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Contents lists available at ScienceDirect

Journal of Trace Elements in Medicine and Biology

jou rn al homepage: www.elsev ier .de / j temb

0th NTES Symposiumeview

he neurotoxicity of iron, copper and manganese in Parkinson’s andilson’s diseases

etr Duseka,b,∗, Per M. Roosc,d, Tomasz Litwine, Susanne A. Schneider f,rond Peder Flateng, Jan Aasethh

Department of Neurology and Center of Clinical Neuroscience, Charles University in Prague,st Faculty of Medicine and General University Hospital in Prague, Czech RepublicInstitute of Neuroradiology, University Medicine Göttingen, Göttingen, GermanyDepartment of Neurology, Division of Clinical Neurophysiology, Oslo University Hospital, Oslo, NorwayInstitute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden2nd Department of Neurology, Institute of Psychiatry and Neurology, Warsaw, PolandNeurology Department, University of Kiel, Kiel, GermanyDepartment of Chemistry, Norwegian University of Science and Technology, Trondheim, NorwayDepartment of Medicine, Innlandet Hospital Trust, Kongsvinger Hospital Division, Kongsvinger, Norway

r t i c l e i n f o

rticle history:eceived 31 January 2014ccepted 22 May 2014

eywords:ronopper

a b s t r a c t

Impaired cellular homeostasis of metals, particularly of Cu, Fe and Mn may trigger neurodegenerationthrough various mechanisms, notably induction of oxidative stress, promotion of �-synuclein aggrega-tion and fibril formation, activation of microglial cells leading to inflammation and impaired productionof metalloproteins. In this article we review available studies concerning Fe, Cu and Mn in Parkinson’sdisease and Wilson’s disease. In Parkinson’s disease local dysregulation of iron metabolism in the subs-tantia nigra (SN) seems to be related to neurodegeneration with an increase in SN iron concentration,

anganesearkinson’s diseaseilson’s disease

helating agents

accompanied by decreased SN Cu and ceruloplasmin concentrations and increased free Cu concentrationsand decreased ferroxidase activity in the cerebrospinal fluid. Available data in Wilson’s disease suggestthat substantial increases in CNS Cu concentrations persist for a long time during chelating treatmentand that local accumulation of Fe in certain brain nuclei may occur during the course of the disease.Consequences for chelating treatment strategies are discussed.

© 2014 Elsevier GmbH. All rights reserved.

ontents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Parkinson’s disease (PD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Environmental exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Dysregulation of brain metal homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Wilson’s disease (WD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Local dysregulation of metal homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author at: Department of Neurology and Center of Clinical Neuroscieospital in Prague, Katerinska 30, 120 00 Praha 2, Czech Republic. Tel.: +420 224 965 528

E-mail addresses: pdusek@gmail.com, petr.dusek@vfn.cz (P. Dusek).

ttp://dx.doi.org/10.1016/j.jtemb.2014.05.007946-672X/© 2014 Elsevier GmbH. All rights reserved.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

nce, Charles University in Prague, 1st Faculty of Medicine and General University.

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ntroduction

Impaired cellular homeostasis of metals may initiate neurode-eneration through various mechanisms. Of these, oxidative stressnduced by the formation of free radicals is the best established [1].ther possible mechanisms are impaired production of metallopro-

eins [2,3], activation of microglial cells leading to inflammation [4]nd promotion of aggregation and fibril formation of �-synuclein,

highly conserved protein with unknown function. This proteins the major constituent of Lewy bodies found as intracytoplas-

atic inclusions in Parkinson’s disease (PD) neurons [5]. Recentn vitro studies have shown that mutant �-synuclein interacts with

etals and that iron (Fe) and copper (Cu) seem to aggravate theoxicity caused by the �-synuclein [6,7]. It has been suggested that-synuclein is a Cu binding protein acting as a cellular ferrireduc-

ase [8,9]. Lewy bodies contain reactive iron along with aggregatedroteins and oxidized lipid material [10].

Our knowledge regarding cellular metal homeostasis hasmproved substantially over the last years. Several proteinsnvolved in cellular import, export, trafficking and storage of Fe, Cund manganese (Mn) have been identified [11–13]. Among those,wo novel Mn carriers involved in intracellular Mn transport intoysosomes and Golgi, ATP13A2 and SLC30A10 respectively, haveeen found. Other recent findings include roles of the copper trans-orter protein 1 (CTR1) in Cu uptake, of Cu chaperones in synthesisf specific cuproproteins, and of ceruloplasmin in cellular Fe export14]. Mechanisms of metal transport across the blood–brain bar-ier (BBB) and the blood–cerebrospinal fluid barrier (BCB) haveeen thoroughly investigated [11,15,16]. While the brain seemso be protected against high plasma Fe concentrations as docu-

