articles Published online: 11 March 2002, DOI: /nn823

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1 Mutant SOD1 auses motor neuron disease independent of opper haperone mediated opper loading Jamuna R. Suramaniam 1, W. Ernest Lyons 1, Jian Liu 2, Thomas B. Bartnikas 3, Jeffrey Rothstein 4,5, Donald L. Prie 1,4,5, Don W. Cleveland 2, Jonathan D. Gitlin 3 and Philip C. Wong 1,5 1 Department of Pathology, The Johns Hopkins University Shool of Mediine, 558 Ross Researh Building, 720 Rutland Avenue, Baltimore, Maryland 21205, USA 2 Ludwig Institute of Caner Researh, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA 3 Department of Pediatris, Washington University Shool of Mediine, St Louis, Missouri 63110, USA 4 Departments of Neurology and 5 Neurosiene, The Johns Hopkins University Shool of Mediine, Baltimore, Maryland 21205, USA Correspondene should e addressed to P.C.W. (wong@jhmi.edu) Pulished online: 11 Marh 2002, DOI: /nn823 Copper-mediated oxidative damage is proposed to play a ritial role in the pathogenesis of Cu/Zn superoxide dismutase (SOD1) linked familial amyotrophi lateral slerosis (FALS). We tested this hypothesis y alating the gene enoding the opper haperone for SOD1 (CCS) in a series of FALSlinked SOD1 mutant mie. Metaoli 64 Cu laeling in SOD1-mutant mie laking the CCS showed that the inorporation of opper into mutant SOD1 was signifiantly diminished in the asene of CCS. Motor neurons in CCS / mie showed inreased rate of death after faial nerve axotomy, a response doumented for SOD1 / mie. Thus, CCS is neessary for the effiient inorporation of opper into SOD1 in motor neurons. Although the asene of CCS led to a signifiant redution in the amount of opper-loaded mutant SOD1, however, it did not modify the onset and progression of motor neuron disease in SOD1-mutant mie. Hene, CCS-dependent opper loading of mutant SOD1 plays no role in the pathogenesis of motor neuron disease in these mouse models. Amyotrophi lateral slerosis (ALS), a progressive, fatal neurodegenerative disease, is haraterized y seletive loss of motor neurons in the spinal ord, rain stem and motor ortex. The sporadi and familial forms of the disease have similar linial and pathologial features. Up to 10% of ases of ALS are familial (FALS), and 25% of these are linked to mutation in Cu/Zn superoxide dismutase, an antioxidant enzyme that protets ells against superoxide toxiity 1,2. A variety of studies, inluding investigations of SOD1-mutant mie that develop motor neuron disease 3 6, estalished that mutant SOD1 auses motor neuron disease through gain of one or more toxi properties 7,8. However, the mehanism(s) wherey mutant SOD1 auses seletive motor neuron death is not yet known. It has een speulated that ell death ould e initiated y aerrant oxidative hemistry atalyzed y the opper atom ound in the mutant SOD1, espeially after loss of zin inding to SOD1 9. Two prominent proposed oxidative hemistries are opper-dependent (i) peroxidation to generate hydroxyl radial 10 or (ii) nitration of tyrosines using peroxynitrite generated from the reation of nitri oxide with superoxide 9,11. In its free form, opper is toxi, and there is virtually no free opper in ells 12. Normally, opper is imported into ells y a plasma memrane ound opper transporter 13,14 and then shuttled to intraellular target proteins y a series of opper haperones 15,16. This is estalished for SOD1 ased on the disovery in yeast of a opper haperone (LYS7) 16 that delivers opper to SOD1 y diret protein protein interation 17. Inativation of the CCS gene in mie has shown 18 that CCS is required for the effiient inorporation of opper into SOD1 in mammals. We have taken advantage of this latter finding to determine whether the inorporation of opper into SOD1 is involved in the pathogenesis of SOD1 mutant mediated motor neuron disease y generating a series of FALS-linked SOD1-mutant mie laking CCS. We show here that SOD1-mutant mie laking CCS have a seletive defet in opper inorporation into wild-type and