Oxidation-induced Misfolding and Aggregation of Superoxide Dismutase and Its Implications for Amyotrophic Lateral Sclerosis

The presence of intracellular aggregates that contain Cu/Zn superoxide dismutase (SOD1) in spinal cord motor neurons is a pathological hallmark of amyotrophic lateral sclerosis (ALS). Although SOD1 is abundant in all cells, its half-life in motor neurons far exceeds that in any other cell type. On the basis of the premise that the long half-life of the protein increases the potential for oxidative damage, we investigated the effects of oxidation on misfolding/aggregation of SOD1 and ALS-associated SOD1 mutants. Zinc-deficient wild-type SOD1 and SOD1 mutants were extremely prone to form visible aggregates upon oxidation as compared with wild-type holo-protein. Oxidation of select histidine residues that bind metals in the active site mediates SOD1 aggregation. Our results provide a plausible model to explain the accumulation of SOD1 aggregates in motor neurons affected in ALS. The presence of intracellular aggregates that contain Cu/Zn superoxide dismutase (SOD1) in spinal cord motor neurons is a pathological hallmark of amyotrophic lateral sclerosis (ALS). Although SOD1 is abundant in all cells, its half-life in motor neurons far exceeds that in any other cell type. On the basis of the premise that the long half-life of the protein increases the potential for oxidative damage, we investigated the effects of oxidation on misfolding/aggregation of SOD1 and ALS-associated SOD1 mutants. Zinc-deficient wild-type SOD1 and SOD1 mutants were extremely prone to form visible aggregates upon oxidation as compared with wild-type holo-protein. Oxidation of select histidine residues that bind metals in the active site mediates SOD1 aggregation. Our results provide a plausible model to explain the accumulation of SOD1 aggregates in motor neurons affected in ALS. ALS 1The abbreviations used are: ALS, amyotrophic lateral sclerosis; FALS, familial amyotrophic lateral sclerosis; SOD1, Cu/Zn superoxide dismutase; CCS, copper chaperone protein; ANS, 8-anilino-1-napthalene-sulfonic acid; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide is a fatal neuromuscular disease that presents as weakness, spasticity, and muscle atrophy. The disease is caused by selective degeneration of motor neurons in the brain, brainstem, and spinal cord. Although ALS presents mostly as a sporadic disease, a familial form of ALS is seen in ∼10% of cases. Twenty percent of familial ALS (FALS) cases are caused by point mutations in the SOD1 gene. More than 90 distinct amino acid mutations spread throughout the sequence of this 153-residue protein have been identified (1Cleveland D.W. Rothstein J.D. Nat. Neurosci. 2001; 2: 806-819Google Scholar). The finding that many FALS-associated SOD1 mutants possess full specific enzyme activity (2Borchelt D.R. Lee M.K. Slunt H.S. Guarnieri M. Xu Z.S. Wong P.C. Brown Jr., R.H. Price D.L. Sisodia S.S. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8292-8296Google Scholar) suggests that the disease is not caused by loss of normal dismutase activity. Further support for this idea has come from transgenic mice studies. Transgenic mice that harbor FALS-associated SOD1mutations develop ALS-like symptoms despite having greater than normal levels of SOD1 activity, including the normal complement of endogenous mouse SOD1 enzyme (3Bruijn L.I. Houseweart M.K. Kato S. Anderson K.L. Anderson S.D. Ohama E. Reaume A.G. Scott R.W. Cleveland D.W. Science. 1998; 281: 1851-1854Google Scholar). Furthermore, SOD1 knockout mice do not develop ALS-like symptoms. Thus, it has been proposed that mutations in SOD1 cause FALS by a gain, rather than a loss, of function (reviewed in Ref. 1Cleveland D.W. Rothstein J.D. Nat. Neurosci. 