ented in hereditary hemochromatosis [17], this is apparently notrue for Mn and Cu. Thus, increased plasma Cu and Mn concen-rations may lead to brain deposits and CNS damage [18]. It wasecently suggested that Mn enters the CNS predominantly throughhe BCB and that high Mn concentration impairs the integrity ofhis barrier [19]. There is ongoing research on the roles of Mnpecies and their transporter molecules in brain Mn influx; Mn-itrate was suggested as the species with the highest influx rate20], and is presumably also the major Mn species in the CSF [21,22].his is in contrast to Fe and Cu which are present in CSF mainly asigh molecular weight species [18]. Mn and Fe share the trans-ort pathway using the transferrin receptor (TfR) [23,24] and itas been suggested that Fe deficit may increase brain accumu-

ation of Mn [25,26]. Some authors argue that Fe deficiency mayead to divalent metal transporter 1 (DMT1) upregulation leadingo increased Mn uptake [27,28]. There is however controversy tohat extent the DMT1 is involved in Mn transport [16,29]. Interest-

ngly, DMT1 is supposedly implicated in Mn uptake by the olfactoryract which is another possible route for brain Mn accumulation30]. In addition to Fe and Mn interdependencies, interactionsetween Fe and Cu [31] as well as between Fe and calcium [32]etabolism have been reported. Comprehensive discussions of Mn,

e and Cu transport across BBB and BCB can be found in recenteviews [11,15,16,33].

Despite these advances, it is still unclear to what extent impairedetal metabolism affects the pathophysiology of particular neu-

odegenerative disorders. The aim of this article is to review currentiterature covering association between metal dyshomeostasis andeurodegeneration, particularly studies measuring concentrationsf Fe, Cu and Mn in the cerebrospinal fluid (CSF) or brain tis-ues of patients with PD and Wilson’s disease (WD) as well astudies examining the pathophysiology of metal abnormalities. PD

s a common neurodegenerative disorder with largely unknownathophysiology, while WD is a rare disorder with well describedathophysiology involving Cu toxicosis where established che-

ating therapy exists.

PRESSedicine and Biology xxx (2014) xxx–xxx

Parkinson’s disease (PD)

Accumulation of several metals, including Fe, Cu and Mn in subs-tantia nigra (SN) has been suggested to play a causative role in thepathophysiology of PD [34]. Metal related PD hypotheses are basedon:

(1) Epidemiological studies assessing the relationship betweenenvironmental exposure to metals and the risk of PD.

(2) Clinical observations that disorders with brain metal accu-mulation such as WD, manganism or the syndromes ofneurodegeneration with brain iron accumulation (NBIA) maymanifest with parkinsonism.

(3) Animal studies examining exposure to high levels of metals andthe effects of chelation treatment.

(4) Measurements of metal concentrations and metal regulatoryprotein expression in post-mortem brain tissue samples.

(5) Measurements of metal concentrations and metal regulatoryproteins in the CSF.

Several of these empirical observations are discussed in the fol-lowing sections.

Environmental exposure

Effects of Fe and Cu exposure have been sparsely studied anda large environmental survey lent little support for an associationbetween environmental exposure to Fe or Cu and PD [35]. How-ever, combined exposure to Cu and Fe [36] or Mn and Fe [37]lasting for two to three decades have been reported to significantlyincrease the risk of PD. Also, toxic effects of Mn have been exten-sively studied [38,39] and excessive Mn intake leads to a specificcondition with motor, cognitive and psychiatric symptoms knownas manganism, with many similarities to PD. Manganism has beendescribed largely in workers in mining, welding and smeltingindustries and in battery factories, but has also been reported incommunities using drinking water with high Mn concentrations.Manganism may also arise from intravenous Mn intake, as in long-term parenteral nutrition or in methcathinon (ephedron) abuserswho use potassium permanganate for the synthesis of the drug [12].Similar clinical symptoms as in manganism have been describedin end-stage liver diseases and due to mutations in the Mn trans-porter gene SLC30A10 associated with insufficient biliary excretionof Mn [40]. Clinical symptoms, neuroimaging findings, pathologicalexamination [41] and lack of response to dopaminergic drugs [42]suggest that manganism is not caused by dysfunction of dopami-nergic neurons in the SN and that it is pathophysiologically differentfrom idiopathic PD. It has been suggested that chronic Mn expo-sure in doses insufficient to induce manganism may be toxic tothe SN rather than the globus pallidus (GP) and can increase therisk of developing idiopathic PD [43]. Several epidemiological stud-ies have found an association between Mn exposure and risk ofidiopathic PD [36,37,44–49], however other studies do not sup-port such an association [35,50,51]. Some reviews [52,53] and onemeta-analysis [54] concluded that available data argue against arelationship between Mn exposure and PD. The latter analysis usedstrict inclusion criteria leading to exclusion of all studies favor-ing the role of Mn in PD pathophysiology and the topic is stillsubject to debate. Efforts to establish a firm conclusion are partlyhampered by difficulties in differentiating between idiopathic PDand manganism-related atypical parkinsonism in large scale epi-demiological studies [55]. Several factors may increase individual

susceptibility to PD-related Mn toxicity, e.g. age, ATP13A2 muta-tions, liver dysfunction or iron deficiency [12]. Epidemiologicalstudies of the relationship between Mn exposure and PD shouldtake these factors into account [43].

