the role of iron in neurodegeneration

The Role of Iron in NeurodegenerationProspects for Pharmacotherapy of Parkinson’s Disease

Kurt A. JellingerLudwig Boltzmann Institute of Clinical Neurobiology, Vienna, Austria

ContentsAbstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

1. Iron and the ‘Oxidative Stress’ Hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1162. Oxidative Stress in Parkinson’s Disease (PD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1173. Iron and PD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1204. Iron-Related Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

4.1 6-Hydroxydopamine Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1234.2 N-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Model . . . . . . . . . . . . . . . . . . . . . . . 1244.3 Iron-Loading Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1254.4 β-Carbolines: The ‘TaClo’ Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1264.5 Overview of Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

5. Iron-Melanin Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1266. Systemic Iron Metabolism in PD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1297. Iron in Other Neurodegenerative Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1298. Current and Future Therapeutic Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

8.1 Neuroprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1298.2 Iron Chelators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1308.3 Lazaroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

9. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

Abstract Although the aetiology of Parkinson’s disease (PD) and related neurode-generative disorders is still unknown, recent evidence from human and experi-mental animal models suggests that a misregulation of iron metabolism, iron-induced oxidative stress and free radical formation are major pathogenic factors.These factors trigger a cascade of deleterious events leading to neuronal deathand the ensuing biochemical disturbances of clinical relevance.

A review of the available data in PD provides the following evidence in supportof this hypothesis: (i) an increase of iron in the brain, which in PD selectivelyinvolves neuromelanin in substantia nigra (SN) neurons; (ii) decreased availabil-ity of glutathione (GSH) and other antioxidant substances; (iii) increase of lipidperoxidation products and reactive oxygen (O2)species (ROS); and (iv) impairedmitochondrial electron transport mechanisms. Most of these changes appear tobe closely related to interactions between iron and neuromelanin, which result inaccumulation of iron and a continuous production of cytotoxic species leading toneuronal death.

Some of these findings have been reproduced in animal models using 6-hydroxydopamine, N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), ironloading and β-carbolines, although none of them is an accurate model for PD in

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humans. Although it is not clear whether iron accumulation and oxidative stressare the initial events causing cell death or consequences of the disease process,therapeutic efforts aimed at preventing or at least delaying disease progressionby reducing the overload of iron and generation of ROS may be beneficial in PDand related neurodegenerative disorders.

Current pharmacotherapy of PD, in addition to symptomatic levodopa treat-ment, includes ‘neuroprotective’ strategies with dopamine agonists, monoamineoxidase-B inhibitors (MAO-B), glutamate antagonists, catechol O-methyltransferaseinhibitors and other antioxidants or free radical scavengers. In the future, theseagents could be used in combination with, or partly replaced by, iron chelatorsand lazaroids that prevent iron-induced generation of deleterious substances. Al-though experimental and preclinical data suggest the therapeutic potential ofthese drugs, their clinical applicability will be a major challenge for future re-search.

Neurodegenerative diseases are inexorably pro-gressive disorders that affect selected vulnerableneuronal populations of the CNS, leading to neuronaldeath and disruption of neuronal circuits. These inturn cause variable biochemical disturbances andspecific clinical deficits. Parkinson’s disease (PD),one of the most frequent neurodegenerative disor-ders of advanced age, is clinically characterised byakinesia, resting tremor and rigidity. Pathologicalfindings include progressive degeneration of thenigrostriatal dopaminergic loop and other subcor-tical neuronal systems, causing striatal dopaminedeficiency and multiple biochemical deficits ofclinical relevance.[1] Loss of nigral neurons correlateswith both the duration of illness and the severity ofmotor dysfunctions (akinesia, rigidity),[2,3] and isassociated with formation of Lewy bodies (LB),the morphological hallmark of PD. These intra-neuronal inclusions, occurring in many subcorticalnuclei and in cerebral cortex, are composed of phos-phorylated neurofilament subunits, α-synuclein,ubiquitin and other cytosolic proteins, and othercellular elements.[4-7]

The causes of neuronal death in PD and otherneurodegenerative disorders are still unclear. How-ever, there is growing evidence for a cascade ofmultiple deleterious factors, including oxidativestress, lipid peroxidation and altered iron metabo-lism, leading to excess free radical formation, mi-tochondrial dysfunction and disturbed calcium ho-meostasis, which in turn result in cytoskeletal

damage and cell death.[8-20] One factor capable ofproducing oxidative stress and mitochondrial dys-function is an excess of intracellular iron, whichmay accelerate oxidative stress and lipid peroxida-tion.[8-23] It remains to be clarified whether oxida-tive stress and deposition of iron are initial eventsthat cause cell death or a consequence of the dis-ease process, how iron gains access to neurons un-dergoing degeneration, and whether these changesare unique to the substantia nigra (SN). It shouldbe noted that neuronal injury, by any mechanism,can lead to iron release and increased free radicalformation, even though the causes of the injurymay be unrelated to these mechanisms.

Although the aetiology of PD is still unknown,the evidence accumulated suggests that the patho-genesis of PD and related neurodegenerative disor-ders may be caused, at least in part, by misregulationof major proteins of iron metabolism,[11,12,20,24-26]

and that iron-induced oxidative stress represents atestable hypothesis in animal models as well as intherapeutic trials.[18,20,27-33]

1. Iron and the ‘Oxidative Stress’ Hypothesis

Oxidative stress is a state of imbalance betweenthe production of reactive oxygen (O2) species(ROS) and antioxidative protective mechanisms,leading to uncontrolled oxidation of critical bio-logical molecules. Excessive formation of hydro-

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gen peroxide (H2O2) and O2-derived free radicalscan result in cell damage due to chain reactions ofmembrane lipid peroxidation or alteration of mem-brane fluidity.[18,20,34,35] H2O2 is relatively inert andnoncytotoxic. However, cell damage occurs whenROS and highly reactive hydroxyl radicals (OH•)are produced by the Fenton reaction, in which areduced transition metal, e.g. Fe++ or complexediron, delivers an electron to H2O2:

H2O2 + Fe++ → OH• + OH– + Fe+++

The Fenton reaction readily converts H2O2 tothe most cytotoxic of the ROS, the OH• radical.Recycling of iron between its oxidised and reducedstates allows iron to act as a catalytic agent, pro-ducing a cascade of free radicals. Recycling of ironto its reduced state can be mediated by tissue ascor-bic acid, glutathione (GSH), dopamine or by super-oxide in the Haber-Weiss reaction. Nitrosylation orhydroxylation may occur via the mechanism of ni-tric oxide (NO) combining with oxygen to formperoxynitrite (ONOO–):

NO + O2 → ONOO–

Protonated peroxynitrite can decompose intohydroxyl and nitrogen dioxide radicals:

Nitration of tyrosine by nitronium ionscatalysed by iron is a major product of peroxyni-trite, the formation of which does not require tran-sition metals. Once formed, peroxynitrite can dif-fuse over several cell diameters to produce celldamage by oxidising lipids, proteins and DNA, andsuperoxide, that is probably formed from dopa-mine oxidation, has been shown to accelerate DNAdamage by increasing free iron levels.[36] NO thatis formed by oxidation of L-arginine by NADPHand molecular oxygen catalysed by the mono-oxygenase NO synthase (NOS),[11] has, therefore,been proposed as a prime candidate for mediatingoxidative damage in vivo.[37] Protein nitration isone of the oxidative mechanisms that has been

demonstrated at the cellular level in neuro-degeneration disorders, indicating that a sequenceof specific oxidative reactions has taken place.Nitration of tyrosine residues has been suggestedto represent a major mechanism of oxidative mod-ifications of proteins, producing dysfunctionalproteins[37,38] and has also been proposed as havinga role in neurodegenerative disease.[13,39,40]