2001; 2: 806-819Google Scholar). One proposed gain of function involves free radical generation by SOD1. Because the dismutase action of SOD1 runs in a reversible catalytic cycle with a number of different possible substrates (4Yim M.B. Kang J. Yim H. Kwak H. Chock P.B. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5709-5714Google Scholar, 5Wiedau-Pazos M. Goto J.J. Rabizadeh S. Gralla E.D. Roe J.A. Valentine J.S. Bredesen D.E. Science. 1996; 271: 515-518Google Scholar, 6Liochev S.I. Fridovich I. Arch. Biochem. Biophys. 2002; 402: 166-171Google Scholar), under some conditions, SOD1 may catalyze the reverse reaction and generate radical species. It has been proposed that certain FALS-associated SOD1 mutants have lower K m values for hydrogen peroxide in the reverse reaction and therefore possess greater free radical generating activity than do wild-type enzymes. This makeup ultimately allows a greater number of cytotoxic peroxidation reactions to occur in these mutants (4Yim M.B. Kang J. Yim H. Kwak H. Chock P.B. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5709-5714Google Scholar, 5Wiedau-Pazos M. Goto J.J. Rabizadeh S. Gralla E.D. Roe J.A. Valentine J.S. Bredesen D.E. Science. 1996; 271: 515-518Google Scholar). The exact species responsible for oxidative damage, however, has recently come under question. Fridovich and co-worker (7Liochev S.I. Fridovich I. J. Biol. Chem. 2002; 277: 34674-34678Google Scholar) showed that the production of hydroxyl radicals would be negligible because of competition with bicarbonate ions for hydroxyl radicals bound to copper in SOD1. Another possible gain of function implicates the formation of zinc-deficient enzyme as the common toxic entity derived from all mutants. One property shared by many FALS-associated SOD1 mutants is a decreased affinity for Zn2+ (8Lyons T.J. Hongbin L. Joy J.G. Nersissian A. Roe J.A. Graden J.A. Café C. Ellerby L.M. Bredesen D.E. Gralla E.B. Valentine J.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12240-12244Google Scholar, 9Crow J.P. Ye Y.Z. Strong M. Kirk M. Barnes S. Beckman J.S. J. Neurochem. 1997; 69: 1936-1944Google Scholar). It has been proposed that reduced Zn2+ binding destabilizes the structure of SOD1, increasing the rate of abnormal reduction of bound Cu2+ to Cu+ by intracellular reducing agents. This reduced form of SOD1 could then catalyze the reverse enzymatic reaction and become a net producer of superoxide anion. In the absence of a well defined protein fold, the electrostatic gradient that is normally present in SOD1 (10Klapper I. Hagstrom R. Fine R. Sharp K. Honig B. Proteins. 1986; 1: 47-59Google Scholar) does not exist to prevent diffusion of the resultant radical anion. Therefore, in the presence of nitric oxide, which reacts five times faster with superoxide than does SOD1 itself, zinc-deficient SOD1 becomes a net producer of peroxynitrite (11Estevez A.G. Crow J.P. Sampson J.B. Reiter C. Zhuang Y. Richardson G.J. Tarpey M.M. Barbeito L. Beckman J.S. Science. 1999; 286: 2498-2500Google Scholar). Thus, the zinc-deficient SOD1 hypothesis maintains that peroxynitrite is the final mediator of oxidative neuronal injury and works by either nitrating and/or oxidizing critical cellular targets. Active site copper plays a critical role in both of the proposed mechanisms for a gain of function of FALS-associated SOD1 mutants described above. A recent study that used transgenic mice that expressed FALS-associated SOD1 mutants but lacked the gene for the copper chaperone protein (CCS) investigated whether alterations in copper loading would affect disease pathobiology (12Subramanium J.R. Lyons E.W. Liu J. Bartnikas T.B. Rothstein J. Price D.L. Cleveland D.W. Gitlin J.D. Wong P.C. Nat. Neurosci. 