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nimal models

A selective dopaminergic loss has been produced by intranigrale infusion in healthy mice [56,57], and also by repeated intraperi-oneal injection of 10 mg iron dextran three times per week duringour weeks in young Wistar rats [58]. In the 1-methyl-4-phenyl-,2,3,6-tetrahydropyridine (MPTP) PD mouse model it was shownhat unilateral MPTP treatment can induce 100% increase in Fe con-entration in the ipsilateral SN after 3–6 months [59,60] along withecreased ceruloplasmin ferroxidase activity [61]. While exam-

ning the timeline of consequences of MPTP treatment, it wasbserved that Fe deposits in SN appeared 4.5 months after treat-ent. Simultaneously a nigral cell loss of 40% had occurred [62]

ndicating that Fe increase in the SN does not precede but mayollow the onset of neurodegenerative changes. Nigral cell dam-ge in the 6-hydroxydopamine (6-OHDA) animal model of PD islso accompanied by increased Fe levels in the SN [63].

Chelator pretreatment reduces degeneration of SN neurons inPTP, 6-OHDA and lactacystin-induced PD animal models [63–69].MT1 upregulation has been documented in animal PD models.ice with dysfunctional DMT1 were partially protected against

D-inducing toxins, stressing the role of this protein in Fe medi-ted degeneration [70]. Ceruloplasmin null mice develop increasedigral Fe levels accompanied by nigral cell loss, both of which wererevented by administration of the Fe binding agent deferiprone61].

Intrastriatal injection of Mn has been shown to cause loss ofopaminergic cells [71]. Long-term exposure to Mn in developingats leads to impaired motor coordination and increased signs ofxidative stress in the striatum [72]. Contrary to results in humans,n intoxication in rodents leads to a 20–75% reduction in the num-

er of SN dopaminergic cells [73,74].Taken together, published data support the notion of Fe and Mn

oxicity directed toward nigral dopaminergic cells and protectiveffects of Fe chelating agents such as deferiprone in animal modelsf PD.

ysregulation of brain metal homeostasis

The majority of autopsy studies have reported 30–100% increasef Fe content in the SN of PD patients compared to age-matchedontrols but several studies did not confirm these findings. Possibleeasons include different analytical methods [75] and pathophysi-logical heterogeneity in PD [76]. One study indicated that the Feontent in the SN was not elevated in mildly affected but only indvanced PD patients [77], suggesting that Fe increase may note involved in the early disease development. The majority oftudies using inductively coupled plasma mass spectrometry (ICP-S) or atomic absorption spectroscopy (AAS) showed increased

e concentration in PD [78–88] (Table 1). It has been argued thategative results yielded by Mössbauer spectroscopy [89,90] are aonsequence of its relatively poor sensitivity compared to othernalytical techniques [91]. Coulometry performed using FerrochemI, which was employed by Loeffler et al. [92], is known for inter-erence with various substances [93] and it cannot be excludedhat interference with some compound in PD SN contributed tohe negative results.

Measurement of metal concentrations in tissues is prone toarious biases such as instability of metal species during sam-le preparation and analysis, sampling contamination, and metal

eaching during tissue storage. In post-mortem studies tissue metalomposition may differ from the living condition. Agonal changes

ncluding ischemia, BBB disruption and cessation of cell respira-ion might alter not only the redox state and labile metal poolroportion but also total tissue metal concentration as has beenocumented for Fe during transient brain ischemia in rats [94].

PRESSedicine and Biology xxx (2014) xxx–xxx 3

The interval between death and tissue freezing or fixation is alsoan important factor influencing the extent of lytic changes thatmight also alter tissue metal composition. Since procedures usedto avoid contamination [95,96] are either not reported or not con-sidered in the majority of studies, it cannot be excluded that errorsduring sample processing contribute to disparate results [91]. Itshould be also noted that routinely used metal analytical methodssuch as AAS or ICP-MS alone cannot distinguish between differentmetal species or oxidation states. Also, differences in total con-centration of a particular metal in a tissue may reflect differencesin the content of metallo-proteins and enzymes (e.g. ceruloplas-min in the case of serum Cu [97] or glutamine synthetase in thecase of brain Mn [98]). Thus, finding of abnormal total tissue metalconcentrations does not automatically implicate abnormal metalstores or abnormal labile (free) metal concentrations. Hyphen-ated techniques, notably high performance liquid chromatography(HPLC) and capillary zone electrophoresis (CZE) online with ICP-MS, have been used for studying Mn species in body compartmentsof healthy subjects [99,100]. These studies documented that mostof the Mn-containing compounds found in liver were Mn-enzymes(for review see Ref. [101]). In order to better understand the role ofaltered metal content in neurodegenerative diseases, similar spe-ciation studies need to be performed in brain tissue and CSF ofpatients.