All of the above reactions involve free radicalattack on biomolecules, leading to a cascade of eventscausing damage to mitochondrial electron trans-port, decompartmentalisation of intracellular Ca++

homeostasis, induction of proteases, increasedmembrane lipid peroxidation and, finally, celldeath.[8,10,12,20,23,29,35,36] A number of mechanismshave evolved to prevent or stop the oxidative cas-cade at different points and thus reduce the likelihoodof subsequent cell damage (fig. 1). These includeferritin, capable of storing iron in an unreactiveform, detoxification of ROS by enzymatic sys-tems, for example superoxide dismutase (SOD) orGSH/glutathione peroxidase (GPO), and free rad-ical scavengers such as ascorbic acid (vitamin C)and α-tocopherol (vitamin E).[18,20,22,23,32,33,41]

2. Oxidative Stress in Parkinson’s Disease (PD)

SN neurons may be at risk for damage by freeradicals because H2O2 is generated as a byproduct

2 H2O2 Catalase

2 H2O + O2

H2O2 + R − (OH)2Peroxidase

2 H2O + RO2

2 GSH GPO

GSSG + H2O

2 O2− + 2 H+

SOD H2O2 + O2

Fig. 1. Reaction pathways preventing formation of oxygen freeradicals. Protective agents are uric acid, α-tocopherol (vitaminE), glutathione, ascorbic acid (vitamin C), iron chelators, selegil-ine and glutathione reductase. GSH = glutathione; GSSG = ox-idised glutathione; GPO = glutathione peroxidase; O2

– =superoxide; SOD = superoxide dismutase; R = radical.

ONOO- + H+ HONOO OH + NO2

NO3

OH. + NO2.

Role of Iron in Neurodegeneration 117


of dopamine metabolism either enzymatically bymonoamine oxidase (MAO) or by oxidative deam-ination/auto-oxidation of dopamine (fig. 2). Undernormal circumstances, H2O2 in the brain is clearedby GSH, a reaction that is catalysed by GPO. How-ever, if H2O2 levels are high or if GSH levels arelow, then H2O2 has the potential to react with re-dox-reactive ferrous iron (Fe++) to generate highlyreactive OH• radicals, one of the prime mediatorsof oxidative damage (fig. 3). The accumulation ofneuromelanin, a polymerised product of oxidiseddopamine, within dopaminergic neurons is an indi-cator for oxidising conditions.[42] In PD, the vul-nerability of neurons from the substantia nigra parscompacta (SNc) appears to be related to their neu-romelanin content,[43] high expression of dopa-mine transporter messenger RNA,[44] and low im-munoreactivity for calbindin, a neuroprotectiveCa++-binding protein.[14,45,46]

Although proof is lacking that oxidative stressis a common mechanism for death of dopaminergicneurons in PD and a contributor to disease prog-ress, a body of evidence from both experimentaland human studies supports this concept (tableI).[12,13,16,18,20,22,23,27-32,35,37,47] This evidence in-cludes the following.• Levels of iron in PD brain tissue are increased,

with a shift of Fe++/Fe+++ ratio.[21,48]

• Antioxidant systems are decreased in SN: thereis a marked depletion of reduced GSH and totalglutathione[49] without a corresponding increasein the oxidised glutathione (GSSG) form,[50-52]

but unchanged or decreased GPO and catalasein SN and striatum.[49,53,54]

• Decreased levels are found of the antioxidanturic acid,[55] of α-ketoglutarate dehydrogenase,a key trichloracetate enzyme,[56] and of gluta-mate dehydrogenase, but the activities of gluta-mylcysteine synthase, the glutathione syntheticenzyme,[57] and of glutathione transferase, whichcatalyses glutathione utilisation via conjugativereactions, appear normal.[53]

• There is a significant increase of SOD, a scav-enger enzyme that catalyses detoxification ofthe superoxide anion to form H2O2 and molec-ular O2,[58-60] and Mg-SOD,[61] an important en-zyme for cellular auto-oxidation defence.

• More direct evidence for oxidative stress is in-creased malondialdehyde (MDA) and lipid per-oxidase,[62,63] and significant increases in lipidhydroperoxidase,[62] 4-hydroxynonenal protein,

Dopamine autoxidation

DA + O2 → SQ· + O2· − + H+

DA + O2· − + 2 H+ → SQ· + H2O2

Dopamine enzymatic oxidation

DA + O2 + H2OMAO

3,4,dihydroxyphenyl-+ acetaldehyde NH3 + H2O2

H2O

2 reaction

H2O2 + 2 GSHGPO

GSSG + 2 H2O

H2O2 + Fe++ GPR OH· + OH− + Fe+++

Fig. 2. Oxidative metabolism of dopamine. DA = dopamine;Fe++ = ferrous iron; Fe+++ = ferric iron; GPO = glutathione perox-idase; GPR = glutathione peroxidase reductase; GSH = gluta-thione; GSSG = oxidized glutathione; MAO = monoamineoxidase; O2

•– = superoxide radical; OH• = hydroxyl radical; OH–

= hydroxyl ion; SQ• = semiquinone.

H2O

GSSG

Gluthathioneperoxidase

GSH

NADPH+ H+

GSSGreductase

NADP+

Melanin/Fe+++Fe++

Fe+++

Fe+++

MelaninOH·

Lipid peroxidation

OH−

DopamineMAO

R-CHO + NH3 + H2O2

Auto-oxidation

Fig. 3. Reaction pathway illustrating the ability of hydrogen per-oxide and melanin to alter the redox state of iron between itstwo valences with formation of cytotoxic hydroxyl radical andinduction of lipid peroxidation (reproduced from Gerlach et al.,[12]

with permission). Fe++ = ferrous iron; Fe+++ = ferric iron; GSH =glutathione; GSSG = oxidised glutathione; MAO = monoamineoxidase; OH• = hydroxyl radical; OH– = hydroxyl ion.

118 Jellinger


a major product of membrane lipid peroxida-tion, exerting a toxic action, and 8-hydroxy-2-oxyguanosine, indicating DNA damage.[64]

Recent studies revealed an increase of 8-hydroxyguanine in the SN of PD brains[65]

which appears to be the first biochemical pa-rameter associated with oxidative stress mea-sure in PD that shows no overlap with otherareas.[29]

These changes are accompanied by decreasedactivity of the mitochondrial electron transportchain enzyme NADH-ubiquinone oxidoreductase(complex I),[66-70] which is encoded by both mito-chondrial and nuclear DNA, suggesting mitochon-drial genetic aetiology.[71] In PD, one or several ofthe complex I mitochondrial genes appear to bedefective; this could lead to important bioenergeticdefects with reduced adenosine triphosphate (ATP)formation, increased O2 free radical productionand sensitisation toward external toxins with a riskof excitotoxic damage even in the presence of nor-mal levels of excitatory amino acids.[27,72]

GSH depletion and the complex I defect areregionally selective for SN in PD, and do not occurin other neurodegenerative disorders exhibitingsimilar SNc cell loss, such as multiple system at-rophy (MSA) and progressive supranuclear palsy(PSP).[52,66] On the other hand, GSH depletion isalready present in incidental Lewy body disease(DLB), which probably represents presympto-matic (subclinical) PD, but without alterations iniron levels and complex I activity. This suggeststhat these latter changes may be secondary to otherbiochemical events,[61] while depletion of GSHmay be the factor which initiates oxidative stress.In vitro studies have shown that upregulation ofcellular GSH evoked by auto-oxidisable agents isassociated with a significant protection of cells.[73]

Recent tissue culture studies provide evidence foran oxidative challenge occurring during inhibitionof energy metabolism by malonate and show thatGSH is an important neuroprotectant for midbrainneurons during situations when energy metabolismis impaired.[74] However, depletion of GSH ap-pears not to be selectively cytotoxic for dopamin-

Table I. Comparison of the cytotoxic action of iron-induced oxidative stress with findings in the substantia nigra of patients with Parkinson’sdisease and in Alzheimer’s disease

Parameter Iron-induced oxidative stress Parkinson’s disease Alzheimer’s disease

Tissue iron ↑ ↑ –/↑Fe++/Fe+++ ratio ? ↑ ?