mutant SOD1. In addition, faial nerve axotomy studies in CCS-defiient mie indiated that CCS is neessary for effiient opper loading into SOD1 in motor neurons. Importantly, we show that the asene of CCS did not impat on the onset and progression of motor neuron disease in a series of FALS-linked SOD1-mutant mie. Taken together, our results demonstrate that mutant SOD1 indues motor neuron disease independent of CCS-mediated opper inorporation into mutant SOD1. RESULTS Amounts of mutant SOD1 are independent of CCS Mie with targeted disruption of one CCS allele (CCS +/ mie) were rossred in two suessive steps with mie expressing either wild-type or mutant (G93A, G37R or G85R) SOD1. This nature neurosiene advane online puliation 1

2 generated ohorts of littermates of either wild-type or mutant (G93A, G37R or G85R) SOD1 mie with or without CCS. As the amounts of mutant SOD1 present orrelate with disease onset and survival 3,4, we first determined the amounts of mutant SOD1 in G37R, G93A or G85R SOD1 mie laking CCS. Immunolot analysis of spinal ord extrats showed that in CCS / mie the steady-state amounts of G37R, G93A or G85R SOD1 are similar to those in the respetive CCS +/+ mutant SOD1 mie (Fig. 1). Thus, the aumulation of mutant SOD1 in spinal ord and rain (data not shown) is independent of CCS. Cu loading into mutant SOD1 is redued without CCS To onfirm that the inorporation of opper into mutant SOD1 is dependent on CCS, we first determined the opper-dependent dismutase ativity of mutant SOD1 from spinal ord extrats. In oth the G37R and G93A mie, the enzyme ativity from spinal ord lysates was markedly redued in the asene of CCS as determined y an in situ gel assay 3 (Fig. 2a,) or a solution assay 19 (Fig. 2), despite the fat that amounts of mutant SOD1 are independent of CCS (Fig. 1). We did not detet any G85R-assoiated enzyme ativity using oth methods (Fig. 2a,). To onfirm that this redution in enzymati ativity is due to a defet in opper inorporation into the atalyti domain, as we a previously estalished for CCS defiient mie 18, and to determine whether opper is ound to any other region of mutant SOD1, we eleted to perform 64 Cu metaoli laeling studies. After injetion of 64 Cu into SOD1 mutant mie with or without CCS, we did not detet 64 Cu-laeled SOD1 in the spinal ords and rains of G37R;CCS / mie, whereas we deteted roust 64 Cu-SOD1 laeling in G37R;CCS +/+ and G37R;CCS +/ mie (Fig. 3a,). As the amount of mutant SOD1 present is at least 10-fold higher than that of endogenous SOD1 3, and 64 Cu-laeled Fig. 1. SOD1 protein levels are not affeted y the asene of CCS. Immunolot analysis of spinal ord extrats from either normal mie, mie expressing human wild-type or mutant (G93A, G37R or G85R) SOD1 with or without CCS. Top, CCS protein levels. Middle, SOD1 protein levels using an antiody that reognizes human and mouse SOD1 with equal affinity. (Upper and lower middle, longer and shorter exposures of the same immunolot, respetively.) Bottom, atin levels. SOD1 an e readily oserved in ontrol mie 18, our failure to detet 64 Cu-laeled SOD1 in mutant SOD1 mie laking CCS implies that there is at least a 90% redution in the inorporation of opper into mutant SOD1. In addition, to assess opper homeostasis in mutant SOD1 mie laking CCS, we examined oth the overall opper ontent and opper traffiking to eruloplasmin, a very sensitive indiator for aerrations in hepati opper status. Copper loading studies of eruloplasmin in G37R mutant mie laking CCS showed no signifiant differenes in opper inorporation into eruloplasmin as ompared to G37R mie with CCS (Fig. 3). In addition, the oxidase ativity of eruloplasmin in the G37R;CCS / mie was similar to that in the G37R;CCS +/+ mie (Fig. 3). Atomi asorption spetrometri analysis showed no signifiant differenes in opper ontent in the rain etween mutant SOD1 mie with and without CCS (Fig. 3d). Thus, these results indiate that the asene of opper loading was seletive for wild-type and mutant SOD1 and that opper homeostasis