Using microscopic in situ imaging techniques with resolution atthe micro- or nanometer scale it was shown that Fe deposits in theSN of PD patients are predominantly associated with neuromelaninin dopaminergic neurons [86,102] and there is evidence that Fe inthese deposits is not strongly bound and thus potentially redoxactive. Suboptimal concentrations of the ferritin protein have beenreported in the SN of PD patients and also in cases of incidentalLewy body disease [78,85,103–105]. Normal ferritin upregulationassociated with increased Fe content during aging was absent inPD patients due to sustained activity of iron regulatory protein 1(IRP1) [106]. Ferritin is the main Fe storage compound in glial cellsand its decreased levels does not necessarily affect Fe metabolism inneurons [107]. Interestingly, ferritin was shown to be a componentof neuromelanin granules suggesting its possible iron-regulatoryfunction also in neurons [108]. Moreover, neuromelanin granulesoverloaded with Fe [10,81,83] exhibit higher level of redox activityin PD [109]. Taken together, these studies suggest that disrup-tion of the ferritin complex along with other disturbances of Feregulation may eventually overwhelm the storage capacity of theneuromelanin system, which may lead to an increased labile Fepool promoting the neurodegenerative process [110]. IncreasedFe3+/Fe2+ ratio in PD was also noted in two studies from the samegroup, using spectrophotometric measurements on samples fromfresh frozen brains pretreated with hydrochloric acid and pepsinwhere ascorbic acid was used to reduce Fe. They concluded that anincreased Fe3+/Fe2+ ratio is a result of ongoing oxidative processes[77,87]. This interpretation is speculative since conditions knownto increase oxidative stress such as ischemic insult or Mn overloadcause the opposite effect, that is an increased Fe2+/Fe3+ ratio [111].Moreover, this finding was disputed by other groups who did notfind any detectable Fe2+ iron in SN using Mössbauer spectroscopy[89] and micro-XANES technique [112]. The abnormally high con-centration of Fe2+ might have been an artifact of the measurementtechnique used by Sofic et al. [91,113]. This is in line with the his-tochemical distribution of Fe3+ and Fe2+ [114] and the finding thatthe major Fe storage molecules in SN, ferritin and neuromelanin,store Fe in the ferric (Fe3+) form [115].

Iron accumulation in PD is confined to the SN, while its lev-

els in the striatum and cerebral cortex are comparable to those ofcontrols or even decreased [116]. Results in the GP are inconclu-sive since both normal [77,87], increased [82,92] and decreased[78,117] concentrations have all been reported. Also CSF studies

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Table 1Concentration of metals in post-mortem basal ganglia and cortical tissue in PD patients.

Reference Method N Main findings (Fe, Cu, Mn content) Comments

Ayton [61] AAS 10 ↑Fe in SN +42%↓Cu in SN −51%=Fe, Cu in frontal GM

↓Ferroxidase activity in SN

Davies [145] SRXFMPIXE 5 ↓Cu in SN (neuromelanin) -50%, LC(neuromelanin) −57%↑Fe in SN (neuromelanin) +37%=Fe, Cu in occipital GM

=Zn in SN, LC, occipital GM↓Ctr1 in SN neurons↑SOD1 in SN

Visanji [105] AAS 3 ↑Fe in SN +100%*

=Fe in Put, pons↑Ferroportin expression in SN↑Ferritin in Put↓TfR in Put

Szczerbowska-Boruchowska (2012,2004) [208], [209]

SRXFM 4 ↑Fe in SN (neurons) +60%=Cu in SN (neurons)=Fe, Cu in SN (non-neuronal tissue)

↑Ca, Zn in SN (neurons)↑Rb, Zn in SN (non-neuronal tissue)

Barapatre (2010) [210] PIXE 6 =Fe in SN (neuromelanin/non-neuronal tissue) ↓Ca in SN (neuromelanin/non-neuronal tissue)Wypijewska (2010)

[90]MS AAS 17 =Fe, Cu in SN ↑labile Fe in SN

=Labile Cu in SNOnly Fe3+ detected in SN

Popescu [117] XFM 1 ↑Fe in SN +40%↓Fe in GP, Put, CN, cGM↓Cu in SN

↓Zn in SN, Gp, Put, CN, GM

Oakley [86] WDXMA 16 ↑Fe in SN (neurons) +100%↑Fe in SN (neuropil) +70%

Reinert (2007) [211] PIXE 1 =Fe in SN (neuromelanin)*

↓Cu in SN (neuromelanin)* −190%↑Ca in SN (neuromelanin)↓Ni in SN (neuromelanin)

Morawski (2005) [212] PIXE 6 ↑Fe in SN (neuromelanin) +100%=Fe (non-significant increase) in SN(cytoplasm, glia)

Griffiths [82,88] AAS EXAFS 6 ↑Fe in SN +100%, GP +43%= Fe in Put, CN, cGM

↑Ferritin loading in SN

Galazka-Friedman [89] MS 6 =Fe in SN Only Fe3+ detected in SNKienzl (1995) [213]

Jellinger [84]EDXMA 3 ↑Fe in SN (neuromelanin)*

=Fe in SN (Lewy bodies, cytoplasm)*Only Fe3+ detected in SN

Loeffler [92] COL 14 =Fe in SN, Put, CN, frontal GM↑Fe in GP*

=Tf in SN, GP, Put, CN, frontal GM

Mann [85] ICP-MS 18 ↑Fe in SN +56% =Zn in SN=ferritin in SN

Good [81] LAMMA 3 ↑Fe in SN (neuromelanin)* +45%=Fe in SN (cytoplasm/neuropil)*

↑Al in SN (neuromelanin)