Antioxidants

H2O2-scavenging glutathione ↓ ↓ ↑ H2O2-scavenging superoxide dismutase ↑ ↑ ↑ glutathione peroxidase ? –/↓ –/↓ catalase ? –/↓ ↓ uric acid ? ↓ ↓ ascorbic acid (vitamin C) ? ↓ ?

H2O2 + OH– ↑ ↑ ↑Lipid peroxidation

malondialdehyde ↑ ↑ ↑ lipid (hydro) peroxidase ↑ ↑↑ ↑ 4-hydroxynonenal protein ? ↑↑ ↑Complex I and II activities of mitochondrialelectron transport

↓ ↓ ↓

Ca++ homeostasis/uptake ↑ ? ?

L-ferritin ↑ ↓/↑/– ↑Transferrin binding sites ? ↓ ↓Lipofuscin ↑ ↑ ↑– = unchanged; ↑ = increased; ↑↑ = strongly increased; ↓ = decreased; ? = unknown.



ergic neurons but may increase their sensitivity to-wards other pathogenic factors such as neurotoxinsor free radicals.[75]

NO may directly affect iron metabolism, since NOand some of its reaction products have been shownto reduce ferritin synthesis and increase transferrinreceptor expression.[76] Electron paramagnetic res-onance (EPR) signals from the iron-sulphur cen-tres, known to act as biosensors of oxidants andiron,[77] of complex I and II are not changed in theglobus pallidus of PD brain. NO radicals are pres-ent in SNc, probably due to nitrosyl-haem (haem-NO) formation.[78] Finally, there is evidence ofoxidative damage to lipids, DNA, proteins andtyrosine-containing molecules, reflecting damageprobably induced by the OH• and NO• radical spe-cies.[28,29,32]

Lipid peroxidation is a rather late event in oxi-dative tissue damage,[79] but the demonstration ofoxidation markers, e.g. heat shock protein 70 andof haem oxygenase-1 (HO-1), an enzyme responsiblefor the conversion of haem into the antioxidant tet-rapyrrole in free iron, as well as of nitrotyrosine-IRin both cortical LB filaments and LB in PD SNneurons suggests either that oxidative stress may beinvolved in the formation of LB[76,80-82] or that theseinclusions are involved in the generation of oxida-tive stress, although other brain areas containingLB do not show evidence of such dysfunction.[16]

The study of increased protein carbonyls and DNAbase products in cortical regions from patients withdiffuse DLB,[83] upregulation of HO-1 in the SN ofpatients with PD and in LBs of patients with DLBand the presence of nitrotyrosine-IR within LBs[82]

support the view that the affected tissue is experi-encing chronic oxidative stress. In addition, exces-sive cellular levels of heme-derived free Fe result-ing from HO-1 overactivity may contribute to thepathogenesis of PD.[76]

Recent studies of normal human brain revealeda 2-fold increase of carbonyl proteins, indicatingprotein oxidation, in SNc compared with basal gan-glia and cortex, suggesting that oxidative stress iselevated in SN and may contribute to neuronal de-generation.[84] These data and the capacity of SNc

neurons to generate NO intraneuronally combinedwith superoxide production from DNA metabo-lism[84] may contribute to neuronal degeneration,which is at least, in part, nitrotyrosine mediated.[82]

Finally, an excess of H2O2 found by using confocalflorescence microscopy in the frontal cortex of apatient with PD and Alzheimer’s disease appears tobe a direct indicator of oxidative stress.[85]

3. Iron and PD

Iron is an essential trace metal that plays a role inmany catalytic and regulatory processes and in celldeath.[20,86] In the CNS, most iron bound to special-ised membranes, e.g. mitochondria or cytoplasm,is complexed/chelated by the protein ferritin,whereas serum transferrin and transferrin receptorsare responsible for iron transport across the blood-brain barrier and into brain cells.[86-89] In humanbrain, iron content is highest in globus pallidus,SN, caudate/putamen and dentate nucleus.[20,87,90,91]

Ferritin is localised in choroid plexus epithelialcells and oligodendroglia, whereas transferrin ismainly found in oligodendroglia and microglia.[92]

Increased levels of iron have been reported in sev-eral neurodegenerative disorders, such as Parkin-son’s, Huntington’s, Hallervorden-Spatz and Alz-heimer’s diseases, amyotrophic lateral sclerosis/Parkinson dementia complex (ALS/PDC) andMSA,[9,11,13,18,20,22,48,85,91] as well as with normalbrain aging.[93]

Much of the current interest in the free radicalhypothesis of PD is based on the finding, in studiesemploying different methodologies, of increasediron in the SNc (table II), and on iron’s known ca-pacity to promote oxidative stress via oxygen freeradical formation (fig. 4). The initial observationof increased iron content in PD brain[114] was con-firmed by Earle,[94] who, using X-ray fluorescentspectroscopy, reported a 2- to 3-fold increase ofiron in PD brain compared with controls. Biochem-ical and histochemical analyses showed 35 to 77%increase of total iron and of Fe+++, but not of Fe++,in SNc of patients with PD.[4,95-98] Although histo-chemistry (Perl stain) revealed a significant in-crease of Fe+++ in SNc, it was not detected in

120 Jellinger


neuromelanin or LB because of its binding to aprotein matrix.[99] Increased iron content in PD SNwas also seen by scanning particle-induced X-rayemission analysis.[100] X-ray microanalysis showeda 3.4-fold increase of iron in SN areas devoid ofmelanin and a 35% increase in neuromelanin.[101]

Energy dispersive X-ray microanalysis (EDX) andlaser microprobe analysis (LAMMA) have shownthat iron consistently accumulates in neuromelaningranules of degenerating SN neurons, whereas noiron increase was found in LB, surrounding neuropiland SN neurons of controls.[85,102,103] Mössbauer

spectroscopy of neuromelanin revealed conflictingresults,[112-117] suggesting that over 90% of iron inSN resembles ferritin, while Fe++ constitutes lessthan 10%.[112,115] Magnetic resonance imaging (MRI)studies in patients with PD also gave evidence ofsignificant increase of iron in SN[107-109,118] andconfirmed biochemical data showing correlationsbetween increased nigral iron and the severity ofPD motor dysfunction.[8,51] However, MRI studies instriatum gave conflicting results.[110,119] Nuclearmagnetic resonance spectroscopy revealed a highiron content concentrated in the melanin/glycidic-

Table II. Iron content in substantia nigra of patients with Parkinson’s disease as found by different investigators using different methodsa

Study Method Total iron Fe+++ Fe++ Region

Earle[94] XFS ↑ ‘Brain tissue"

Sofic et al.[48] SP ↑ ↑ – SN

Sofic et al.[95] SP ↑ ↑ – SNc

– – – SNr

Dexter et al.[96] CPS ↑ SN

Dexter et al.[97] CPS ↑ SN

Uitti et al.[98] AA/AES – SN

Jellinger et al.[99] PIH ↑ ND SNc

– ND SNr

Aotsuka et al.[100] PIXE ↑ SN

Hirsch et al.[101] EDX ↑ SN

↑ LB

Jellinger et al.[102] EDX ↑ ↑ ND SNc/melanin

– – ND LB

Good et al.[103] LAMMA ↑ SN/melanin

Griffiths & Crossman[104] AASP ↑ SN, lateral PALL

Rutlege et al.[105] HFMRI ↑ SN

↑ PUT

De Volder et al.[106] HFMRI ↑ PUT

Chen et al.[107] HFMRI ↑ (33%) PUT

Ordidge et al.[108] HFMRI ↑ SN

Gorell et al.[109] HFMRI ↑ SN

Ye et al.[110] HFMRI ↑ SN

Aima et al.[111] NMR ↑ SN/melanin

Bauminger et al.[112] MS – – ND SN

Galazka-Friedman et al.[113] MS – (>10%) ↑ >5% SN

a Some of the conflicting data may be due to differences in analytical methods or techniques of brain tissue dissection, or to the use of un-suitable techniques.

