CHAPTER 11

Metal Toxicity andMetallostasis in AmyotrophicLateral Sclerosis

H. L. LELIE,a J. P. WHITELEGGE,b D. R. BORCHELTc

AND J. S. VALENTINEa*

aUCLA Department of Chemistry and Biochemistry, Box, 951569,607 Charles E. Young Drive, Los Angeles, CA 90025-1569, USA; b ThePasarow Mass Spectrometry Laboratory, Box 42 NPI-Semel Institute, DavidGeffen School of Medicine, UCLA, 760 Westwood Plaza, Los Angeles, CA90024-1759, USA; cDepartment of Neuroscience, McKnight Brain Institute,SantaFe HealthCare Alzheimer’s Disease Research Center, University ofFlorida, 100 Newell Drive, Gainesville, FL 32611, USA

11.1 Introduction

Metals have long been suspected to play a role in the motor neuron diseaseamyotrophic lateral sclerosis (ALS) (see Figure 11.1). Thus early epidemiologicalcase studies linked ALS with exposure to lead,1–3 selenium,4 mercury,5,6 manga-nese,7 aluminium,7 or cadmium,8 and more recently it was reported that sig-nificantly increased levels of iron,9 copper,10 zinc,9 calcium,11 aluminium,12

cadmium,8 selenium,4 and bromine;13 and decreased levels of manganese,13 mag-nesium,13 cesium9 and sodium13were present in tissue, serum, and bone fromALSpatients and from mouse models of familial ALS. The earlier studies listed abovewere based on whole tissue analysis, but more recently, with the advent of more

RSC Drug Discovery Series No. 7

Neurodegeneration: Metallostasis and Proteostasis

Edited by Danilo Milardi and Enrico Rizzarelli

r Royal Society of Chemistry 2011

Published by the Royal Society of Chemistry, www.rsc.org

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powerful and precise instrumentation as well as the availability of mouse modelsof ALS, it has been determined that subcellular changes are occurring in thedistributions of iron, copper and zinc ions in ALS diseased tissue.14–16

A major discovery that further linked metals to ALS was the discovery in1993 that mutations in SOD1, the gene coding for the metalloenzyme copper–zinc superoxide dismutase, caused many cases of familial ALS.17 Subsequent tothat discovery, various theories were advanced to explain how copper and/orzinc might play major roles in the SOD1-linked ALS disease. These theoriesrange from pro-oxidant effects of copper bound to mutant SOD1 proteins toprotein aggregation caused by abnormal amounts of metal-deficient mutantSOD1 proteins, but no single theory has been entirely ruled in or ruled out atthis time.18,19

The major purpose of this chapter is to review the evidence for the invol-vement of metals in ALS. This involves two main areas of research, one wheremetals in general were shown to have an effect on all forms of ALS and asecond area in which specifically copper and zinc are suspected to play a directrole in mutant SOD1 mediated ALS. The implications for the possibility ofabnormalities in metallostasis and a role for copper and zinc in the toxicconversion of mutant SOD1 are explored.

11.2 Background

ALS has an incidence rate of about 2 per 100 000 and a lifetime risk of 1 in1000, making it the most commonly occurring motor neuron disease.20 Theearliest symptoms of ALS are weakness, muscle twitching, and cramping.21

These symptoms give way to the gradual spreading of muscle weakness and

Figure 11.1 Heavy metals and other elements associated with ALS.

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paralysis of all the voluntary muscles. Death typically occurs within 2–3 yearsafter diagnosis, although longer (and shorter) survival times are possible. Bothupper motor neurons, which originate from the brain and run down the trackof the spinal cord, and lower motor neurons, which originate in the ventralhorn of the spinal cord and connect to the specific innervated muscle, areaffected. Upper motor neurons transmit signals from the brain to lower motorneurons, which are responsible for sending the impulses that control musclemovement. These motor neurons represent some of the largest cells in the body,with some extending processes up to 1 m long, and they bear a strong electricalload due to the necessity of sending rapid and frequent action potentials.22

Motor neurons are not all equally affected by ALS and can be divided intotwo subtypes on this basis. The ALS-resistant motor neurons, which controlsphincter and ocular function, contain lower densities of calcium permeableglutamate receptors and increased calcium buffering capacity as compared tothe ALS-vulnerable motor neurons,23 possibly rendering those cell types moreresistant to calcium-mediated excitotoxicity. It is interesting in this regardthat riluzole, the only drug that is currently approved by the US FDA forALS, increases lifespan, although only slightly, by targeting calcium-mediatedexcitotoxicity.ALS has a complex etiology, and environmental and genetic factors have

both been linked to disease development. Epidemiological studies on ALS havelong concluded that external environmental factors must play an importantrole in the disease, and excessive exercise, military service, geography, age,smoking, spinal trauma, viruses, and exposure to environmental toxins such asheavy metals and chemical agents such as pesticides have all been suggested aspossible risk factors for disease.24–27 Nevertheless, no one specific class ofagents has been definitively identified.Ninety percent of all ALS cases are sporadic and only ten percent are