Dexter (1989)[214,78,79]

ICP-MS 27 ↑Fe in SN +35%↓Fe in GP −29%↓Cu in SN −34%=Mn in SN, Put, CN, cGM=Cu in GP, Put, CN, cGM=Fe in Put, CN, cGM

↑Zn in SN, Put, CN=Pb in SN, cGM

Hirsch [83] WDXMA 5 ↑Fe in SN (non-neuromelanin) +240%↑Fe in SN (Lewy bodies) +400%↓Fe in SN (neuromelanin) -35%

=Zn in SN↑Al in SN (Lewy bodies)

Riederer [77] AAS 13 ↑Fe in SN (only in severe degeneration) +35%↑Cu in RN=Fe in GP, Put, CN, RN, cGM=Cu in SN, GP, Put, CN, cGM

↑Zn in RN=Ca, Mg, Zn in SN, GP, Put, CN, RN, cGM↑Ferritin in SN↑Fe3+/Fe2+ ratio

Uitti [146] AAS, AES 9 ↓Cu in SN=Fe in SN, CN=Mn in SN, CN

↓Mg in CN= Ag, Al, As, B, Be, Ca, Cd, Co, Cr, K, Pb, Mn, Mo,Na, Ni, P, Se, Ti, V, W, Zn in SN, CD

Sofic [87] SPF 8 ↑Fe in SN +76%=Fe in Put, GP, cGM

↑Fe3+/Fe2+ ratio

Earle [80] XFS 11 ↑Fe in SN, BG +100%=Mn in SN, BG

=Zn in SN, BG

AAS – atomic absorption spectroscopy, AES – atomic emission spectroscopy, ICP-AES – inductively coupled plasma-atomic emission spectrometry, ICP-MS – inductivelycoupled plasma-mass spectrometry, SPF -spectrophotometry, MS – Mössbauer spectroscopy, (SR)XFM – (synchrotron radiation) X-ray fluorescence microscopy, PIXE –particle induced X-ray emission, WDXMA – wavelength dispersive X-ray microanalysis, XFS – X-ray fluorescence spectroscopy, LAMMA – Laser Microprobe Mass Analysis,E y micrn transN

hc(nhFdP

XAFS – Extended X-ray absorption fine structure, EDXMA – energy dispersive X-raucleus, RN – red nucleus, BG – basal ganglia, (c)GM – (cortical) gray matter, Tf(R) –

= number of patients.* Not statistically significant or statistical analysis not performed.

ave reported both normal [118–121] and decreased [122–124] Feoncentration in PD suggesting no overall Fe increase in the CNSTable 2). Here it is important to note that speciation analysis wasot performed in these CSF studies and neither abnormal cellular Fe

andling, Fe2+/Fe3+ dysbalance nor increased proportion of labilee can be excluded. Alteration in Fe balance can be suspected sinceecreased CSF ferroxidase activity (see below) has been found inD [125].

oanalysis, SN – substantia nigra, LC – locus coeruleus, Put – putamen, CN – caudateferrin (receptor).

The cause of metal regulatory protein dysfunction in PD SN isunknown. Several genes involved in the causation of PD are relatedto the metabolism of metals [126] and mutations in DMT1, cerulo-plasmin and transferrin were found to be weakly associated with

the risk of developing PD [127–129]. Several studies have docu-mented abnormalities in ceruloplasmin function in PD. Specifically,decreased ferroxidase activity was found in plasma [130,131],CSF [125,132] and post-mortem SN tissue [61]. Dysfunction of

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Table 2Concentration of metals in the CSF of PD patients.

Reference Method N Main findings Comments

Hozumi [119] ICP-MS 20 ↑Cu +80%, Mn +70%=Fe

↑Zn +174%=Mg

Boll [132] AAS 22 ↑Cu +230% (free) Free Cu correlated with motor impairmentAlimonti [122] ICP-AESSF-ICP-MS 42 ↓Fe −20%

=Cu, Mn↓Co, Cr, Pb, Si, Sn=Al, Ba, Be, Bi, Ca, Cd, Hg, Li, Mg, Mo, Ni, Sr, Tl, V, W, Zn, Zr

Bocca [123] ICP-AESSF-ICP-MS 91 ↓Fe −37%, =Cu, Mn ↓SiForte [124] ICP-AESSF-ICP-MS 26 ↓Fe* −55%

=Cu, Mn↓Si=Al, Ca, Mg, Zn

Boll [125] AAS 49 =Cu (total) Ferroxidase activity/total Cu inversely related to PD stageJimenez-Jimenez [121] AAS 37 =Fe, Mn, Cu ↓ZnGazzaniga [118] AAS 11 =Fe, Mn, CuPall [120] AAS 31 =Fe, Mn

↑Cu↑Cu (free) confirmed byphenantroline-copper assay

AAS – atomic absorption spectroscopy, AES – atomic emission spectroscopy, ICP-AES – inductively coupled plasma-atomic emission spectrometry, ICP-MS – inductivelycN

cttmOpeawFctmccslriLa[dwhth

biutblocPcI[isleiib

oupled plasma-mass spectrometry. = number of patients.* Not statistically significant or statistical analysis not performed.