AA/AES = atomic adsorption/atomic emission spectroscopy; AASP = atomic absorption spectrophotometry; CPS = coupled plasmaspectroscopy; EDX = energy dispersive X-ray microanalysis; HFMRI = high field magnetic resonance tomography; LAMMA = lasermicroprobe mass analysis; LB = Lewy body; MS = Mössbauer spectroscopy; ND = not detectable; NMR = nuclear magnetic resonancetomography; PALL = pallidum; PIH = Perl’s histochemistry; PIXE = particle-induced x-ray emission; PUT = putamen; SN = substantia nigra;SNc = substantia nigra, pars compacta; SNr = substantia nigra, pars reticulata; SP = spectrophotometry; XFS = X-ray fluorescentspectroscopy; ↑ = increased compared with controls; – = unchanged.



lipid matrix of neuromelanin,[111] suggesting thatmost of the iron is chelated by insoluble melaninand confirming both the EDX and LAMMA stud-ies. The latter technique also revealed an increaseof aluminium in neuromelanin granules of SN insome PD brains, and a significant increase of iron-related signals in ALS/PDC.[18] Increased levels ofiron were also reported in SN in PSP and MSA,[96]

suggesting that these changes are not a cause butrather a result of the neurodegenerative process.[15,16]

On the other hand, an increase of Fe-containingperoxidase-positive astrocytes in the aging SN (ofrats) detected mainly in nigral gliosomes may pro-mote the biological action of dopamine to neuro-toxic free radical intermediates, thus predisposingthe aging CNS to PD.[120]

The elevated iron level of SN neurons in PDcould be related to dysfunction of the homeostasisor transport of iron, which is regulated by the fer-

ritin and transferrin proteins.[26] The expression oftheir receptors is regulated by the intracellular ironconcentration: when iron levels are high, ferritinmRNA is translated and transferrin receptor mRNAis degraded, leading to iron storage and preventingiron uptake.[121] Ferritin measurements remaincontroversial, showing decreased, unchanged orslightly increased levels in the SN of patients withPD.[21,96,97,99,122,123] Ferritin is co-localised withabnormal tau protein filaments in PSP, suggestingthat ferritin Fe could modulate these aggregates inparkinson-like disorders.[124] Autoradiographicstudies of transferrin binding sites revealed a de-crease[125,126] or no significant change[127] in PDmesencephalon, with similar conflicting results forthe striatum,[125,127] whereas the transferrin/iron ra-tio, a measure of iron mobilisation capacity, wasdecreased in globus pallidus and caudate in bothPD and Alzheimer’s disease, suggesting reduced

Fig. 4. Diagram of the interacting biochemical events of nitric oxide and iron in causing neurotoxicity in Parkinson’s disease (repro-duced from Gerlach et al.,[12] with permission). ATP = adenosine triphosphate; Fe++ = ferrous iron; Fe+++ = ferric iron; NO• = nitric oxideradical; O2

• – = superoxide radical; OH– = hydroxyl ion; ONOO– = peroxynitrite ion; TF = transferrin.

122 Jellinger


mobilisation of iron by transferrin.[128] However, asignificant decrease in [3H]transferrin receptor andH-chain ferritin mRNA in SN was seen in latestages of PD, apparently reflecting the loss ofmelanised neurons.[126,129] Increased levels of L-ferritin may be associated with activated microgliain PD-SNc.[99] Autoradiographic studies showedthat the densities of ferrotransferrin binding siteswere increased in the putamen and caudate of PDbrain, suggesting that they may be located on dopa-minergic terminals and that iron may be retro-gradely transported to the perikarya of SN neurons.The increase of ferrotransferrin binding sites in PDmay reflect an increase in the iron uptake mecha-nism located on dopaminergic nerve endings in thestriatum, or may be the result of changes in cerebralmicrovessels.[130] Positron emission tomographystudies of 52Fe citrate movements showed an in-crease of ferrotransferrin uptake in half of a groupof patients with PD as compared with controls.[131]

There is increased immunoreactivity of theiron-binding protein lactoferrin and of lactoferrinreceptors, particularly in regions with severe neu-ronal loss in PD.[132,133] These data and recent find-ings of decreased transferrin binding sites on SNcneurons in PD[134] may represent a compensatorychange in response to increased Fe in these cells.On the one hand, they suggest that L-ferritin recep-tors on vulnerable neurons may increase the intra-cellular Fe levels and that impaired Fe homeostasiscould promote excessive Fe accumulation leadingto increased production of OH• and, thus, to oxida-tive damage. On the other hand, stress-related trap-ping of non-haem iron by astroglial mitochondriamay be an important mechanism underlying thepathological accumulation of redox-active ironand the electron transport deficits observed inPD.[76,135]

4. Iron-Related Animal Models

In some experimental animal models of PDwhere degeneration of nigrostriatal dopaminergicneurons has been observed there is evidence foriron-induced oxidative stress as a pathogenic fac-tor.[23] These are: (i) the 6-hydroxydopamine (6-

OHDA) model in the rat; (ii) the N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) modelin mouse and monkey; (iii) the Fe+++ model in therat and mouse; (iv) the β-carboline/‘TaClo’ model.

4.1 6-Hydroxydopamine Model

6-OHDA is selectively transported into the ter-minals of catecholaminergic neurons. Its neurotox-icity is based on 2 mechanisms of action: (i) itforms free radicals, and (ii) it is a potent reversibleinhibitor of complex I and IV.

6-OHDA is rapidly oxidised to form cytotoxicsemiquinones and quinones, with concurrentproduction of H2O2 and OH• free radicals.[30,136]

This reaction is probably initiated by transitionmetals, e.g. iron, since iron is increased in thestriatum.[110,137] In the SNc of 6-OHDA–treatedrats there is an almost complete loss of neurons thatare immunoreactive for tyrosine hydroxylase, in-dicating their disruption.[138,139] 6-OHDA releasesiron from ferritin in vitro and promotes ferritin-de-pendent lipid peroxidation.[140,141] Indicators foroxidative stress caused by 6-OHDA in striatum arereductions of GSH levels and SOD activity, andincreases of MDA and of conjugated dienes.[142]

Recent studies have shown that generation of quin-ones from 6-OHDA requires the presence of iron,either in the ferrous or ferric form, and is unaffectedby peroxidase, catalase, hydroxyl radical scaven-gers or biologically relevant antioxidants such asascorbate or glutathione. Metal chelators, includ-ing EDTA and bipyridyl, markedly suppress qui-none formation without, however, inhibiting dopa-mine oxidation.[143] These and other results suggesta hydroxyl radical–independent hydroxylation/oxidation mechanism basically different from theFenton reaction, involving direct interaction ofH2O2 with a dopamine-Fe+++ chelate generatedduring the process. The demonstration that iron-deficient rats are protected from the neurotoxic ac-tion of 6-OHDA suggests the involvement of ironin the mechanism of action of this neurotoxin.[144,145]

The partial or complete prevention of the neuro-toxic effects of 6-OHDA and iron by prior admin-istration of the iron chelator desferrioxamine



(deferoxamine),[146] α-tocopherol,[147] desferrioxam-ine and α-tocopherol[148] and the monoamine oxidase-B (MAO-B) inhibitor selegiline (deprenyl) providefurther evidence for an oxidative mechanism.