familial, but genetic studies on inherited ALS have nonetheless provided thegreatest breakthroughs in the field and have set the framework for under-standing the disease. In a landmark report published in 1993, a genetic linkagebetween mutations in the SOD1 gene and ALS was reported,17 and it is nowestablished that 20% of genetic cases are due to mutations in the antioxidantenzyme copper–zinc superoxide dismutase (SOD1). This discovery in 1993 wasparticularly significant since it led to the creation in 1994 of the first transgenicmouse models of the disease, expressing mutant human SOD1.28 Studies basedupon these mouse models of ALS have increased our understanding of themany pathological abnormalities associated with the disease, including theroles of oxidative stress, apoptosis, excitotoxicity, neurofilament disorganiza-tion, mitochondrial dysfunction, proteasome inhibition, impaired axonaltransport, reactive astrocytes, and protein aggregation (Figure 11.2).19,29

The normal primary function of SOD1 is thought to be acting as amajor antioxidant enzyme in cells by reducing levels of intracellular superoxideradical. However, the targeted deletion of the SOD1 gene in mice doesnot produce catastrophic abnormalities or cause ALS-like symptoms.30

Thus SOD1-mediated ALS is not apparently related to a loss in this antioxidant

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function, but rather the ALS mutations render the mutant SOD1 proteinstoxic. More recently, mutations in the RNA processing proteins, TAR DNAbinding protein (TDP-43), and FUS (FUsed in Sarcoma) were discovered toalso be associated with ALS.31–33 Understanding how these DNA/RNAbinding proteins can lead to motor neuron death and how those processes arerelated to those elicited by mutant SOD1 promises to be a rewarding avenue ofresearch in the future.34

11.3 Toxic Metals and ALS

Decades before any genes involved in familial ALS were identified, the searchfor clues concerning ALS came mainly from epidemiological studies. Some ofthe earliest clues about the causes of ALS originated in the remote westernPacific region of the world in the Micronesian islands, the largest of which isGuam. In the 1950’s, it was observed that the frequency of ALS in this regionwas fifty times higher than in the rest of the world. ALS in these patients wasoften also associated with a Parkinsonism dementia complex. Initially, it wasbelieved that the extreme prevalence was due to a genetic factor, but shortlyafter the 1950’s, the incidence began to decline dramatically suggesting that astrong environmental factor may have been responsible. One of the many early

Figure 11.2 Pathogenic mechanisms in ALS.

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candidates considered for this environmental factor was excessive exposure tothe transition metal manganese. This theory was supported by the observationthat volcanic formations in Guam contain high levels of manganese, leading tohigh manganese content in soil and drinking water as measured by neutronactivation analysis (NAA).12,35,36 Excessive manganese exposure can lead tosymptoms similar to those of ALS, and epidemiological studies revealed acluster of ALS cases among manganese miners in Guam. When spinal nervoustissue from Guam patients was analyzed, an increase in the content of man-ganese in the areas of degeneration was reported.12 Interestingly, an increase inother metals including aluminium and calcium was also observed in those sameaffected tissues. Loss of bone density and further tissue studies revealeddramatic variation in calcium levels in tissue subsections, supporting aninvolvement of calcium homeostasis in the disease.4,7 Other indications ofuniversal disturbances in metallostasis were observed in blood samples wherelower levels of manganese and selenium were reported for patients versuscontrols.4 These early studies appeared hopeful and provided a lot of promisetoward understanding ALS pathology.Over time, however, follow-up studies on patient exposure and tissue metal

levels proved inconsistent. For example, one study on the drinking water andsoil in Guamanian villages where large clusters of ALS cases existed did notshow high levels of manganese present as before but actually indicated thecomplete opposite, a lack of both manganese and calcium.37 Moreover newinvestigations reported difficulty in reproducing the earlier findings concerningtissue metal levels,38 while others implicated a completely different set ofmetals.39 Studies as recent as 2001, where blood metal levels were measured tovalidate earlier findings of changes associated with motor neuron disease, didnot confirm earlier studies with a larger sample size of sporadic ALS cases andin fact led to contradictory findings.8 Taken together, these studies demonstratethat fluctuations in metal levels are very frequently associated with ALS inGuam, but the specific metals vary and the extent of the fluctuationsremains unclear. Currently, the view on the reports of metals in Guam ALS andALS–Parkinsonism dementia patients is inconclusive. While some level ofinvolvement of metallic elements appears probable, determining which ones arerelevant remains elusive due to variation in the methodologies used in theearly studies, the relatively small pool of patients, and inconsistent results.Nevertheless, these studies laid the groundwork for future studies on theassociation of metals and ALS.The Guam studies inspired several subsequent studies of metals and ALS. In

a 1983 clinical survey of metal exposure of ALS patients at the Mayo Clinic, itwas discovered that ALS patients had a slightly increased exposure to heavymetals relative to controls.40 Follow-up investigations with a large sample sizeof male ALS patients revealed that they were frequently employed in blue collarjobs involving welding and soldering, thus implicating increased leadexposure.3 Very recent studies showed that sporadic ALS patients had a higherthan normal exposure to lead on average, which led to higher levels in bloodand bone.2,41 These findings support a pathological role for increases in