was normal in the CCS / animals. Taken together, these results estalish that CCS is neessary for the effiient inorporation of opper into mutant SOD1 in mie expressing ALS-linked SOD1 mutants. It still is possile, however, that the residual SOD1 ativity (Fig. 2a ) seen in SOD1-mutant mie laking CCS is the result of seletive, CCSindependent opper loading into SOD1 in motor neurons and that in tissue extrats ontaining the ontents of several ell types, our methods are not sensitive enough to assess this magnitude Fig. 2. Redued SOD1 ativity in mutant SOD1 expressing mie laking CCS. (a) SOD1 ativity gel of 30 µg protein extrats from spinal ords of normal (Ntg), human wildtype SOD1-overexpressing (WT), G37R and G93A mutant SOD1 mie with (+/+) or without CCS ( / ). Lanes 1 and 2, endogenous mouse SOD1 ativity in the wild-type and CCS-null mie. Ativity standards (Sigma; U, units of enzyme ativity) are provided y different amounts of purified human SOD1 (lanes 10 14). Lane 9, apo-sod1 (300 ng, without opper and zin). Bottom, similar analysis as top, exept for mie expressing G85R mutant SOD1. () CCS-dependent redution in gel SOD1 ativity assay in G93A (n = 3; redution, 84.5%) and G37R mie (n = 3; redution, 85%). () CCS-dependent redution in solution SOD1 ativity assay (n = 3) for nontransgeni mie, G85R, G93A, G37R and human wild-type SOD1-expressing mie. 2 nature neurosiene advane online puliation

3 Fig. 3. Diminished 64 Cu inorporation into the mutant SOD1 protein in the asene of CCS. (a) Protein extrats of spinal ord were otained from CCS +/+ ;G37R, CCS +/ ;G37R and CCS / ;G37R mie after 64 Cu injetion. Top, 64 Cu inorporation into G37R SOD1; middle, SDS- PAGE gel of spinal ord, immunolotted with CCS antiody; ottom, same as middle, immunolotted with human/mouse SOD1 antiody. () Speifiity of SOD1 protein. Protein extrats of spinal ord from wild-type and SOD1 / mie were resolved in a parallel native PAGE as in (a), followed y immunolot analysis using h/msod1 antiody. () Normal opper inorporation and ativity of eruloplasmin (Cp) in the presene or asene of CCS. Spinal ord lysate of the G37R CCS +/+ and CCS / mie were used. Left, 64 Cu inorporation in SOD1(top), a parallel G37R SOD1 ativity native PAGE to show the speifiity of SOD1 (top middle) and immunolots proed for CCS (ottom middle) and human/mouse SOD1 (ottom). Right, 64 Cu inorporation into Cp of the plasma (top), ativity of Cp (middle) and immunolot for Cp protein levels (ottom) in G37R mie in the presene and asene of CCS or in mie laking Cp (Cp / ) in the plasma. (d) Total opper ontent is unaltered in the asene of CCS in non-transgeni, mutant G37R/G85R rain tissue. Copper ontent (n = 3) is expressed as mirograms of opper per gram wet weight of rain tissue. of SOD1 ativity in these ells. As inreased motor neuron death ours after faial nerve axotomy in SOD1 / mie relative to normal mie 20, a similar vulneraility to axotomy would e expeted in CCS / mie if opper inorporation into SOD1 in motor neurons is defiient. To test if CCS is neessary for SOD1 ativity in motor neurons, uniased stereology 21 was used to assess the magnitude of death after axotomy of motor neurons of the faial nulei 22 in the rain stem of CCS / mie as ompared to CCS +/+ littermates. The CCS / mie showed an inreased ( 20%) loss of motor neurons (Fig. 4) ompared to CCS +/+ mie. This is the same magnitude of inreased sensitivity to axotomy-indued motor neuron death oserved in faial nulei of SOD1 / mie as ompared to ontrols 20. These results provide evidene that CCS is required for effiient inorporation of opper into SOD1 within motor neurons. a d Mutant SOD1 indued disease proeeds without CCS To determine the influene of CCS on the onset and progression of motor neuron disease in SOD1-mutant mie, we ompared the time of disease onset and the length of survival of G37R, G93A or G85R SOD1 mie to these parameters in the respetive SOD1-mutant mie laking CCS. Onset of disease (Tale 1) was not signifiantly different in the G37R or G93A