6-OHDA further reversibly inhibits complex I(NADH dehydrogenase) of brain mitochondrial re-spiratory chain[144] and is even more toxic than 1-methyl-4-phenylpyridinium (MPP+), the probableneurotoxic form of MPTP that causes productionof superoxide free radicals, H2O2 and OH• radi-cals.[149,150] Electron-dense reaction products ofH2O2 on mitochondrial membranes in damaged SNneurons after intraventricular administration of 6-OHDA to mice provide evidence for oxidative in-activation of the mitochondrial electron transportchain.[151] Fe+++ decreases the inhibition of respi-ratory enzymes by stimulating the nonenzymic ox-idation of 6-OHDA.[152] The inhibition of respira-tory enzymes by 6-OHDA is insensitive towardsiron chelators, with the exception of desferrioxam-ine.[144,146] These results suggest that ferritin-Fe re-lease contributes to free radical–induced cell dam-age in vivo.[140]

4.2 N-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Model

The dopaminergic neurotoxin MPTP causes asyndrome in primates and humans which, at leastin part, mimics PD in clinical, pathological andbiochemical findings.[153,154] The MPTP model isone of the most widely used and the best investi-gated model of PD.[30] The neurotoxic cascade ofMPTP-induced neuronal death involves severalsteps, a first being its conversion in the glia byMAO-B into MPP+, the probable ultimate toxicagent, and the second the selective uptake of MPP+

by dopaminergic terminals in the striatum and sub-sequent vesicular storage. The mechanism of itsneurotoxic action is an inhibition of complex I ac-tivity in the mitochondrial electron transport chainin dopaminergic neurons via an alteration in mito-chondrial Ca++ homeostasis, resulting in depletionof intracellular ATP and neuronal death.[136,155] In-crease of intracellular Ca++ damages the mitochon-drial electron transport chain via generation of ox-

ygen free radicals, resulting in increased lipid per-oxidation.[136,156]

Indirect evidence for the presence of oxidativestress in MPTP-treated rodents involves increasedlipid peroxidation and decreased antioxidant, ascor-bic acid, uric acid and GSH levels.[34,157] Alterationsof dopamine uptake in rat striatal slices have beenshown not to be related to lipid peroxidation.[158]

MPTP treatment of mice, and exposure to MPP+ ofhuman neuroblastoma cells, increases oxygen freeradical production and antioxidant enzyme activi-ties. These characteristics are similar to those of‘cybrid’ cells created by transfer of PD mitochon-dria into cell culture, although the antioxidant en-zyme activities of these cybrid cells are not furtherincreased by exposure to MPP+.[159] In the marmo-set also, no significant increase of GSH and ubiqui-nol, the reduced form of coenzyme Q, and no inhi-bition of complex I were found 1 week after toxinexposure,[160] suggesting that acute MPTP treatmentproduces no changes in mitochondrial complex ac-tivities and indices of oxidative damage.[160] MPP+

does not deplete GSH from primary mesencephaliccell cultures, although a low level of GSH in dopa-minergic cells potentiates their susceptibility toMPP+.[161] These data suggest that, although de-creased GSH content may be involved in dopamin-ergic neuron loss, other factors such as oxidativestress may be involved in MPTP neurotoxicity inprimates.

Recent studies have shown that NO• enhancesMPP+ inhibition of complex I in rat brain mito-chondria[162] and that NO is involved in the produc-tion of hydroxylradicals resulting from MPP+-induced dopamine afflux in the striatum[29] whichcan be reduced by neuronal NOS inhibition,[163]

while NOS knock-out mice show decreased MPTPsusceptibility.[164] These data support the notion ofa biphasic mechanism in the toxic action of MPTP:a reversible inhibition of complex I causing ATPstarvation, followed by inhibition of electron trans-port in the respiratory chain producing a secondarypermanent deactivation of complex I via excessivefree radical formation.

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The interaction of MPP+ with complex I leadsto liberation of iron, which increases in the SNc ofMPTP-treated monkeys.[165,166] Ferritin immuno-staining suggests that iron accumulation is not re-lated to altered metabolism of ferritin in thismodel.[167] A decrease in ferritin transporters and atendency for decreased levels of transferrin bind-ing in rat striatum following MPTP administrationsuggest the acute loss of these proteins in striataldopaminergic terminals,[125,128] leading to releaseof iron in SNc.[166] Ferric lactate and ferric ATP areinvolved in the iron-induced process of modifica-tion of Ca++ homeostasis[168] that also involves glu-tamate and N-methyl-D-aspartate (NMDA) recep-tors.[30] This is supported by the protective effectof the iron chelator desferrioxamine[169,170] and ofNMDA receptor agonists[171] against MPTP toxic-ity,[29,171] while the neuroprotective action ofselegiline against MPP+ neurotoxicity in dopamin-ergic cells may be independent of its MAO-B in-hibitory activity.[172] In a primate model of pro-gressive parkinsonism caused by chronic exposureof cynomolgus monkeys to MPTP, the antioxidantand free radical scavenger OPC-14117 (7-hydroxy-1-[3-methoxy-phenyl]-1-piperazynal acetylamino-2,2,4,6-tetramethyllindan) had no protective ef-fect.[173]

4.3 Iron-Loading Models

4.3.1 Fe+++ Infusion ModelUnilateral injection or infusion of Fe+++ into the

SN of rats causes an increase of iron in this region.Also observed are dose-dependent selective neu-ron loss and reactive gliosis in the SNc, progres-sive SN atrophy associated with progressive deple-tion of striatal dopamine, 3,4-dihydroxyphenylaceticacid and homovanillic acid[174-176] and elevation ofdopamine turnover by 50%, suggesting an upregu-lation of surviving SN neurons. There is also a pro-gressive increase in apomorphine-induced rotationbehaviour confirming damage to the SN.[174,177,178]

Although large amounts of iron also result in dam-age to the pars reticulata of the SN, other neuro-chemical striatal markers, e.g. noradrenaline, sero-tonin and 5-hydroxyindoleacetic acid, remain

unchanged.[174,177,179,180] The mechanism of actionof intranigrally injected Fe+++ is probably mediatedby uptake into neurons and glia by transferrin re-ceptors, where it is reduced to Fe++, which has beenshown to have cytotoxic actions in mesencephalicneuronal cultures.[181] Similar to MPP+, free iron isknown to compromise mitochondrial Ca++ homeo-stasis,[168] and to damage the mitochondrial elec-tron transport chain via generation of hydroxyl freeradicals from H2O2, which is an endogenous prod-uct of enzymatic dopamine metabolism.

Increased intracellular Ca++ and increased lipidperoxidation may be an early sign of damage toSNc neurons and glia where the iron accumulates.Acute increases of MDA and of thiobarbituricacid-reactive compounds (TBAR), a measure oflipid peroxidation, in SN at 1 hour and 1 day post-infusion support the possibility that iron-inducedlipid peroxidation and transient oxidative damagecontribute to neuronal death in SNc.[176,182] Prog-ress of the histologically demonstrated diffusion ofthe iron in the post-infusion period from the ini-tially involved neurons to reactive astroglia and,finally, to oligodendroglia indicates a shift of in-fused iron to a more bound/stable and unreactiveform, thus explaining why only short-term in-creases in lipid peroxidation are observed.

The progression of SNc atrophy, striatal dopa-mine deficiency and abnormal rotation behaviourfollowing intranigral infusion of iron have shownfor the first time that a toxic insult results in chronicprogressive lesions in an animal model of PD; thepathogenic mechanisms for the chronic progres-sive course await further elucidation. The lesion-ing activity of iron, like that of 6-OHDA, can beprevented by the selective iron chelator des-ferrioxamine, an inhibitor of radical formation.[10]

s-Nitroso-glutathione can protect dopaminergicneurons from Fe-induced oxidative stress and de-generation, suggesting that s-nitrosylation of GSHby NO and oxygen may be part of the antioxidativecellular defense system.[183] The atypical antioxi-dative properties of manganese may protect SNneurons from Fe-induced oxidative stress.[175]



4.3.2 Iron-Feeding ModelIron loading by feeding a high-iron diet to wean-

ing mice, causing a significant increase of total ironin the striatum, is associated with a significant in-crease of GSSG, decrease in GSSG + GSH, in-crease in the GSSG/GSSG + GSH ratio and in-crease in OH+ levels in striatum and brainstem.[184]

Although excessive iron alone did not change eitherdopamine or MDA concentrations in striatum, asingle injection of MPTP into the iron-loaded micegreatly enhanced all biochemical abnormalities, in-cluding a significant increase of MDA formation;there was no increase in antioxidative enzyme ac-tivities, GSH levels being even lower than in con-trols. These data suggest that excess iron accumu-lation in the brain is a potential risk factor forneuronal damage.