eruloplasmin may be a consequence of oxidative changes inhis enzyme [133]. Decreased ferroxidase activity was foundo be associated with earlier disease onset [134,135] and with

arkers of iron deposition in the SN of PD patients [129,136,137].ther studies have reported abnormal expression of Fe regulatoryroteins. Nigral cells behave as if they were Fe deficient in PD;xpression of proteins involved in iron uptake as DMT1, TfR2nd lactotransferrin receptor (LTFR) is upregulated [138,139],hereas expression of proteins involved in iron efflux such as

PN1 is downregulated [140]. SN areas with the most severeell degeneration showed an increased LTFR expression [139]. Athe cellular level, regulation of Fe metabolism is dependent on

itochondria where Fe is incorporated into iron-sulfur (Fe S)lusters by the action of a complex of multiple enzymes. Fe Slusters are prosthetic groups necessary for proper function ofeveral proteins such as Complexes I–III of oxidative phosphory-ation, ubiquinone and aconitase. The latter is also known as ironesponsive element binding protein 1 (IREB1) since it is involvedn the regulation of the transcription of Fe transport proteins.ow level of Fe S cluster production leads to low cytoplasmicconitase activity and increased transcription of TfR and DMT1141]. Upregulation of Fe uptake may be related to mitochondrialysfunction associated with decreased Fe S cluster synthesis,hich was documented in nigral cells in PD [142]. Alternatively, itas been show that iron regulatory protein 2 (IRP2) is degraded byhe ubiquitin–proteasome system and its dysfunction may lead toigh IRP2 levels which in turn stimulate production of DMT1 [143].

Concerning Cu, greater variation in Cu metabolic profiles haseen shown in PD compared to healthy controls using 65Cu

sotope tracer. Most importantly, higher peak plasma Cu val-es were documented suggesting more efficient absorption inhe gut [144]. Increased concentration of redox available Cu haseen reported in PD CSF [120] and its concentration was corre-

ated with motor impairment [132]. The SN contains high levelsf Cu in healthy subjects [145]. The decrease in total Cu con-entrations reported in the SN in the majority of studies inD patients [61,78,102,146] may be a reflection of loss of theuproproteins superoxide dismutase 1 (SOD1) or ceruloplasmin.n post-mortem PD SN tissue normal SOD1 activity was found102]. Moreover, decreased Cu levels are not accompanied by sim-lar decreases in zinc (Zn) levels in the majority of post-mortemtudies (Table 1) which would be expected in the case of SOD1oss. Currently, it seems more likely that decreased SN Cu lev-

ls is a consequence of reduced ceruloplasmin content whichs in line with the low ceruloplasmin ferroxidase activity foundn SN [61]. Increased labile Cu in the CSF of PD patients maye caused by its impaired incorporation into cuproproteins or

dysfunction of the blood–CSF barrier similar to the situation inWD [147].

Abnormal concentrations of Mn have not been reported inPD brains [78–80,146], but all published studies are rather old.The majority of studies also found normal Mn levels in the CSF[118,120–124].

Wilson’s disease (WD)

WD is a genetic disorder caused by mutation of the ATP7Bprotein which is mostly expressed in the liver [148]. Dysfunc-tion of ATP7B leads to disturbed Cu metabolism with a gradualaccumulation of Cu in the body and reduced synthesis of variouscuproproteins, in particular ceruloplasmin. The traditional view onthe pathophysiology of neurologic symptoms in WD holds thatoverwhelming of the liver storage capacity for Cu leads to itsspillage into plasma and CSF, leading to cellular uptake and oxida-tive damage in the CNS [149], although alternative theories havebeen proposed [150]. However, ATP7B is expressed also in the brain,namely in the choroid plexus, basal ganglia, cerebellum and cor-tex [145] and its dysfunction may lead to neurological symptomsthrough other mechanisms than just overflow of hepatic Cu. ATP7Bdysfunction in astrocytes may hypothetically lead to impairedproduction of ceruloplasmin with the possible consequence ofimpaired Fe efflux, similar to the situation in aceruloplasminemia[151]. It has been shown that ATP7B is expressed in the choroidplexus and its dysfunction may hamper the elimination of excessCu across the blood–CSF barrier [152].

The combined evidence of animal, neuroimaging, CSF and post-mortem metal studies (see below) suggests that metals other thanCu may accumulate in WD.

Local dysregulation of metal homeostasis

In patients with the neuropsychiatric form of WD, measure-ments of Cu in CSF and post-mortem brain tissue clearly show thatCu concentrations are diffusely increased in the CNS up to 850% ofnormal levels [153] (Table 3). With chelating treatment it takesup to 4 years for CSF Cu concentrations to normalize [154,155]and high levels are a marker of non-compliance [156]. ProfoundCu accumulation is still persistent in all brain regions after 10months of chelating treatment as shown by post-mortem studies

[157–159]. Another post-portem study showed abnormal brain Cudeposits even after several years of chelating treatment, and alsothat the severity of neuropathological findings was correlated withcerebral copper content [160].