2002; 5: 301-307Google Scholar). CCS facilitates the incorporation of Cu2+ into SOD1 in vivo (13Culotta V.C. Klomp L.W. Strain J. Casareno R.L. Krems B. Gitlin J.D. J. Biol. Chem. 1997; 272: 23469-23472Google Scholar,14Wong P.C. Waggoner D. Subramaniam J.R. Tessarollo L. Bartnikas T.B. Culotta V.C. Price D.L. Rothstein J. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2886-2891Google Scholar), and copper is essential for normal dismutase activity as well as for any gained functions that are oxidant-mediated. The transgenic study found that knocking out the CCS gene reduced copper incorporation into FALS-associated SOD1 mutants; however, disease onset and progression in the mouse model was largely unaffected. The fact that 20–30% of total SOD1 activity remained in the absence of CCS prevents this study (12Subramanium J.R. Lyons E.W. Liu J. Bartnikas T.B. Rothstein J. Price D.L. Cleveland D.W. Gitlin J.D. Wong P.C. Nat. Neurosci. 2002; 5: 301-307Google Scholar) from completely ruling out copper-mediated mechanisms of toxicity in SOD1 transgenic mice, but it does suggest that other mechanisms such as protein aggregation may play an important role in the overall cytotoxicity. Another dramatic gain of function exhibited by SOD1 mutants is a very high propensity to aggregate (3Bruijn L.I. Houseweart M.K. Kato S. Anderson K.L. Anderson S.D. Ohama E. Reaume A.G. Scott R.W. Cleveland D.W. Science. 1998; 281: 1851-1854Google Scholar, 15Koide T. Igarashi S. Kikugawa K. Nakano R. Inuzuka T. Yamada M. Takahashi H. Tsuji S. Neurosci. Lett. 1998; 257: 29-32Google Scholar). COS7 cells transfected with FALS-associated SOD1 mutants produce cytoplasmic aggregates composed of the SOD1 mutant protein; transfections of wild-type SOD1, on the other hand, do not cause such cellular alterations (15Koide T. Igarashi S. Kikugawa K. Nakano R. Inuzuka T. Yamada M. Takahashi H. Tsuji S. Neurosci. Lett. 1998; 257: 29-32Google Scholar). A number of transgenic mice, all of which expressed a particular FALS-associated SOD1 mutant and co-expressed different amounts of wild-typeSOD1, were shown to uniformly exhibit intracellular SOD aggregation in neural tissue as well as ALS-like symptoms regardless of whether wild-type SOD1 expression was elevated or eliminated (3Bruijn L.I. Houseweart M.K. Kato S. Anderson K.L. Anderson S.D. Ohama E. Reaume A.G. Scott R.W. Cleveland D.W. Science. 1998; 281: 1851-1854Google Scholar). SOD1 aggregates have been proposed to produce toxicity by interference with normal proteasome function (16Johnston J.A. Dalton M.J. Gurney M.E. Kopito R.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12571-12576Google Scholar) or by altering chaperone (e.g. heat shock protein 70) (17Bruening W. Roy J. Giasson B. Figlewicz D.A. Mushynski W.E. Durham H.D. J. Neurochem. 1999; 72: 693-699Google Scholar, 18Okado-Matsumoto A. Fridovich I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9010-9014Google Scholar) activity. In the present study, we sought to elucidate physiologically relevant environmental factors that may trigger aggregation of SOD1 in motor neurons. SOD1 aggregates seen in ALS patients and transgenic mouse models are limited to neural tissue (motor neurons and, occasionally, neighboring astrocytes) and are not seen in other cell types. Given that SOD1 is present in high concentrations in all cells, an environmental factor must exist within motor neurons that induces aggregation specifically in this cell type. Two differences between SOD1 molecules in motor neurons and other cells are its long half-life and higher concentration. Concentration of SOD1 is greater in motor neurons than in other neurons and glial cells, and it is found not only in the cell body of motor neurons but also within axons and nerve termini (19Pardo C.A. Xu Z. Borchelt D.R. Price D.L. Sisodia S. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 954-958Google Scholar). To reach the nerve termini, SOD1 is transported through the axon by using the slow component b of the anterograde axonal transport system (20Borchelt D.R. Wong P.C. Becher M.W. Pardo C.A. Lee M.K. Xu Z.S. Thinakaran G. Jenkins N.A. Copeland N.G. Sisodia S.S. Cleveland D.W. Price D.L. Hoffman P.N. Neurobiol. Dis. 1998; 5: 27-35Google Scholar), which has a rate of 2–8 mm/day. Thus, the transport time for motor neurons with a meter-long axon could approach 500 days, and the life span of the protein must exceed the transport time. The long life span of this protein increases the chances of oxidative modification by reactive oxygen species; one possible byproduct of oxidative modification is induction of protein aggregation. The greater life span of SOD1 in motor neurons means that it would have more opportunity to accumulate oxidative modifications and to be altered in ways that could increase its own production of abnormal oxidants (i.e. to become zinc-deficient and catalyze the formation of peroxynitrite). 2M. J. Strong, W. L. Strong, B. P. He, M. M. Sopper, and J. P. Crow, personal communication.Oxidative damage to SOD1, either self-induced or the result of other oxidant sources, in turn may trigger aggregation. In support of this hypothesis, markers of oxidative damage were shown to be significantly elevated in neural tissue of ALS patients as compared with controls (21Bowling A.C. Schulz J.B. Brown Jr., R.H. Beal M.F. J. Neurochem. 1993; 61: 2322-2325Google Scholar, 22Ferrante R.J. Browne S.E. Shinobu L.A. Bowling A.C. Baik M.J. MacGarvey U. Kowall N.W. Brown Jr., R.H. Beal M.F. J. Neurochem. 1997; 69: 2064-2074Google Scholar). To explore the possibility that oxidation triggers SOD1 aggregation, we examined the effects of oxidation on fully metallated wild-type SOD1 (holo-SOD1), on zinc-deficient SOD1, and on four SOD1 mutants. Wild-type Cu-Zn SOD1 from human erythrocytes was obtained from Sigma. Mutant and zinc-deficient SODs were prepared as described previously (9Crow J.P. Ye Y.Z. Strong M. Kirk M. Barnes S. Beckman J.S. J. Neurochem. 1997; 69: 1936-1944Google Scholar). Oxidation reactions consisted of 10 μm SOD1, 4 mm ascorbic acid, and 0.2 mm CuCl2in 10 mm Tris, 10 mm acetate buffer, whereas control reactions were 10 μm SOD1 in buffer. Reactions were incubated at 37 °C for 48 h. The pH was 7.0 unless stated otherwise. To readily recognize inhibition of SOD1 aggregation, the most aggregation-prone SOD1 species (zinc-deficient SOD1) was used. SOD1 aggregation mixtures (10 μm SOD1, 4 mm ascorbate, 0.2 mmCuCl2, 10 mm Tris acetate, pH 7) were incubated with 2 mm EDTA, 10 mm mannitol, or 10 mm DMPO as probes for the reactive oxygen species. Anaerobic conditions were achieved by degassing all solutions and oxidizing them under vacuum (37 °C) in a vacuum hydrolysis tube (Pierce). Light scattering measurements were made with a Photon Technology International QM-1 fluorescence spectrophotometer. Excitation and emission wavelengths were set to 350 nm (bandpass = 4 nm). All images were obtained by using a Digital Instruments NanoScope III© atomic force microscope. Samples were deposited and dried onto freshly cleaved mica under positive pressure. Contact-mode images were obtained by using a Si3N4 tip (Digital Instruments) with a nominal spring constant of 0.12 N/m. were on 10 of SOD1 with acetate and examined in an acid of and control SOD1 were by using the which acid hydrolysis and either with or with were by using a to a by were by and ions were and in a was with 10 μm SOD1 in 10 mm Tris acetate was incubated for with μm or μm emission at and SOD1 aggregates were to a final of μm and incubated with μm for and visible Zinc-deficient SOD1 aggregates were for at and the was and with mm buffer, pH were then by and were on an model at used oxidation with and ascorbic acid to generate reactive oxygen species because of the of this oxidation is the of hydroxyl radicals under normal conditions E.R. Chem. 1997; Scholar), and it is important under conditions of oxidative E.R. Biochem. 