4.4 β-Carbolines: The ‘TaClo’ Model

As a result of their structural similarity to MPTP,β-carbolines (norharmanes) have been suggestedas endogenous toxins leading to parkinsonism. In-tranigral injection of the N-methyl β-carboline 2-methyl-norharman results in depletion of striataldopamine and its metabolism, but the lesion ap-pears nonspecific, producing large lesions in themidbrain and also affecting nondopaminergic fi-bres.[30] By contrast, 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo), a putative in vivocondensation product of chloral hydrate and trypta-mine,[185-187] causes cell loss and reduction of do-pamine uptake in neuronal and glial cultures,[188]

enhanced apomorphine-induced behavioural ef-fects after subchronic (medium-term) intraperitonealinjection in rats,[189] and, after intranigral injectionof TaClo, reduced dopamine metabolism in the ip-silateral striatum.[190] Like MPP+, TaClo is a powerfulinhibitor of complex I, inhibiting electron transferto ubiquinone at concentrations 10 times lowerthan MPP+; however, like other β-carbolines, italso inhibits complex II.[191,192] Thus, the neuro-toxic effect of β-carbolines may be related to in-hibition of mitochondrial respiration. Like the‘iron’ model, the TaClo model shows a chronic pro-gressive course of behavioural and biochemical

changes,[30] although there is recent evidence thatsystemic application of TaClo has no toxic effecton the nigrostriatal system.[193] Hence, the basicmechanisms of neurotoxicity and the role of ironin this model require further elucidation.

4.5 Overview of Models

Most of the models described in sections 4.1 to4.5 showing experimentally induced destruction ofdopaminergic nigrostriatal neurons have bothsimilarities and dissimilarities with human PD.Chronic progressive degeneration of nigrostriataldopamine neurons, the primary pathogenic featureof PD, has been observed in the 6-OHDA model,the iron model and the, still questionable, TaClomodel. Such degeneration has been observed in arodent model following injection of 6-OHDA intothe striatum, the terminal field of the striatonigralprojection, causing progressive loss of SN neu-rons.[194] In some of these models, as in human PD,there is an increase of iron content and of lipidperoxidation in SNc, but also selective involve-ment of SN neurons with low immunoreactivity forthe Ca++-binding protein calbindin.[46] Althoughmost of these models do not prove a direct involve-ment of iron in the neurodegenerative process ofPD, they suggest that accumulation of iron in thebrain is a potential risk factor for neuronal damagethat, together with other triggering factors, maylead to a cascade of free radical reactions finallyleading to SN cell death. This interaction betweeniron and neuromelanin appears to be highly rele-vant to this process.

5. Iron-Melanin Interaction

As well as storage of iron in ferritin, large amountsof iron, particularly in the basal ganglia and SN,are bound to proteins or catecholamines. Neuro-melanin in SN is a mixed melanin, a complex copoly-mer of dopamine, cysteine and glutathione[195-197]

which also contains oxidation products derivedfrom both dopamine and cysteinyldopamine.[198]

Neuromelanin has redox properties and the capac-ity to chelate or bind large amounts of metals, inparticular, iron.[195-199] EPR and X-ray fluores-

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cence spectroscopy studies of several human brainregions showed that only the SN, and to a lesser ex-tent the locus coeruleus, had a free-radical signalconsistent with that of neuromelanin, with somedifferences in the structure of this pigment in the 2regions.[200]

Synthetic dopamine-melanin binds specificallyto Fe+++, and a significant amount of bound ironhas been reported in neuromelanin isolated fromthe SN.[196,197] 57Fe Mössbauer spectroscopy ofpurified neuromelanin from human SN revealedan Fe+++ content of 2.8 ± 1.4%,[115] while EPRspectroscopy, a useful method for determining theamount of paramagnetic Fe+++ bound to neuro-melanin,[201] revealed about 0.9%.[200] A wide rangeof values (48 to 360 μg/g wet tissue) for the con-centration of iron in normal human SN have beenreported using different techniques (atomic ab-sorption spectroscopy, neutron activation analysis,X-ray fluorescent spectroscopy).[51,199] EPR spec-tra of neuromelanin extracted from normal humanSN clearly indicate that iron is present as polynu-clear oxyhydroxy ferric aggregates and isolatedFe+++ centres. Although there are differences be-tween the 2 domains, they show similar pathwaysof formation and inclusion of the polynuclear ironoxide in neuromelanin and in model systems.[111]

However, in vitro studies have shown that themechanism of dopamine oxidation leading to po-lymerisation is independent of metal ions such asFe+++ or Cu++.[202-204]

At physiological pH, neuromelanin is an effi-cient antioxidant.[205] Synthetic neuromelanin hasbeen shown to reduce lipid peroxidation in rat cor-tical homogenates as indicated by reduced concen-trations of TBARs.[146] Studies using the electronspin resonance trapping technique confirmed theantioxidant properties of dopamine-neuromelaninby showing that the 1-electron reduction potentialof Fe+++ was lowered by complexation.[206] Oncebound to neuromelanin, Fe+++ ions are protectedagainst redox activation. Significant generation offree OH• radicals was detected only when neuro-melanin became saturated with large amounts of

Fe+++ ions. Bound Fe+++ can be displaced only bycompounds with iron-chelating capacity.

On the other hand, neuromelanin may potenti-ate the formation of oxygen-derived radicals whenoverloaded with Fe+++ by overwhelming the anti-oxidative defence mechanisms.[146] In an experi-mental cell culture model, increased intracellulariron resulted in elevated MDA and decreased glu-tathione levels, consistent with oxidative dam-age.[207] In vitro studies have shown that: (i) un-coupled iron and H2O2 convert dopamine into thenontoxic 5-OHDA and into the cytotoxic 6-OHDAvia a Fenton-like reaction,[203,208] both substancesbeing detectable in the urine of human patientswith PD, particularly in those receiving levodopatreatment;[209] and (ii) that the oxidation of 6-OHDA by Fe+++ proceeds without prior formationof a metal-ligand complex. The release of iron asFe++ from ferritin by 6-OHDA is an ‘outer sphere’electron transfer reaction, while with other cate-cholamines it is an ‘inner sphere’ reaction.[210] Thisimplies that 6-OHDA alone is capable of removingiron – as Fe++ – from complexes such as ferritin.[85]

On the other hand, radical reductants, such as su-peroxide, H2O2, NO• and hydroxyl, with the abilityto mobilise ferritin-bound iron[10] can be formedboth endogenously or from exogenously derivedneurotoxins such as 6-OHDA, MPTP or paraquat;this may be the cause of the increased iron levelsin both PD and related animal models.