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exposure to heavy metals leading to increased levels in the body. Ultimately,however, no retrospective study has yet conclusively defined epidemiologicaland environmental factors that lead to ALS, largely due to inconsistencies inthese reports.5 It should be noted, however, that inconsistencies in the literaturemay be partially explained by the fact that tissue investigations on ALS aregenerally limited to post-mortem end-stage samples, rendering it difficult toassess disease-relevant changes. Recent investigations implementing moreadvanced techniques with the ability to measure more localized microscopicregions of tissue have reinvigorated the possibility that metal ions and otherelements such as iron, manganese, copper, zinc, bromine, sodium, magnesium,and calcium homeostasis are affected in neurodegeneration.13,42–44 Ross et al.have proposed a model in which multi-metal toxicity is responsible for ALSpathology.45 In this model, each metal can contribute to toxicity in its own way,either directly through aberrant redox chemistry, perturbed homeostasis,changes in metalloenzyme activities, or altered signaling, and so forth. Asanalytical technology advances, we anticipate future successes in this currentlyinconclusive area of ALS research.

11.4 Metallothioneins and ALS

Metallothioneins are small (6–7 kDa) peptides responsible for scavenging andtransporting mono- and divalent metal ions.46 Four main species exist inhumans, MT-I to MT-IV, and in nervous tissue, MT-I and MT-II are localizedto astrocytes and MT-III is present in neurons. It is generally believed thatthese proteins play a protective role against disease by regulating dangerouslyhigh levels of metals and scavenging ROS.47,48 This protective role wasconfirmed when knockout mice for either the astrocyte specific metallothioneins(I and II) or the neuronal metallothioneins (MT-III) were crossed with G93ASOD1 ALS mice resulting in acceleration of disease.49 Gene expression profilesfound that MT-I and -II were up-regulated prior to symptoms in spinal cord ofG93A SOD1 mice but MT-III was up-regulated later, suggestive of an initialglial response.50,51 In later stages, it appears that MT-I becomes up-regulatedin other tissues such as liver, kidney, and skeletal muscle of transgenic mice,indicating that a systemic response to metal homeostasis and oxidative stressexist in ALS.52,53

Metallothioneins, which play important roles in normal metallostasis aswell as in providing protection from toxic metals in human tissues, have beenobserved to be elevated in liver, kidney, spinal cord gray matter and astrogliafrom ALS patients relative to non-ALS controls, indicating a systemic break-down in metallostasis associated with the disease.54 Metallothioneins have beenshown to mediate tissue expression of SOD1 when copper is present,55 and ALSpatients have been shown to have decreased expression of metallothioneinsrelative to controls.56 It has further been suggested that one of the mechanismsby which exercise helps patients is through increased expression of metal-lothioneins.57 Intriguingly, a single nucleotide polymorphism in the

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metallothionein gene, MT-1, was associated with some sporadic ALS patientsthat had high exposure to environmental toxicants, possibly linking a geneticsusceptibility to metal toxicity.58 The combination of the transgenic mice stu-dies with the reports on human patients suggest that metallothioneins may playa role in countering disease, possibly due to their function in maintainingmetallostasis.

11.5 SOD1 and ALS

11.5.1 SOD1 and Familial ALS

SOD1, originally termed hemocuprein, hepatocuprein, or erythrocuprein, wasfirst isolated from red blood cells in 1938 and identified as a major copper-binding protein in erythrocytes. After its superoxide dismutase activity wasdiscovered by McCord and Fridovich in 1969,59 it was renamed copper–zincsuperoxide dismutase. Research in subsequent years led to extensive char-acterization of the protein, and we now have considerable knowledge of itsphysical, chemical, and biological properties.18

SOD1 is a 153-amino acid homodimeric enzyme that adopts a beta barrel fold.Each subunit contains two large loops that encase a zinc and copper ion, thelatter a prerequisite for activity. In the human SOD1 protein, there are fourcysteines, two of which are involved in an intramolecular disulfide bond. Thefully metallated, disulfide-oxidized, dimeric protein is extremely stable, a prop-erty that is apparent from its high melting temperature (above 90 1C), and itsextreme resistance to proteolytic degradation and denaturation by 10 M urea or4% SDS.18,60

SOD1 catalyzes a two-step disproportionation reaction of two superoxideanions to produce one molecule of hydrogen peroxide and one molecule ofmolecular oxygen as the copper ion is alternately reduced and oxidized (eqns.11.1–3).