SOD1-mutant mie laking CCS as ompared to the respetive mutant SOD1 in either the CCS +/+ or CCS +/ akground (Fig. 5a,). In addition, the mean survival time of the G37R, G93A or G85R mie (Tale 1) was similar to that of the respetive SOD1-mutant mie laking CCS (Fig. 5 e). Therefore, CCS-dependent opper loading has no impat on the onset and progression of motor neuron disease in SOD1-mutant mie. To onfirm that the SOD1-mutant mie laking CCS showed the features of motor neuron disease seen in the SOD1-mutant mie, we arried out oth linial and neuropathologial analysis of these ohorts of mie. Clinially, G37R, G93A or G85R mie laking CCS were indistinguishale from the respetive SOD1- mutant mie expressing CCS. Affeted mie showed impaired extension of hind lims when suspended y the tail, tremors, altered gait, oarse oat due to improper grooming, musle wasting along the flanks, progressive slowness in the movement, and, near the end stage of the disease, inaility to reah for food or turn over when plaed on the ak. Neuropathologially, the SOD1-mutant mie with or without CCS in the late stage of disease showed severe degeneration and loss of motor neurons of the spinal ord (Fig. 6a d). In mie Fig. 4. Inreased axotomy-indued death of motor neurons of faial nulei in CCS / mie. Perent motor neuron survival is the ratio of surviving motor neurons of the axotomized to the numer determined in the ontralateral non-axotomized faial nuleus of CCS +/+ (n = 7) and CCS / (n = 7) mie. There is 20% redution in neuronal survival after axotomy in the CCS / mie ompared to normal mie (CCS +/+, 84 ± 5%; CCS /, 66 ± 7; p < 0.001; unpaired Student t-test). Error ars, s.e.m. nature neurosiene advane online puliation 3

4 Tale 1. Motor neuron disease onset and proaility of survival of mutant SOD1 mie in the presene or asene of CCS. Mutant SOD1 Onset of motor neuron disease (days) Mean life span (days) CCS +/+ CCS +/ CCS / CCS +/+ CCS +/ CCS / G37R 108 ± 13 (n = 4) 110 ± 0 (n = 3) 163 ± 17 (n = 10) 155 ± 15 (n = 15) 146 ± 15 (n = 11) G93A 98 ± 10 (n = 5) 96 ± 6 (n = 2) 142 ± 13 (n = 13) 136 ± 10 (n = 33) 134 ± 9 (n = 10) G85R ND ND 310 ± 45 (n = 9) 324 ± 66 (n = 13) 353 ± 66 (n = 10) ND, not determined; n, numer of mie. expressing G37R or G93A SOD1, there was evidene of vauolation of perikarya, axons and dendrites. When stained for SOD1 with an antiody reognizing mouse or human SOD1 with equal avidity, SOD1 was found in the ytoplasm surrounding the vauoles (Fig. 6g,h). Motor neuron ell odies, axons and dendrites also harored aggregates of SOD1, uiquitin and phosphorylated neurofilament protein (Fig. 6g l). Within these spinal ord setions, reative astrogliosis was deteted on the asis of GFAP immunoreativity (Fig. 6m,n). In the mutant G85R SOD1 mie laking CCS, there was severe degeneration of motor neurons in the spinal ord (Fig. 6d) and aggregates ontaining SOD1, uiquitin, phosphorylated neurofilament protein and reative astrogliosis (data not shown). In mutant mie with CCS, CCS was present in the motor neurons and astroytes (data not shown) and oasionally in the neuropil, and CCS was seen in aggregates (Fig. 6e). As expeted, in the CCS / spinal ord tissue, no CCS immunoreativity was oserved (Fig. 6f), ut the asene of CCS did not aolish the other harateristi features of the neuropathology. At end stage of disease, SOD1, uiquitin and phosphorylated neurofilament aggregates were oserved in the spinal ord of all three lines of SOD1-mutant mie (G37R, G93A and G85R) irrespetive of the presene or asene of CCS. Taken together, these results estalish that CCS- a d mediated opper loading does not partiipate in the pathogenesis of motor neuron degeneration indued y mutant SOD1. DISCUSSION Whether an oxidative mehanism involving opper underlies the pathogenesis of ALS linked to mutant SOD1 is still a sujet of deate 23. In vitro studies showing that either wild-type or mutant Zn-defiient SOD1 an ause peroxynitrite-mediated death of motor neurons in ulture provide support for suh a role of opper 