will markedly influence SOR activity since SOR activity depends on the [E ⫺ Cu(II)] in the steady state. Changing [O⫺ 2 ] will change the ratio of reduced to oxidized enzyme, and thus the SOR activity; and, as can be seen from the above equation, at high [O⫺ 2 ] this ratio will depend only on ka/kb. Finally, it follows that the ratio of reduced to oxidized enzyme also will be responsive to [RH2] and to kf. Similar conclusions apply to activities other than the SOR activity of Cu,Zn SOD, including, for example, the O⫺ 2 -dependent peroxidase activity of the H48Q mSOD, which depends on the generation of a bound oxidant from the reaction of E ⫺ Cu(I) with H2O2. This activity depends on the steady state [E ⫺ Cu (I)]. Since, in the weakly active H48Q mSOD, ka is probably less than kb, then the proportion of the enzyme in the [E ⫺ Cu(I)] state will be small. This limits its O⫺ 2 -dependent peroxidase activity, but would favor SOR activity. Hopefully the foregoing explanations clarify some of the complexities of which the Cu,Zn SOD is capable and may help others to interpret otherwise puzzling observations. CONCLUSIONS

Despite considerable effort, the nature of the toxic gain of function of the FALS-associated mutant SODs remains undecided. The proposal that Zn(II) depletion, due to decreased affinity for this metal, is the problem deserves more attention from the viewpoint that increased SOR activity is the culprit rather than O⫺ 2 production. In this regard, the recent report of a sporadic case of FALS due to a mutation of one of the Zn(II) ligands of SOD is very exciting [54]. It is entirely possible that multiple oxidative activities of mSODs contribute to their toxic gain of function. Thus, some

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mSODs, such as H48Q, could be damaging to motor neurons because of O⫺ 2 -dependent peroxidase activity, while others, or their Zn(II)-depleted forms, could be toxic because of SOR or thiol oxidase activities. The proposal based on the interaction of the mSOD with other proteins, such as the heat shock proteins, is another promising avenue to learning the causation of FALS. Acknowledgements — This work was supported by research grants from the Amyotrophic Lateral Sclerosis Association and the National Institutes of Health (RO1 DK 59868).

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