O2� þ CuðIIÞZnSOD! O2 þ CuðIÞZnSOD ð11:1Þ

O2� þ CuðIÞZnSODþ 2Hþ ! H2O2 þ CuðIIÞZnSOD ð11:2Þ

Sum : 2O2� þ 2Hþ ! H2O2 þO2 ð11:3Þ

SOD1-linked ALS is not due to a loss of SOD activity. This conclusion is basedon several key pieces of evidence. Firstly, the majority of SOD1 ALS mutationsdo not result in loss of SOD activity.61 Additionally, sod1 knockout mice do notdevelop the disease.30 Furthermore, with the exception of northern Europeanfamilies that inherit the D90A mutation, the disease shows a dominant patternof inheritance. Thus, mutations in SOD1 that are associated with ALS arethought to instill a toxic property to the protein.

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Several hypotheses have been put forth for this toxic gain-of-function bymutant SOD1, including a conversion to pro-oxidant activity of enzyme-boundcopper,62 but the currently most widely accepted hypothesis for this gain-of-function is the accumulation of misfolded and abnormally oligomerized mutantSOD1.60,63 This hypothesis was derived from the presence of prominent cyto-plasmic inclusions containing SOD1 that were identified within the motorneurons of mouse models of ALS and in the spinal cords of human ALSpatients.60,64 It is also possible that some combination of oxidative damage andprotein aggregation could be involved in SOD1-linked ALS. For example,oxidative modification of histidine residues at the active site was shown to makethe protein more prone to aggregation,65 and the inclusions found in ALStissues show signs of post-translational modifications such as glycosylation andside chain modification66 and are also commonly co-localized with regionsof lipid peroxidation, suggesting that oxidative damage could play some sort ofrole in their formation or toxic action.67

Understanding how SOD1 aggregates has been an area of intense study.Some important clues were derived from studies on the composition of SOD1aggregates isolated from transgenic mice spinal cord.68 Counter to severalhypotheses, aggregated SOD1 was not post-translationally modified, nor did itco-aggregate with any prominent binding partners. However, a recent study byWang et al. of mice that express mutant SOD1 fused to yellow fluorescentprotein (YFP) reported that aggregates of the mutant fusion protein containHSP70.69

The structure of the large aggregates that form inclusions has been anotherarea of interest, particularly whether the mutant SOD1 produces amyloid oramyloid-like structures. Recent studies of human SOD1-linked ALS have notfound convincing evidence for amyloid structures.70 However, purified apo WTSOD1, even under physiologically relevant conditions, has a propensity to formamyloid-like fibers.71,72 Amyloid-like fibers have also been seen in crystalstructures of some ALS mutant SOD1 proteins,73,74 and fibrillar structures thatbind Thioflavin-S (a dye that selectively binds amyloid fibrils) have beendetected in tissues of symptomatic transgenic mice that express mutantSOD1.75 These findings correlate with the detection of detergent-insoluble andsedimentable forms of mutant SOD1 in spinal cord tissues of symptomatictransgenic mice.76 Interestingly, in the mouse models, these large aggregates ofmutant protein accumulate largely in the interval between the onset of the firstnoticeable symptoms and paralysis, indicating that forms of the mutant proteinother than large aggregates are likely to be involved in initiating the disease. Atpresent, there are many unresolved questions regarding the structure of theaggregates in vivo and the relative contribution of different types of aggregatesto disease pathogenesis.It is unclear how aggregates may be toxic, but several hypotheses exist. It has

been suggested that aggregates can mediate aberrant chemical reactionsfollowing misfolding of mutant SOD1, or they can sequester essential cellularcomponents.34,77 They may also monopolize chaperones and overburdenprotein degradation pathways. Aggregates have also been found to interfere

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with mitochondrial functions,78 induce endoplasmic reticulum (ER) stress,79

and inhibit axonal transport.80

Despite their associations with many neurodegenerative diseases, it is notknown if the formation of the visible aggregates themselves is toxic, correlative,or beneficial; with the latter being attributed to the clearing of otherwise toxiccomponents. For instance, in the case of Alzheimer’s disease, it is believed thatthe smaller oligomers of amyloid beta peptide harbor greater neurotoxicitythan the large amyloid plaques that represent large extracellular aggregates ofamyloid peptide.81 A similar scenario is possible for mutant SOD1 with smalleroligomeric assemblies of protein harboring greater toxicity than the largeraggregates that are visible as pathologic inclusions. However, it is entirelypossible that the smaller oligomeric assemblies and the larger aggregatesimpinge on different cellular processes with each exacting some type of toxicityto the motor nervous system.