9. However, our findings demonstrating that a series of SOD1- mutant mie laking CCS develop motor neuron disease provides ompelling in vivo evidene that CCS-dependent opper loading onto SOD1 is not required for motor neuron disease mediated y mutant SOD1. It is also possile that suh protetive effet of loking opper delivery to mutant enzymes may e offset y deleterious effets of as-yet-unidentified targets that may require CCS for opper loading. However, our demonstration that opper inorporation into eruloplasmin is unaltered in SOD1- mutant mie laking CCS (Fig. 3) and our oservation that CCS-null mie showed no anormalities in opper uptake, distriution or loading into other uproenzyme(s) leads us to elieve it unlikely that other ritial CCS-dependent targets exist. Although it ould e argued that the apparent (10 20%) residual SOD1 ativity oserved in vitro from analysis of spinal ord extrats of G37R or G93A SOD1 mie laking CCS (Fig. 2a,) reflets some CCS-independent opper inorporation, diret in vivo laeling with radioative opper showed undetetale opper loading even onto mutant SOD1-G37R, whih was previously reported to retain full dismutase ativity 24 although the failure to lael the mutant enzyme might reflet a stale pool of holosod1 that is insensitive to opper exhange. In addition, previous studies in yeast have shown that G85R SOD1 (unlike other human SOD1 mutants) does not omplement lysine auxotrophy in sod1 /, lys7 / yeast ells 25, indiating that opper annot e effiiently loaded e Fig. 5. Asene of CCS does not affet the onset of motor neuron disease or lifespan in G37R, G93A and G85R mutant mie. (a, ) Disease onset is not altered y the asene of CCS. The onset of disease is determined y motor funtion defiit seen in rotorod performane in G37R (a) and G93A () SOD1 mie in the presene (+/+ and +/ ) and asene ( / ) of CCS. Motor funtions were tested on the rotorod at an aelerated speed from 5 to10 rpm for 7 minutes. G37R mie, +/+ and +/ (n = 4; lak irles); / (n = 3; lak squares); G93A mie, +/+ and +/ (n = 5; lak irles); / (n = 2; lak squares). ( e) The lifespan of mutant SOD1 mie is not altered y the asene of CCS. Kaplan Meyer survival urves of G93A, G37R and G85R SOD1 mie in the presene (+/+ and +/, red and lue, respetively) and asene ( /, green) of CCS. G37R, +/+ (n = 10); +/ (n = 15); / (n = 11); G93A, +/+ (n = 13); +/ (n = 33); / (n = 10); G85R, +/+ (n = 9); +/ (n = 13); / (n = 10). 4 nature neurosiene advane online puliation

5 a e f Fig. 6. Asene of CCS does not alter neuropathologial anormalities in end-stage G37R and G93A mutant SOD1 mie. Ventral horn of lumar spinal ord setions stained with hematoxylin and eosin (a d) revealed no gross differenes in histologial strutures in G37R and G85R mie with or without CCS (CCS +/+, CCS / ). Sale ar, 30 µm. Arrowhead, vauoles in G37R mutant SOD1 mie. Immunoytohemial analysis of the ventral horn stained with antiodies to CCS (e, f), SOD1 (g, h), uiquitin (i, j); phosphorylated neurofilament (P-NF; k, l); and GFAP (m, n). A surviving motor neuron with vauoles is indiated y a lue arrow in (f). Astrogliosis was revealed y GFAP immunostaining (m, n). Sale ar, 12.5 µm. onto the G85R SOD1 in the asene of CCS. Thus, given that the intraellular free opper level is very limiting 12, it seems most likely that part or all of the apparent in vitro ativity of the G37R and G93A mutants in the asene of CCS may arise from opper aquisition y SOD1 during tissue homogenization or through another CCSindependent pathway. In addition, we found that motor neurons themselves were dependent in vivo on CCS for opper inorporation into SOD1, as faial motor neurons in CCS / mie had a similarly inreased sensitivity to death after faial nerve injury as did those from the SOD1 defiient mie (Fig. 4). Even if we assume that there exists in the ell a CCS-independent pathway for loading opper onto SOD1 that may e responsile for the opper-mediated mutant SOD1 toxiity, CCS deletion in SOD1-mutant mie should favor an inrease in CCSindependent