6-OHDA is readily regenerated by hydroxyla-tion of dopamine. The kinetic data suggest thatFe++ in the basal ganglia promotes not only dopa-mine biosynthesis but also, under as yet unknownphysiological conditions, initiates both dopamineauto-oxidation and hydroxylation via a Fenton-like cycle chain mechanism. This results in accu-mulation of free iron and a continuous productionof cytotoxic species, thus triggering a cascade ofreactions finally leading to SN cell death (fig. 5).Recent in vitro studies, however, suggest that a lowoxidation potential and an ortho-dihydroxyphenylstructure are important in the mechanism by whichferritin-iron is mobilised. In the presence of ferri-tin, both 6-OHDA and 1,2,4-trihydroxybenzene



strongly stimulate lipid peroxidation, an effectabolished by the addition of the iron chelatordesferrioxamine.[140] These data also suggest thatrelease of iron from ferritin contributes to free-radical–induced cell damage, while intranigral in-fusion of ferrous citrate in rats resulting in hy-droxyl radical formation and lipid peroxidation canbe inhibited by exogenous NO acting as a radicalscavenger and thus protecting neurons from oxida-tive injury.[140]

Iron is neurotoxic to cultured dopaminergicneurons and their mitochondrial fraction in a dose-dependent manner, inducing significant inhibitionof complex I.[188,211] However, in vitro studies ofrat nigrostriatal co-cultures revealed a more pro-nounced neurotoxic effect of iron-melanin com-plex.[212] This may be related to the increased lipidperoxidation that has been shown to occur in thepresence of iron and dopamine, which shows bothpro- and anti-oxidant activity.[54] Neuromelanin,the oxidised form of dopamine, is able to induceapoptosis (programmed cell death) in dopamin-

ergic PC12 cells;[213] iron significantly increaseddopamine/neuromelanin toxicity, and the iron che-lator desferrioxamine totally abolished the addi-tional iron toxicity. Antioxidants such as reducedGSH, catalase or SOD could only produce limitedinhibition of dopamine-neuromelanin toxicity, incontrast to their effect on dopamine toxicity.[214]

There is now a large body of evidence indicatingthat dopamine can participate in neurotoxic events.This has been attributed, at least in part, to the re-active quinone form of the oxidised molecule andother conjugates.[42,215,216] In vitro studies showedthat iron increases the cytotoxicity of dopamine-induced apoptosis and lipid peroxidation, althoughthere is no causal link between the 2 reactions.[213]

These data suggest that iron may increase the cy-totoxicity of neuromelanin and dopamine by in-creasing their rates of oxidation, although theremay be differential protective effects of antioxidantsagainst the toxicity of dopamine and dopamine-neuromelanin.[214] In addition, the sensitivity ofdopaminergic neurons to iron toxicity could be the

H2O2

O2 O2

Dopamine

OH·

Fe+++

Fe++

Fe++ 6-OHDA

Ferritin/Fe+++

6-OHDA-Q

Iron-containingneuromelanin

DopaminochromeDopamino-

quinone

Fig. 5. Reaction routes of dopamine and 6-hydroxydopamine (6-OHDA) in dopaminergic neurons (reproduced from Gerlach et al.,[12]

with permission). Fe++ = ferrous iron; Fe+++ = ferric iron; OH• = hydroxyl radical; 6-OHDA-Q = 6-hydroxydopaminoquinone.

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result of different uptake mechanisms or intracel-lular storage of this element in these cells.[211]

6. Systemic Iron Metabolism in PD

Very little information exists about systemiciron metabolism in PD, and in many cases the evi-dence is conflicting. Although previous studiesfound either a slight increase or a significant reduc-tion in serum iron and ferritin levels,[217,218] recentstudies reported a significant decrease of circula-ting iron, ferritin and transferrin, total iron bindingcapacity and transferrin saturation in patients withPD.[24] Transferrin receptor changes were stronglycorrelated with mortality in patients with PD, butnot in controls.[26] This increase of serum transfer-rin receptor concentrations before death suggeststhat the previously observed perturbation in ironmetabolism may continue throughout the diseasecourse.[26] The decreased levels of systemic ferritinand transferrin and the decreased total iron bindingcapacity parallel the findings in PD brain and sug-gest a defect in the systems that regulate the syn-thesis of the main proteins of iron metabolism inboth the liver and the brain in PD. This may, overtime, expedite entry of iron into the brain and de-crease iron in the extracellular compartment. An-other recent study reported significant reduction oftransferrin binding in PD as compared with con-trols; transferrin binding was significantly greaterin patients treated with levodopa than in untreatedones.[219]

These data also support the hypothesis that oxi-dation reactions may be of pathogenic significancein PD. However, recent analysis of several serumproducts relevant to oxidative stress, includingMDA, 5-S-cysteinyl-dopa, α-tocopherol and uricacid, failed to show any abnormalities in patientswith PD as compared with controls and, thus, didnot confirm any systemic defect of oxidative me-tabolism outside the CNS.[220] In addition, popula-tion-based studies showed that a high dietary in-take of Fe is not associated with an increased riskof PD.[221]

7. Iron in Other Neurodegenerative Disorders

Although there is no direct evidence for the in-volvement of iron and free radicals in the aetiologyof other major neurodegenerative disorders such asAlzheimer’s disease or ALS, increasing evidencesupports the hypothesis that these alterations maycontribute to the pathogenesis of neuronal death inat least some of these disorders.[13,17,20,22,27,222-225]

Iron changes in ALS and ALS/PDC have recentlybeen reviewed.[18]

8. Current and Future Therapeutic Strategies

The evidence accumulated from the experimen-tal and human studies reviewed in the precedingsections suggests that changes in cerebral iron maytrigger a cascade of noxious events leading to celldeath in PD and in at least some related neuro-degenerative disorders. This was the rationale forextensive therapeutic efforts to prevent or, at least,to slow such deleterious events by reducing theoverload of iron and the generation of ROS andother cytotoxic substances.[18,37,41,148,225,226] In ad-dition to levodopa administration, still consideredthe mainstay of PD treatment,[226-228] these strate-gies include ‘neuroprotective’ agents, iron chela-tors and lazaroids. All these approaches have beenextensively tested in preclinical studies, and somehave reached clinical trials.

8.1 Neuroprotection

Current pharmacotherapy of PD includes a num-ber of ‘neuroprotective’ strategies via dopamine-sparing agents, antioxidant compounds and freeradical scavengers.[31-33]

Dopamine agonists are believed to have bothsymptomatic and neuroprotective effects, mainlydue to dopamine sparing.[229-235] These includeapomorphine, which has been shown to be a highlypotent iron chelator, antioxidant and free radicalscavenger,[236-239] and which protects mesence-phalic neurons form glutamate neurotoxicity.[240]



MAO-B inhibitors include selegiline, which isnot only a potent inhibitor of MPTP toxicity[30,241-244]

and of glutamate receptor–mediated dopaminergicneurotoxicity,[244,245] but has been shown to delaythe emergence of disability and to slow the progres-sion of clinical deficits in PD.[18,31,32,246-249] Un-fortunately, the ‘neuroprotective’ effect seen in theDeprenyl and Tocopherol Antioxidative Therapy ofParkinsonism (DATATOP) trial of selegiline andtocopherol is not apparent on longer follow-up.[250-252]

Other MAO-B inhibitors are in various stages ofdevelopment or clinical use.[253]

Glutamate/NMDA receptor antagonists havebeen shown to reverse experimental models of PDand are suggested to show clinically relevant neuro-protective effects.[254] These include aminoaman-tadanes (amantadine or memantine)[255-261] andriluzole.[262,263]

Other agents tested include: inhibitors of cate-chol O-methyltransferase as adjuncts to levo-dopa therapy (dopamine sparing effect);[263-266] O-methylation inducing products and melatonin,suggested to show protective effects against freeradical formation;[267-269] and α-tocopherol andascorbic acid, both being effective antioxidantsand radical scavengers.[147,148,270,271] In experi-mental models and in clinical trials, α-tocopheroland ascorbic acid have shown controversial re-sults.[30,147,148,249,252,271-273]

Most of these substances have been reported tobe associated with clinical improvement and pro-longed survival in patients with PD,[31,32,256,273,274]

although their effect on mortality is still a matter ofdiscussion.[275-279]