11.5.2 SOD1 and Sporadic ALS

It is unknown if SOD1 also plays a role in the pathogenesis of sporadic ALS,but several lines of evidence suggest that it may. SOD1-linked familial ALShas a very similar clinical pathology to sporadic ALS, although treatmentsdeveloped using the G93A-SOD1 ALS mouse model, in which the proteinis highly over-expressed, have not been successfully translated to humanpatients.82 Inclusions containing SOD1 have recently been identified insporadic ALS patients.83 Smaller, soluble, oligomeric forms of WT SOD1 mayalso be neurotoxic entities.76 It is interesting to note that wild-type SOD1, in itsmetal-free form, does have a propensity to oligomerize in vitro under physio-logical conditions71,72 and thus might be able to take on a toxic form in vivo andplay a role in sporadic ALS. Interestingly, oxidatively damaged wild-typeSOD1 has been reported to misfold and acquire toxic properties similar tothose of mutant SOD1 proteins, suggesting the additional possibility thatoxidized wild-type SOD1 could be involved in sporadic ALS.84,85 A 2007 articlefrom Gruzman et al. reported the identification of a 32-kDa covalently cross-linked SOD1 dimer associated with both sporadic and familial ALS patienttissues specifically.86 On a related note, a recent study shows that sporadic ALSpatients have increased SOD1 mRNA transcripts in affected tissues therebylinking sporadic ALS, SOD1, and possibly aggregation.87

It is also possible that toxic exposure to metallic elements could influence thelevels of SOD1 expressed. Exposure of yeast cells to exogenous Cu, Ca, Cd, Co,Cr, Li, Mn, Zn, K, Na, Ni, Rb or Fe led to the dramatic and consistentup-regulation of SOD1. Zinc resulted in the highest SOD1 induction, whileother antioxidant proteins, such as thioredoxin peroxidase, cytochrome cperoxidase, and heat shock proteins all were down-regulated.88 It wasconcluded that SOD1 plays some role, apart from its role as a superoxidedismutase, in detoxification of heavy metals. Studies using the human HepG2hepatoma cell model revealed that mammalian systems recapitulate the yeast

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results; heavy metals were found to induce SOD1 through a metal responsiveelement (MRE) that is 273 to 276 base pairs upstream of the SOD1 gene.89 Onepossible explanation is that SOD1 acts in the buffering and maintenance ofmetallostasis of these various metals.Recent investigations further suggest that a strong link may exist between the

SOD1 protein and tissue metallostasis.90 Synchrotron based X-ray fluorescenceimaging carried out on SOD1 transgenic mouse spinal cord sections revealed adramatic effect of over-expression of SOD1 on the levels of metals in thesetissues. Over-expression of either ALS-mutant or wild-type SOD1 resulted in adramatic shift in the localization of copper and iron from the spinal cord whitematter to the spinal cord gray matter without affecting the total metal levels.90

Copper levels reflected the distribution of SOD1, which was higher in the graymatter than the white matter in transgenic mice, and iron levels were correlatedwith copper levels, possibly reflecting the linked metabolism of these twometals. Interestingly, zinc levels were much higher in the spinal cord whitematter of diseased animals (i.e., mutants G93A SOD1 and H46R/H48Q SOD1)compared to the non-transgenic and wild-type SOD1 controls. Deciphering therelationship between SOD1 and both normal metallostasis and toxic metalexposures may provide clues about the disease process in ALS.

11.5.3 Stabilization of the SOD1 Protein by Zinc and Copper

In any discussion of metals in ALS and SOD1, it is pertinent to understand thecritical roles played by metals in the maturation, structure and function ofSOD1. SOD1 comprises up to 1% of all soluble cytosolic proteins in neurons.91

Maturation of SOD1 requires removal of the initial methionine, N-terminalacetylation, acquisition of zinc and copper ions, oxidative formation of thedisulfide bonds in each subunit, and dimerization, but the timing of these post-translational modifications is not yet known. A recent focus in the SOD1–ALSfield has been on investigating these maturation steps, in particular metalacquisition, since metal ion binding contributes significantly to the stability andother characteristics of the mature protein.The mechanism of zinc acquisition by SOD1 in vivo is not well understood.

Some have proposed that it can occur through simple diffusion or that zinc isdelivered by metallothionein (reviewed by Potter and Valentine, 2003),92 whileothers suggest that it may be assisted by the copper chaperone for SOD1,known as CCS.93 Tight zinc binding is believed to be an early step inmaturation and is required for the proper folding of SOD1.94 Zinc coordinatesto SOD1 via His 63, His 71, His 80, and Asp 83, binding in a distorted tetra-hedral geometry. Its binding significantly stabilizes the structure of SOD1.95

Copper insertion is better characterized than zinc insertion and is thought tooccur along two main pathways in humans: a CCS-dependent and a CCS-independent pathway.96 In the CCS-dependent pathway, CCS, which is presentin less than one tenth the levels of SOD1,93,97 docks with SOD1, deliveringcopper and facilitating formation of the intramolecular disulfide bond in an