opper loading. Our findings that disease onset and progression are not aelerated in SOD1-mutant mie laking CCS are inonsistent with the view that suh a CCSindependent opper loading pathway plays a ruial role in disease pathogenesis in the SOD1-mutant mie. A strong predition onerning the hypothesis of oppermediated oxidative damage is that marked redution of opper inorporation into SOD1 (to as little as 20% of normal) would signifiantly delay motor neuron disease onset or inrease lifespan, or oth. However, we did not see suh an outome. Even though in vitro ell ulture studies have suggested that oppermediated toxiity, partiularly after loss of zin from SOD1, plays a ruial role in disease indued y mutant SOD1 9,26, we found no differenes in the linial features of disease in the asene of CCS-dependent opper loading. Reent studies also provide evidene that is inonsistent with the possiility that peroxynitrite is a primary mediator of neurotoxiity in mutant SOD Although initial studies 10,29,30 indiated that a opper-dependent mutant SOD1 indued peroxidase reation ould mediate toxiity, susequent reports provide evidene against suh a view 31,32. d g i k m It has also een proposed that opper ound to adventitious sites of mutant SOD1 ould indue opper-mediated toxiity 33,34. Although our findings doumenting that signifiant diminution in opper loading to SOD1 did not ameliorate motor neuron disease in mutant SOD1 laking CCS are inonsistent with the view that opper-dependent peroxidase mediated toxiity plays a major role in the degeneration of motor neurons, they do not formally rule out suh an hypothesis. Thus, we onlude that CCS-dependent aerrant opper hemistry is not involved in the pathogenesis of mutant SOD1 indued FALS. Although the moleular mehanisms underlying mutant SOD1 linked FALS remain unlear, several pathogeni mehanisms other than the opper hypothesis have een proposed. Mutant SOD1 ontaining aggregates have een impliated as partiipating in the pathogenesis of SOD1-linked FALS Although the diret role of these aggregates in disease pathogenesis is not yet lear, the identifiation of mutant SOD1- ontaining omplexes as well as uiquitin-positive inlusions as early hanges in mutant SOD1 mie 38,39 supports the importane of mutant SOD1 assoiated aggregates in the pathogenesis of SOD1-linked FALS. METHODS Mouse reeding. A two-step reeding strategy was employed to otain mie without CCS. Initially, the mutant or wild-type (WT) human SOD1 mie were rossred with CCS +/ mie to generate mutant or WT SOD1 h j l n nature neurosiene advane online puliation 5

6 with a single funtional CCS allele. These mie were then rossred with CCS +/ mie to generate mutant or WT SOD1 mie with two (CCS +/+ ), one (CCS +/ ) or no (CCS / ) funtional CCS alleles. All mie were housed at the Johns Hopkins University mouse faility under a 12 h light/12 h dark yle. Food and water were provided ad liitum, and all are was given in ompliane with National Institute of Health guidelines on the use of laoratory and experimental animals. Determination of SOD1 ativity. Spinal ord tissues from 2.5-month mie (n = 2 of eah genotype) or terminally sik mie (n = 2) were homogenized in 20 mm Tris (ph 7), 1% Triton X-100, 1 mm EDTA, 1 mm athouproinedisulfoni aid (BCS) and protease inhiitor mixture (50 µg/ml leupeptin, 50 µg/ml pepstatin, 10 µg/ml aprotinin and 0.25 mm phenylmethylsulfonyl fluoride). After entrifugation at 10,000g for 10 min, 30 µg of the supernatant was frationated y native polyarylamide gel eletrophoresis (PAGE) with 7.5% polyarylamide and with 100 µm EDTA in the gel and the running uffer, and the SOD1 ativity was determined using nitrolue tetrazolium redution. The SOD1 ativity was determined y densitometry. For immunolot analysis, 2 µg of the same spinal ord tissue extrats in SDS sample uffer were separated on 16% SDS-PAGE, lotted to nitroellulose memranes and proed with antiody against CCS or human or H/M SOD1 or antiody against atin. Copper loading studies in mie. 64 Cu