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Table 3Concentration of metals in the CFS and brain tissue in WD.

Reference Method N Type of tissue Main findings Comments

Kodama [153] AAS 4 (2 neu, 2 hep) CSF ↑Cu in neuro WD +280%=Cu in hepato WD

=Zn↓Cp concentrationCu decreases with therapy

Stuerenburg [155] AAS 4 (neu) CSF ↑Cu WN Cu decreases with therapyWeisner [154] AAS 5 (neu) CSF ↑Cu +330% ↓Cp concentration

Cu decreases with therapyCumings (1948) [158] COL 3 Brain ↑Cu* in CN(+160%), Put(+600%), thal(+380%), GP(+60%),

WM(290%), GM(+320%)↑Fe* in CN(+100%), Put(+20%), thal(+160%), WM(+38%),GM(+140%)=Fe in GP

Faa [159] ICP-AES 1 Brain ↑Cu** in all brain regions↓Fe** in all brain regions

↓Mg, Zn, Ca in all brainregions

Litwin [157] ICP-MS 12 (10 neu, 2 hep) Brain ↑Cu in Put(+540%), DN(+590%), pons(+540%), frontalGM(+850%)↑Fe in ND(+74%)=Fe in Put, pons, frontal GM=Mn in Put, DN, pons, frontal GM

=Zn in Put, DN, pons, frontalGM

AAS – atomic absorption spectroscopy, COL – colorimetry, ICP-AES – inductively coupled plasma-atomic emission spectrometry, ICP-MS – inductively coupled plasma-massspectrometry.SN – substantia nigra, DN – dentate nucleus, Put – putamen, CN – caudate nucleus, GM – gray matter, WM – white matter.Neu – neuropsychiatric form, hep – hepatic form, N = number of patients.

m[bilitg[GwtadhiitPiv

ptFTmlWpdtammctSo

* No statistical analysis performed.** No control group, compared to published reference values.

Increased Fe content has been reported in the striatum, grayatter, white matter [158] and dentate nucleus of the cerebellum

157] in post-mortem studies. The latter study reported normalrain Mn concentrations, but Mn was not measured in the GP which

s the structure with the greatest predisposition toward accumu-ation of Mn and other divalent metals [26]. In fact, MRI studiesndicate that elements such as Fe and Mn may also accumulate inhe brain over the course of WD. T1 hyperintensities in GP are sug-estive of Mn deposits in patients with the hepatic form of WD161,162]. Several MRI studies documented T2 hypointensities inP and other brains regions in WD patients suggestive of depositsith paramagnetic properties [163–167]. It has been suggested that

he GP changes might reflect Cu deposits [168], since Cu2+ speciesre paramagnetic. However, little is known of which form Cu iseposited in the WD CNS, and another possibility is that these T2ypointensities correspond to the paramagnetic effect of ferritin-

ron. It has been documented that the area of T2 hypointensitiesncreases during chelating treatment but it seems unlikely thathis is due to increased brain Cu concentrations [165]. Indeed, aET study with radioactively labeled Fe in human subjects showedncreased brain Fe uptake in WD patients compared to healthyolunteers [169].

Iron accumulation in WD may be a consequence of low cerulo-lasmin levels. However, it has been reported that as little as 10% ofhe normal plasma level of ceruloplasmin was sufficient for normale metabolism in a pig model [170] and in liver preparations [171].his finding was confirmed in WD patients in whom defective Feetabolism was observed only in those who had ceruloplasmin

evels less than 5% of the normal range [172]. The fact that mostD patients have more than 10% of the normal level of cerulo-

lasmin also argues against gross changes in Fe metabolism in thisisease. Although some authors reported elevated liver Fe concen-ration in WD patients [151], results of the largest published studyrgue against a clinically relevant elevation of Fe in the liver in theajority of patients [173]. This, however, pertains to peripheral Feetabolism and CNS may be more susceptible to oxidative damage

aused by decreased ceruloplasmin function. Another factor con-ributing to the risk of Fe accumulation may be mode of treatment.tandard penicillamine chelation does not remove Fe from therganism; conversely it may lead to higher tissue Fe accumulation

because it decreases the availability of Cu for ceruloplasminproduction. Zinc treatment can have opposite effects [174]. Nodifference in brain Fe concentration was however found betweenpatients treated with Zn and penicillamine [157]. Ceruloplasminalso interacts with Mn and its decreased activity can alter the neu-rotoxicity of Mn [175].

Altogether, these data suggest that profoundly increased Cu con-centrations in the CNS of WD patients persist for a long time duringchelating treatment and that local accumulation of Fe in certainbrain nuclei may occur during the course of WD.