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oxygen-dependent mechanism.91 SOD1 has four cysteines, Cys 6, Cys 57, Cys111, and Cys 146, with Cys 57 and Cys 146 forming the native intramoleculardisulfide bond in each subunit of the mature protein. The formation of thedisulfide bond is important in stabilizing the dimer interface by anchoring aloop of the protein (Glu 49 to Asn 53).98 The free thiols, C6 and C111, serve anunknown role or no role at all, however many mutations have been associatedwith these sites including C6S, C6F, C6G, C111Y.Some organisms such as Saccharomyces cerevisiae, wild-type yeast cells,

require CCS while other organisms such as Caenorhabditis elegans do not evenhave an endogenous copper chaperone for SOD1.96 Therefore, when the ccs1gene is removed from yeast, yeast SOD1 does not become activated. However,human SOD1 can be activated even when expressed in a ccs1D null yeast cellvia a CCS-independent pathway, and this pathway includes the oxidation ofthe disulfide without a requirement for oxygen.99 The precise loadingmechanism of the CCS-independent pathway has not yet been determined.It is unclear whether SOD1 dimerization occurs before or after the other

post-translational modifications, but we do know that the dimer is quite astable state for all forms of the protein except the metal-free, disulfide-reducedspecies.100–102 Thus dimerization could occur at any point along the maturationpathway, possibly depending on the local steady state concentrations of SOD1,CCS, and glutathione. Fully mature dimeric SOD1, with metals bound anddisulfide bonds intact, has an extremely stable quaternary structure with amelting temperature of above 90 1C.100

Even without the metal ions bound, the wild-type SOD1 disulfide oxidizedapoprotein is relatively stable, with a melting temperature around 52 1C.Nevertheless, apo SOD1, with the disulfide bond intact or reduced, has beenfound to form amyloid-like species readily under relatively mild condi-tions,63,71,103,104 and it has been suggested that the increased propensity towardaggregation of some of the mutant SOD1 proteins may be due to their reducedability to bind metals in vivo. In order to address this question, new analyticalmethodology was developed to assess the metallation state of soluble andaggregated SOD1 proteins in vivo in the SOD1-ALS transgenic mice.90

Metallation levels of mutant SOD1-containing aggregates isolated from mousespinal cord were found to be very low. By contrast, the soluble mutant andwild-type SOD1 from the mouse spinal cords was found to be highly metallatedin most cases, with a total metallation state of about four metal ions per SOD1dimer (usually around three zinc ions and about one copper ion per dimer).90

These results suggest that aggregated SOD1 is derived from immature SOD1prior to metallation. Several other lines of evidence also suggest that the toxicform of SOD1 is derived from nascent metal-free SOD1 species (seeSeetharaman et al. for a review of immature SOD1 and ALS).105

Some of the ALS-mutant SOD1 proteins are severely destabilized in theirapo states while others have stabilities very similar to those of wild-type SOD1in both the apo and metallated states regardless of the status of the disulfidebond,106 and ALS-mutant SOD1 proteins that are highly stable whenexpressed, isolated, and biophysically characterized106 are nonetheless found to

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aggregate in vivo in the transgenic mice and/or in cell culture assays whereas thewild-type SOD1 protein is not.107–109 These results suggest that the kinetics offolding and maturation of the mutant SOD1 proteins may be altered relative towild-type SOD1 and that aggregation may occur from a folding intermediate ofthe protein. This hypothesis is supported by experiments carried out by Kopitoand coworkers who used a cell-free reticulocyte lysate expression system anddemonstrated that the kinetics of metal acquisition to a hyperstable state wereimpaired for ALS-mutant SOD1 relative to wild-type SOD1.94 Zinc appears tobe especially important in this folding process since it is likely to be an earlyevent and its binding is kinetically more labile than that of copper, allowinggreater freedom across the energy field landscape of folding.110,111 From allof these data, it seems possible that the key to mutant SOD1 mediatedtoxicity may lie in its initial misfolding when coming off the ribosome, possiblyexacerbated by slow or improper zinc binding.

11.6 ALS and Some Specific Metals

11.6.1 Copper

Cellular metabolism of copper is tightly regulated, probably owing to itspotential for promoting metal-mediated oxidative stress.112 Copper ion import,transport, and insertion into proteins are tightly regulated, and storage andscavenging is accomplished by both glutathione and the metallothioneins.Copper distribution is believed to follow a gradient whereby increased copperaffinity determines its localization, with SOD1 and metallothioneins havingthe highest affinities for copper,113 suggesting that SOD1 can play a role instoring copper in a non-toxic form, particularly if intracellular copper levels gettoo high.Disruption of the role of SOD1 in copper buffering has been suggested as