8.2 Iron Chelators

Human and animal studies suggest that promis-ing approaches to PD and other neurodegenerativedisorders could be the development of iron chela-tors that prevent iron-induced generation of freeradicals and ROS.[8,146,148,240,280]

Desferrioxamine, a powerful iron-chelating agentthat displaces iron from neuromelanin, has been ex-tensively studied in animal models.[8,30,146,148,280-282]

Pretreatment with desferrioxamine has been shown

to prevent, or at least reduce, the SN degeneration,striatal dopamine deficiency and elevation of do-pamine release induced by 6-OHDA[146,282] or ironinfusion.[8,282] Its neuroprotective effect againstMPTP and MPP+ has been demonstrated by micro-dialysis.[169,170] In a mouse model of long-termMPTP administration, desferrioxamine plus α-tocopherol inhibited iron accumulation, reversedthe increase in GSSG/GSSG + GSH ratio, OH• andlipid peroxidation levels, and increased striatal do-pamine concentrations to normal values.[148] Thepossible antioxidative effect of desferrioxamine, sug-gested by effects similar to those of α-tocopherol[147]

and MAO-B inhibitors[30] in animal models, could beindependent of its iron-chelation properties,[283]

since previous studies have shown the antioxida-tive chain-breaking ability of desferrioxamine[284]

and its possible ability to scavenge peroxyl radi-cals[285] and the oxidising effect of peroxynitrite.[286]

However, from the animal experiments cited above,the iron-chelating effect of desferrioxamine may bethe primary protective mechanism because of itscritical role in inhibiting excessive iron-initiatedfree radical reaction.[148]

Although these and other data suggest a thera-peutic potential of iron-chelating substances, theefficacy of the best established chelating drugdesferrioxamine is limited because of its high cere-bral and ocular toxicity and its inability to cross theblood-brain barrier.[280] New bioactive iron-chelatingagents are currently being developed that are lipidsoluble and thus able to penetrate the brain, areprobably less toxic, and can flexibly respond to anincrease of iron-induced free radical formation[unpublished work].

8.3 Lazaroids

Lazaroids (21-aminosteroids) are nongluco-corticoid lipophilic inhibitors of iron-catalysedlipid peroxidation, iron chelators and oxygen freeradical scavengers.[287-292] Some of these compounds,e.g. U-74500A, tirilazad mesylate (U-74006F), U-74389F (15-desmethyl tirilazad), U-83836E (a 2-methylamine chroman) and U-748390, have beenshown to intercalate into the lipid layers of the cell

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membrane, where they preserve the function of theendogenous membrane-bound free radical scavengerα-tocopherol and increase cell membrane viscos-ity.[288,289] Tirilazad mesylate also protects the cellmembrane by directly inactivating iron-catalysedlipid peroxidation, preventing the formation of hy-droxyl radicals.[287-290]

U-74389G and U-83836F have recently beenshown to increase the survival of cultured dopa-minergic neurons subjected to serum-free condi-tions or oxidative stress through depletion of glu-tathione[293,294] or exposure to NO congeners.[295]

Furthermore, these 2 lazaroids prolong the timeduring which dissociated mesencephalic cells ex-hibit a high viability in vitro[294-296] and increasethe survival of dopaminergic neurons grafted intorat brain.[295,297] Tirilazad mesylate has recentlybeen shown to promote the survival of both rat andhuman embryonic mesencephalic neurons invitro,[297] but does not enhance TH-positive nervefibre growth into striatal portions of the grafts, anddid not, as expected, increase target innerva-tion.[298] The pathophysiological mechanisms un-derlying the low survival rate of embryonic dopa-minergic neurons after transplantation into thebrain may be related to free radical formation,since lazaroid treatment[297] and overexpression ofthe scavenger enzyme SOD[299] are capable of re-ducing the death of grafted neurons. Recent studiesindicate that the administration of tirilazad mesyl-ate may be a useful strategy in enhancing dopamin-ergic neuron graft survival in patients with PD.[300]

The lazaroids U-74500A and tirilazad mesylateare not only inhibitors of monoamine oxidase-Aand MAO-B but, like desferrioxamine, they alsoinhibit binding of Fe+++ to melanin[8] as well as theformation of free radicals.[301] Tirilazad mesylateand U-74389G have been shown to prevent iron-induced SN neurotoxicity and striatal dopaminedeprivation.[164,302] Iron chelators, lazaroids andcalcium antagonists, e.g. nifedipine, either givensimultaneously or separately, inhibit the uptake ofCa++ and iron-induced lipid peroxidation[8,289] andcould interfere with dysfunctions of the mitochon-

drial electron transport chain arising as a conse-quence of iron toxicity.[8,303]

The only lazaroid in current clinical use is tirilazadmesylate, a synthetic nonhormonal 21-aminosteroidwithout glucocorticoid activity.[287,291,292] However,several of the newly developed lazaroids that havebeen shown to increase the survival of dopamin-ergic neurons in neurotoxic models of PD, and ofembryonic dopaminergic neurons to be graftedinto the brain, may be useful for future treatmentof human PD.

9. Conclusions

Although the aetiology of PD and related neuro-degenerative disorders is still unknown, accumu-lating evidence from both human and animalstudies suggests that a misregulation of iron meta-bolism, iron-induced oxidative stress and free rad-ical formation are major pathogenic factors. Thesetrigger a cascade of deleterious events leading tocell death, particularly of dopaminergic SN neu-rons that are highly vulnerable as a result of theirneuromelanin content and low expression of pro-tective Ca++-binding proteins.

At present, many important questions aboutiron and oxidative stress remain unresolved. Arethey initial events that trigger cell death, or a con-sequence of the disease process, and how does ex-cess iron get access to vulnerable neuronal popu-lations in pathological conditions such as PD? Ironaccumulation within neuromelanin, as shown byseveral techniques, could explain the vulnerabilityof dopaminergic SN neurons, while excess ironwithin (micro)glial cells would suggest their par-ticipation in the neurodegenerative process, al-though these changes could also be secondary toneuronal death.

These events might also account for the findingin PD of a profound reduction in glutathione-related mechanisms of free radical defence, whichare primarily localised in glial cells.[304] Accumu-lation of iron, particularly in a perivascular distri-bution, might also suggest that breakdown of theblood-brain barrier is a pathogenic factor. On theother hand, iron changes are not selectively asso-



ciated with SN cell damage or PD, whereas gluta-thione depletion in PD has not been observed inother PD-like neurodegenerative disorders such asMSA and PSP and, thus, in PD, may precede bothiron changes and the dysfunction of the mitochon-drial electron transport chain.

The hypothesis that accumulation of iron, closelyrelated to the interaction between iron and neuro-melanin, is likely to be a risk factor for neuronaldamage in PD is the rationale for therapeutic effortsto prevent or to delay these deleterious mecha-nisms by: (i) reducing the overload of iron and thegeneration of ROS and other cytotoxic compounds,and thus stopping the production of harmful oxy-and hydroxy-radicals, e.g. 6-OHDA;[305] and (ii)preventing the effects of such changes by ‘neuro-protective’ strategies. Future prospects for pharma-cotherapy of PD and related neurodegenerative dis-orders, in addition to symptomatic levodopa andlevodopa-sparing strategies, will be the developmentof potent and well-tolerated iron chelators andlazaroids that prevent iron-induced free radical andROS generation. This represents an importantchallenge for future pharmacological and clinicalresearch.

Acknowledgements

The study was supported by a grant from the AustrianFederal Ministry of Science and Transport and by COSTAction D8L ‘Chemistry of Metals in Medicine of the EU’.Thanks are due to Mr K. Paukner for secretarial work andillustrations.

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Correspondence and reprints: Prof. Dr Kurt A. Jellinger,Ludwig Boltzmann Institute of Clinical Neurobiology,PKH/B-Building, Baumgartner Höhe 1, A-1140 Vienna,Austria.E-mail: [email protected]

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