1993; Scholar). The concentrations of ascorbic acid used in this study are well within the normal found in neurons and glial cells M. Neurosci. 2000; Scholar). examined the effects of oxidation on different ALS-associated mutants of and as well as a mutant that has decreased affinity L. I. D.E. J. Biochem. Scholar) and as a model of zinc-deficient SOD1. is the most common that FALS, a form of FALS (1Cleveland D.W. Rothstein J.D. Nat. Neurosci. 2001; 2: 806-819Google Scholar), and is the mutant most used for the transgenic mouse model of ALS. examined the of oxidation on the zinc-deficient form of wild-type SOD1, because this species has been in with ALS (11Estevez A.G. Crow J.P. Sampson J.B. Reiter C. Zhuang Y. Richardson G.J. Tarpey M.M. Barbeito L. Beckman J.S. Science. 1999; 286: 2498-2500Google Scholar) and because it to produce superoxide and hydrogen peroxide that at a oxidation of of the SOD1 mutants and zinc-deficient wild-type SOD1 induces the formation of aggregates that The zinc-deficient protein the most aggregation reaction and, the that an form of FALS, the of aggregate Oxidation of wild-type SOD1 under conditions not the formation of aggregates by scattering (i.e. visible aggregates nm in the of zinc-deficient SOD1, aggregates not form in control that lacked The of aggregate in control of zinc-deficient protein suggests that this form of the protein has an aggregation The aggregation reaction distinct pH with reduced aggregation at pH pH has been in the aggregation of human in which oxidation of a for the pH M. 2000; Scholar). of the oxidation reaction under conditions or in the presence of aggregation and that copper and oxygen are an for aggregation On the other hand, the of the free radical and DMPO not aggregation results have been obtained with aggregation of both human S. C. 1995; Scholar) and protein J.R. D. G. Stadtman E.R. R.L. Proc. Natl. Acad. Sci. U. S. A. 2001; Scholar). The to free radical and the pH of the aggregation are with the oxidation This a binding site that is in to the modification E.R. Chem. 1997; Scholar). In this of oxidation very residues are acid was on wild-type protein and on and zinc-deficient SOD1 The most of the amino acid of both of protein was the loss of histidine acid that of the histidine residues of the SOD1 were It is that oxidation of to of histidine residues to or E.R. Chem. 1997; Scholar). Because the and do not to be altered by it is that have been largely to Further support for the to was obtained by of wild-type SOD1 by The of were by which is with the formation of that both and contain an these residues are at the and copper binding of SOD1 of amino acid of to control in a amino of of of SOD1 in a of of of SOD1 The results that oxidation of select residues induces and aggregation of SOD1. the do these in aggregates aggregates seen in of ALS by and have shown them to be a of ALS and distinct from the and seen in disease and the intracellular seen in disease K. S. T. Y. Neurosci. Lett. Scholar, Scholar, D. 1993; Scholar). In ALS are not by the K. S. T. Y. Neurosci. Lett. Scholar). the seen in COS7 cells that ALS mutants of SOD1 (15Koide T. Igarashi S. Kikugawa K. Nakano R. Inuzuka T. Yamada M. Takahashi H. Tsuji S. Neurosci. Lett. 1998; 257: 29-32Google Scholar), transgenic mouse models of ALS (3Bruijn L.I. Houseweart M.K. Kato S. Anderson K.L. Anderson S.D. Ohama E. Reaume A.G. Scott R.W. Cleveland D.W. Science. 