Animal models

Established animal models for WD are the Long Evans Cinnamon(LEC) rat [176], the toxic milk mouse [177] and the Atp7b(−/−)mouse [178]. In LEC rats markedly increased concentrations ofloosely bound Cu were found in liver and plasma when total Cu con-centration exceeded the capacity of liver metallothionein synthesis[179–181]. Increased brain Fe levels have been demonstrated inLEC rats [182] and low Fe diet as well as Fe removal by phlebotomywas beneficial in terms of decreased oxidative stress markers inthe liver [183,184]. Treatment with Zn not only reduced the liverdamage through induction of metallothionein expression but alsoled to decreased liver Cu and Fe concentrations in LEC rats [185].In the toxic milk mouse, increased Cu stores were found in all tis-sues, but elevated Fe was present only in the kidneys, possibly as aresult of hemolysis [186]. The brain Fe content was even decreasedin the hippocampus [187]. Penicillamine treatment in these miceincreased labile Cu in plasma and brain tissue as a result of itsmobilization from the large tissue deposits and enhanced oxida-tive stress, which may explain the initial worsening seen in humanpatients [188].

In the Atp7b(−/−) mouse imaging of Cu metabolism usingingested 64Cu showed Cu accumulation in liver but not in brain[189]. In the same WD model decreased ferroxidase activity ofceruloplasmin was related to decreased levels of serum Fe and

transferrin saturation [190]. In contrast, Cu intoxication itself doesnot cause Fe accumulation in liver or brain of rats [191]. This find-ing suggests indirectly that decreased ceruloplasmin, rather thanCu toxicity, affects impaired Fe homeostasis in WD.

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oncluding remarks

Current data from studies measuring metal concentrations inrain tissue and CSF do not support the idea of a general metalccumulation in the entire CNS of PD patients. Local dysregulationf Fe metabolism seems to occur in the SN, at least in a sub-roup of PD patients. The pathophysiological relation of iron toeurodegeneration and why it selectively affects the SN remainsnanswered. It has been suggested that dopamine producing cellsre particularly vulnerable to metallotoxic effects [192]. One of theulnerable enzymes in the dopamine synthesis pathway may beyrosine hydroxylase, which requires appropriate amounts of ironnd molecular oxygen as cofactors. Animal data as well as follow-p of patients with manganism gives some support to the idea ofxposure to Mn or Cu as aggravating factors in PD [193,194].

Abundant evidence suggests that decreased activity of cerulo-lasmin in CSF and SN is contributing to Fe accumulation in PD anday be relevant to its pathophysiology. It should not be forgot-

en that in contrast to Fe deposition, neurodegenerative changesn PD are not restricted to dopaminergic neurons in the SN [195].his argues against the hypothesis that Fe accumulation alone ishe trigger of neurodegeneration in PD. Nevertheless, based onhe likely presence of labile Fe in the SN, labile Cu in the CSF andesults from animal studies, we suggest along with other authors196] that a therapeutic trial with metal chelating agents is jus-ified in PD. Several points, however, need to be considered. Theose of the chelating drug should be rather low, aiming at targetedetal detoxification and redistribution of iron to tissues capable

f reutilizing it [197]. Due to the risk of increased concentrationsf labile Cu, a drug with Cu chelating capabilities may be pre-erred in future studies. The Fe chelator deferiprone is currentlyeing tested in early stage PD (Clinicaltrials.gov NCT01539837 andCT00943748). Results from the latter study document that a mod-rate dose of deferiprone (30 mg/kg/day) given for 12 months isafe and efficient in Fe removal from the SN. Moreover, using theelayed-start paradigm this study indicated a potential disease-odifying effect of deferiprone in PD [69]. Since oxidative stress

nd glutathione depletion apparently accompany the progressionf PD treatments with antioxidants have been examined. Althoughontroversial results of such treatments have been reported, e.g.sing vitamin E or vitamin C [198–200], the glutathione preser-ing agent lipoic acid appears to possess a therapeutic potentialccording to an animal study [201].

Regarding WD, little is known about accumulation of otheretals beside Cu, but dysfunction of ceruloplasmin may lead to Fe

ccumulation while liver dysfunction may lead to Mn accumula-ion in the CNS. Both metals may contribute to neurodegeneration,specially while acting synergistically with Cu. The rationale oftudying synergistic metal toxicity is further strengthened byecent studies of multiple metals with neurotoxic properties in theSF in other neurodegenerative disorders [202,203]. More stud-

es of toxic interactions among metals are needed, because suchtudies may produce invaluable information for the optimal choicef therapeutic metal chelating agents. Disodium calcium edetateCaNa2–EDTA) has been successfully used to decrease tissue man-anese levels and improve neurological symptoms in manganism204,205]. EDTA chelation has often been administered togetherith high doses of vitamin C in order to decrease oxidative stress

n several disorders. This approach, however, does not seem toe substantiated by recent clinical observations, and paradoxicallyitamin C may enhance oxidative stress [206]. Unlike penicillamine,rientine not only chelates Cu but also Fe and Mn, although the sta-

ility constants of these complexes are lower than that with Cu207]. Thus, trientine may be useful in patients with signs of Fe and

n accumulation in combination with copper. For this reason, theres a need for further studies examining free metal concentrations in

PRESSedicine and Biology xxx (2014) xxx–xxx 7

larger groups of WD patients and studies validating the use of brainMRI in determining the regional distribution of specific metals.

Conflict of interest

The authors have no conflict of interest.

Acknowledgments

This work was supported by the Czech Ministry of Education,research project PRVOUKP26/LF1/4 and by the Czech-NorwegianResearch Programme CZ09.

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