inorporation was arried out in 2.5-month mie y intraperitoneal and intraranial injetion of 250 µci of 64 Cu as desried previously 18. Twenty-four hours later, mie were anesthetized and plasma was harvested y olleting lood from tail-vein leeds in EDTA and entrifuging it at 3,000g for 10 min at 4 C. Spinal ord tissue homogenate was prepared in 50 mm HEPES (ph 7.6), 250 mm NaCl, 0.1% Nonidet P-40, 5 mm EDTA and a protease inhiitor mixture (Sigma, St. Louis, Missouri). Protein extrats (75 µg), otained as supernatant after entrifugation at 25,000g, 10 min, 4 C, were separated y 10% native PAGE and exposed to PhosphorImager (Amersham, Sunnyvale, California) to detet holosod1 as desried previously 16,18. To detet 64 Cu laeling in eruloplasmin, 2.5 µl of plasma was resolved y 4 20% native PAGE followed y exposure to PhosphorImager 18. The oxidase ativity of eruloplasmin was determined y resolving 2.5 µl of plasma y 4 20% native PAGE and inuating the gels in 4.6 mm p-phenylenediamine/0.1 M sodium aetate, ph 5.2, at 37 C for 2 h in the dark 41. Ceruloplasmin protein level was determined y immunolot analysis with antisera to human eruloplasmin. Determination of opper ontent. Whole rains of 2.5-month nontransgeni and mutant SOD1 (G37R/G85R) mie with or without CCS were weighed and digested in onentrated nitri aid aording to previously estalished methods 42. Copper ontent was measured using a graphite furnae atomi asorption spetrophotometer (Perkin Elmer, Shelton, Connetiut). Faial nerve axotomy. Mie (3 months old) were anesthetized with a mixture of oxygen, nitri oxide, and 2% enflurane (Aott Las, Aott Park, Illinois) through a speially designed nose adapter, and were operated on under sterile onditions. The faial nerve was transeted just distal to the exit from the stylomastoid foramen and the proximal stump was ligated to prevent regeneration. After 5 weeks, the mie were deeply anesthetized with hloral hydrate and perfused with old phosphate-uffered saline (PBS), ph 7.4, followed y 4% paraformaldehyde in PBS. Brains were removed and post-fixed for 2 3 days and ryoproteted; then 40-µm-thik serial setions were ut on a freezing mirotome and stained with resyl violet. Total numer of motor neurons in the faial nulei was ounted using uniased stereologial methods. Determination of disease onset and progression. Onset time was assigned as the age at whih motor defiits were manifested as the inaility of the mie to remain on the rotorod (Columus Instruments, Columus, Ohio) for 7 min at different speeds (5 10 rpm, rpm, and rpm). Initially, the mie were trained for a total of 30 min on the rotorod at the three speeds (7 10 min per speed) every day for 5 days. Performane was then determined at intervals of 1 week or 3 4 days throughout disease progression. Terminal illness was defined y either the inaility of a mouse to right itself in 10 se when left on its ak or the inaility to reah for food or water. Histology and immunohistohemistry. Terminally sik mie were perfused with PBS followed y 4% paraformaldehyde in PBS. The spinal ord was removed, emedded in paraffin, setioned (10 µm), deparaffinized using standard protools and stained with hematoxylin and eosin or with speifi antiodies. Aknowledgements We thank G. Cristostomo for support in histology, E. Corpus for mouse maintenane, L. Jensen for assistane in opper ontent determination, T. O Halloran for aposod1 and D. Borhelt and V. Culotta for disussions. This work has een supported y grants from the National Institute of Health (P.C.W., D.L.P., D.W.C. and J.D.G.), Amyotrophi Lateral Slerosis Assoiation (P.C.W.) and The Spinal Cord Researh Foundation (J.L.). RECEIVED 21 AUGUST 2001; ACCEPTED 12 FEBRUARY Rosen, D. R. et al. Mutations in Cu/Zn superoxide dismutase gene are assoiated with familial amyotrophi lateral slerosis. Nature 362, (1993). 2. Deng, H. X. et al. Amyotrophi lateral slerosis and strutural defets in Cu,Zn superoxide dismutase. Siene 261, (1993). 3. 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