1998; 281: 1851-1854Google Scholar, L.I. Becher M.W. Lee M.K. Anderson K.L. Jenkins N.A. Copeland N.G. Sisodia S.S. Rothstein J.D. Borchelt D.R. Price D.L. Cleveland D.W. 1997; Scholar), and ALS patients S. M. Y. K. Takahashi K. Ohama E. J. 1996; Scholar, S. H. K. E. Kato M. A. Nakano I. K. Ohama E. J. 1997; Scholar, S. S. K. A. Nakano I. M. Kato M. K. Ohama E. 1999; 97: Scholar, S. K. S. R. Cleveland D.W. Liu J. A. M. Kato M. Nakano I. S. K. Ohama E. 2001; Scholar) are all composed of a of aggregates and some as compared with the seen in D.A. H. A. M. Proc. Natl. Acad. Sci. U. S. A. Scholar). Our atomic force of aggregates by oxidation of zinc-deficient SOD1 aggregates μm that were composed of μm of in vivo S. M. Y. K. Takahashi K. Ohama E. J. 1996; Scholar, S. H. K. E. Kato M. A. Nakano I. K. Ohama E. J. 1997; Scholar, S. S. K. A. Nakano I. M. Kato M. K. Ohama E. 1999; 97: Scholar, S. K. S. R. Cleveland D.W. Liu J. A. M. Kato M. Nakano I. S. K. Ohama E. 2001; Scholar). of wild-type protein at pH a number of aggregates that could be by aggregates were composed of aggregates with aggregates that were nm in and long aggregates are than the by the which are in D.A. H. A. M. Proc. Natl. Acad. Sci. U. S. A. Scholar). binding with and as well as were also used to whether the SOD1 aggregates A of fluorescence was with the aggregates from zinc-deficient SOD1 4 however, the fluorescence seen with is of higher H. Sci. 1993; 2: Scholar). On binding to very increase was seen in or which would have been the aggregates in fact been W.E. 1999; Scholar). This of increase is in with the of to bind SOD in vivo K. S. T. Y. Neurosci. Lett. Scholar). The of SOD1 a on aggregation the of SOD1 aggregates rather than the of Thus, it that oxidative damage of SOD1 results in and aggregation, the resultant aggregates do not to be the of the aggregates in in this study with that of SOD1 in ALS models and To whether to aggregation of zinc-deficient SOD1 and SOD1 mutants results from an altered binding were on protein binding is a of in Zinc-deficient SOD1 bound the most ANS, wild-type SOD1 not any and the SOD1 mutants of binding It is that the bound in SOD1 the structure of the active site and is not in and of destabilizes the enzyme J.A. Valentine J.S. Roe J.A. A. Brown Jr., R.H. J. Biol. Chem. 2002; 277: Scholar). The binding that in to an in that to of is also with The levels of binding with the SOD1 mutants may have from an altered of the protein in as has been by the of mutant SOD1 M. Nersissian Gralla E.B. Valentine J.S. D. Sci. 1998; Scholar). the binding may result from in the of the in which mutant that the binding contain of metallated of which could be have shown that zinc-deficient SOD1, a mutant with binding and FALS-associated SOD1 is more to aggregation than the fully metallated wild-type with the long half-life of SOD1 in motor neurons and the high levels of oxidative damage that are to occur in neural of ALS patients (21Bowling A.C. Schulz J.B. Brown Jr., R.H. Beal M.F. J. Neurochem. 1993; 61: 2322-2325Google Scholar), provide a possible for the SOD1 aggregates in ALS. Although it to be whether the SOD1 aggregates are is that protein aggregates exhibit a toxicity that is of the function of the protein in its M. E. L. J. G. M. 2002; Scholar). Our are also with the recent model by and Fridovich A. Fridovich I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9010-9014Google Scholar) in which factors such as heat shock are by abundant such as SOD1 or other by to and for and and for and Zhuang for and to produce SOD1 that to studies. also I. Fridovich for critical of this

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