a problem that could occur in mutant SOD1-mediated ALS.114 Rae et al.calculated that a normal yeast cell has roughly 390 000 copper ions, about 13%(50 000) of which are bound to SOD1.112 In mutant SOD1-ALS mice, SOD1 isover-expressed to levels that are at least sixfold and up to tenfold higher thannormal. Recent studies have measured the metallation state of SOD1 derivedfrom the spinal cord of such mice.90 An individual dimer of SOD1 that is fullymetallated contains four metal ions, two copper and two zinc, and the tissuemetallation state of SOD1 represents the average number of copper and zincions bound to each dimer of SOD1 averaged over the whole tissue. The resultsrevealed that, while the copper levels were close to two per dimer for theendogenous mouse SOD1 (with two zincs in the zinc site), the copper metal-lation state is only half, at about one copper per dimer for wild-type and G93Aand G37R SOD1 mutants over-expressed in these mice (each with now aboutthree zincs per dimer) relative to the endogenous mouse SOD1. Since the totaltissue metal levels do not change dramatically, these data imply that the mutantand wild-type SOD1 bind 30–40% of total cellular copper. The fact that the

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wild-type mice do not fall ill despite the obviously large impact on coppermetabolism suggests that altered copper metallostasis is unlikely to play amajor role in ALS pathology in the transgenic mice.An early theory concerning the toxic gain-of-function of ALS-mutant SOD1

proteins was aberrant pro-oxidant copper chemistry from copper bound tomutant SOD1 proteins.18,115,116 However, studies on transgenic mice expressinga mutant SOD1 incapable of binding metals at the normal copper sites haveshown that copper is not required for the mouse to fall ill,107 and investigationsusing a mouse knockout of the copper chaperone for SOD1 (CCS) revealedthat the lack of copper insertion into SOD1 did not change onset or progressionof the disease.117 These and subsequent studies appear to eliminate a major rolefor copper in SOD1-mediated toxicity.107,117–119

At this point, it seems clear that copper chemistry can be ruled out asthe major factor causing SOD1-linked ALS. Nevertheless, copper has beenimplicated in other neurodegenerative disorders, such as Alzheimer’s diseaseand prion encephalopathies, and there are many reports suggesting that coppermay be somehow linked to ALS. For example, excess copper was found inerythrocytes of SOD1-associated familial ALS patients,120–123 and a cross of acopper-deficient mouse with an SOD1-ALS mouse led to delayed onset ofdisease but did not decrease mortality.124 Administration of a copper chelatorprolonged the lifespan of G93A SOD1 transgenic mice,16,124 and further studiesrevealed that mutant SOD1 but not wild-type SOD1 led to a dramatic up-shiftin proteins responsible for copper homeostasis in the spinal cords of thesemice.16 These results suggest that while copper chemistry is an unlikely factor inpathology, disruptions in its normal homeostasis may be relevant.

11.6.2 Iron

Iron is a redox-active metal that, even more than copper, has the potential toact as a pro-oxidant and its metabolism is tightly linked to that of copper asiron transport into cells relies on the multi-copper oxidase, ceruloplasmin.114

Increased iron levels in spinal cord and brain and increased levels of the ironstorage protein, ferritin, during later stages of disease have been reproduciblyshown in samples from ALS patients.1,9 Interestingly, increased mitochondrialferritin levels were detected in a G37R mouse model of ALS, confirming alterediron metabolism, and treatment with an iron chelator prolonged survival forthis mouse strain by a very significant five weeks.14 More studies are needed todetermine whether iron homeostasis plays a significant role in ALS pathology.

11.6.3 Zinc

Zinc is the second most abundant metal in biology, after iron, and it is vital forprotein stability, DNA replication, regulation of transcription, protein trans-lation, metabolic protein function, signaling pathways, and as a cofactor inmany enzymatic reactions. Zinc is also well known to play major roles in the

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central nervous system, and neurons have been shown to contain threedistinct pools of zinc, 80% bound to protein, 5–15% in vesicles ofglutaminergic synapses, and 5% free labile zinc.125 Zinc is not a redox-activemetal, but its levels are nonetheless tightly regulated, and alterations in zinclevels could impart adverse effects due to its role in various signalingpathways.46 Zinc levels within spinal cord and serum were not significantlydifferent in ALS patients versus controls.120 However, zinc deficiencyaccelerated disease processes in a G93A hSOD1 mouse.126 The effects of zincsupplementation on the mice models have been mixed, with low levelsappearing beneficial while high levels were toxic.126 Interestingly, as mentionedearlier, increased levels of the zinc-responsive metallothioneins MT-I andMT-II have been associated with early stages of disease in transgenic mice,127

suggestive of increased zinc levels. More recently it was discovered that thetransgenic mice spinal cords have excess labile zinc localized in neuronsand astrocytes when they are undergoing neurodegeneration as measured bycolocalization of HNE (an endogenous neurotoxic agent) and the zinc probeTFL-Zn.15 Increased levels of chelatable zinc are observed in cell culturesof immune cells undergoing apoptosis and in neurons which underwentischemia or seizure activity.48 Excessive zinc levels are observed in a number ofpathologies associated with neurodegeneration, ranging from oxidative stressand protein aggregation to apoptosis (for a comprehensive review of zinc inneurodegenerative diseases, see Smith et al.).46 In the case of ALS, zinc levelsappear to increase in spinal cord white matter as a result of disease but theoverall levels remain unchanged.90 The studies thus far on zinc suggest animportant link between zinc and ALS; however no specific role for zinc inpathogenesis has been defined.Approximately 10% of all zinc in the central nervous system is located in

vesicles within the presynaptic cleft of glutaminergic neurons. Release of thiszinc into the synaptic cleft is important for signaling as it binds many receptorsincluding N-methyl-D-aspartate (NMDA) and g-aminobutyric acid (GABA)receptors and voltage-gated calcium channels.128 It has been postulated thatone of the mechanisms of aging involves the aberrant uptake of zinc throughvoltage-gated calcium channels following glutamate receptor activation andthat this zinc can lead progressively to mitochondrial dysfunction and even-tually apoptosis.125 A similar mechanism was proposed in ALS,15 in whichmutant SOD1 could also contribute to a breakdown in zinc homeostasis,leading to mitochondrial dysfunction and apoptosis of motor neurons. Thismechanism was modeled in spinal motor neuron culture in which it was foundthat exposure to zinc led to an acutely toxic effect, which they found to bemediated by the activation of calcium-permeable AMPA/kainite channels.129

Based on these findings, one would expect the use of a zinc chelator to helpcounter disease. To test this, 20mg kg�1 day�1 of the zinc chelator, TPEN, wasadministered to G93A SOD1 mice, resulting in a modest beneficial effect with afive to ten day increase in survival time.15 These results combined with the linkbetween neuronal zinc and other neurodegenerative diseases indicate a need foradditional studies to clarify the role of zinc in ALS.

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11.6.4 Calcium

The role of calcium in the molecular pathology of motor neurons in ALS hasbeen very well characterized. Excess calcium influx into the neurons and poorcalcium buffering contribute to mitochondrial degeneration and apoptotic celldeath. During normal neurotransmission, glutamate is released from apresynaptic neuron and binds to the AMPA receptors of a postsynaptic neu-ron, leading to a large influx of calcium ions. Normally, the glutamate ispromptly removed from the synaptic cleft by the excitatory amino acid trans-porter (EAAT2) on glial cells. However in ALS, EAAT2 levels are diminished,which in turn leads to over-activation of AMPA receptors and hence excessivecalcium influx.130 Interestingly, a knock down of EAAT2 in rats led to adeteriorating motor syndrome with ALS-like symptoms.131 Relative to otherneurons, motor neurons express lower levels of GluR2, which cause AMPAreceptors to be more permeable to calcium.132 Furthermore, diseased motorneurons have a particularly difficult time recovering to basal levels after AMPAreceptor activation, and the excitotoxic pathway is thus enhanced.11 Beta-N-methylamino alanine (BMAA), which is one of the candidates for the neuro-toxic ALS-causing agent in Guam, has been shown to activate AMPAreceptors, in turn propagating the same calcium influx mechanism.133 The ALSdrug riluzole acts to inhibit tetrodotoxin sodium channels, thereby reducingcalcium influx and countering the effects of overstimulation of glutamatereceptors.134 Further contribution to improper calcium handling in the motorneuron may result from decreased expression of the calcium binding proteinscalbindin D-28k and parvalbumin.135 Furthermore, calmodulin has also beenfound to coaggregate with mutant SOD1, possibly leading to lower levels ofthis major calcium buffering protein.68 The studies on calcium provide a clearexample of how metal ions and metallostasis function specifically in thisneurodegenerative disease.

11.7 Conclusions

Neurodegenerative diseases are frequently associated with the appearance ofabnormal protein deposits in nervous tissue, and it has been proposed that thispathological hallmark is indicative of an underlying proteopathy and thatfailures of normal proteostasis may be the root cause of many of these dis-eases.136,137 More recently, it has been appreciated that maintaining the delicatebalance of different metal ion concentrations present in the cell may also beimportant in maintaining normal proteostasis, and it is now clear that failuresin metallostasis could also play an important role in disease. In the case of ALS,both sporadic and familial, metals have long been linked to the disease,although in many different ways, and any proposed pathogenic mechanismshould certainly address their potential involvement. In the case of SOD1-linked familial ALS, more studies are needed to determine how SOD1 proteinsbecome toxic to motor neurons. As analytical technologies for detectionof metals continue to improve, we can expect to learn more about how

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metallostasis and proteostasis are linked under normal conditions and howdysfunction in either might lead to ALS. It is also clear that involvement oftoxic metals in either sporadic or familial ALS cannot be ruled out and shouldcontinue to be considered.

Acknowledgements

The authors wish to thank Dr Edith B. Gralla and Dr Sadaf Sehati for helpfuldiscussions and review of this chapter. They also would like to thank thesupporting NINDS grant P01NS049. HL was supported by the NIH ChemistryBiology Interface Training Program; Grant number: 5T32GM008496.

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