Review: iron metabolism and the role of iron in neurodegenerative disorders.

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Iron in neurodegeneration

Neuropathology and Applied Neurobiology

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Iron metabolism and the role of iron in neurodegenerative disorders1 -review articleRevised version Maya Hadzhieva1, Elmar Kirches1,2 and Christian Mawrin1,2#

1

Department of Neuropathology and 2Center for Behavioural Brain Sciences (CBBS), Otto-von-Guericke-University Magdeburg, Germany;

Running title: Iron in neurodegeneration Key words: neurodegeneration, ROS, iron, mitoferrin, TfR, DMT1, ferritin, frataxin, IscU

#

Correspondence:

Christian Mawrin, MD Department of Neuropathology Otto-von-Guericke University Leipziger Strasse 44 D-39120 Magdeburg Tel: +49 391 67 158282 Fax. +49 391 6713300 e-mail: [email protected]

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/nan.12096

1 This article is protected by copyright. All rights reserved.

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Abstract

Iron plays a role for the biogenesis of two important redox-reactive prosthetic groups of enzymes, iron sulphur clusters (ISC) and heme. A part of these biosynthetic pathways takes plays in the mitochondria. While several important proteins of cellular iron uptake and storage and of mitochondrial iron metabolism are well-characterized, limited knowledge exists regarding the mitochondrial iron importers (mitoferrins). A disturbed distribution of iron, hampered Fe-dependent biosynthetic pathways and eventually oxidative stress resulting from an increased labile iron pool are suggested to play a role in several neurodegenerative diseases. Friedreich’s ataxia is associated with mitochondrial iron accumulation and hampered ISC/heme biogenesis due to reduced frataxin expression, thus representing a monogenic mitochondrial disorder, which is clearly elicited solely by a disturbed iron metabolism. Less clear are the controversially discussed impacts of iron dysregulation and iron-dependent oxidative stress in the most common neurodegenerative disorders, i.e. Alzheimer´s disease (AD) and Parkinson’s disease (PD). Amyotrophic lateral sclerosis (ALS) may be viewed as a disease offering a better support for a direct link between iron, oxidative stress and regional neurodegeneration. Alltogether, despite significant progress in molecular knowledge, the true impact of iron on the sporadic forms of AD, PD and ALS is still uncertain. Here we summarize the current knowledge of iron metabolism disturbances in neurodegenerative disorders.


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INTRODUCTION

With the steadily increasing life expectancy in western countries, the social and economic burden of neurodegenerative disease is an increasing challenge for medicine

and

science.

This

applies

especially

to

the

most

common

neurodegenerative disorders in the elderly population, Alzheimer’s (AD) and Parkinson’s disease (PD). These two disorders and amyotrophic lateral sclerosis (ALS), the most common cause of rapid motoneuron loss, are all characterized by

progressive neuronal degeneration. Most cases are considered idiopathic, although some weak genetic risk factors may influence disease manifestation such as the allelic status of apolipoprotein E in AD [1]. Inherited (familial) forms of AD, PD and ALS have been identified, but represent only a minor percentage of cases. Inherited AD has been linked to defects in genes located on chromosomes 1, 14, 19 and 21 [2], which account for 1-5% of AD patients. Heritable monogenic PD is associated with mutations of six different genes (SNCA, LRRK2, Parkin, PINK1, DJ-1 and ATP13A2), which explain approximately 3-5% of the morbidity rate [3]. A somewhat higher impact of a primary genetic pathogenesis is seen in ALS, where familial forms account for 5-10% of all cases and can be associated with mutations in the SOD1, ALS2, SETX, VAPB and Dynactin genes [4]. In the last five years, a few other genes

have been discussed in ALS. The transcriptional regulator TDP-43 (transactive

response DNA-binding protein 43) was implicated in the development of sporadic

ALS [5, 6] as well as in the development of inherited ALS without SOD1-mutations [7] and of other neurodegenerative disorders, which can exhibit TDP-43 immunopositive inclusions, such as FTLD-U (frontotemporal lobar degeneration with ubiquitin inclusions) [6]. In familial ALS, several mutations in the gene have been identified [7]. TDP-43 inclusions partially co-localize with the well-known Bunina bodies in ALS 3 This article is protected by copyright. All rights reserved.

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brains [8], and the pathogenetic impact of TDP-43 overexpression and brain accumulation for the process of neurodegeneration has been demonstrated in a mouse model [9].

The gene for the RNA-processing protein FUS (fused in sarcoma) is another

candidate, mutations of which are likely to explain a fraction of familial ALS cases [10-12]. An impairment of motor activity by either mutant or quantitatively reduced FUS has been shown recently in a Zebrafish model [13].

Mutations in an open reading frame on chromosome 9 (C9ORF72), which

encodes a cytoplasmic neuronal protein of as yet uncharacterized function, were found recently in several neurodegenerative disorders, seem to explain about twice as many familial ALS cases as SOD-1 mutations, and occur much more frequently than the above mentioned TDP-43 and FUS mutations (summarized in [14]). For all these proteins the pathogenetic mechanism is not clear, but no relation to neuronal iron metabolism has come into focus.

Compared to AD and PD, strictly inherited neurodegenerative diseases are

relatively rare. Numerous autosomal forms have in common the extension of a polyglutamine repeat, which is suggested to yield misfolded neurotoxic proteins or neurotoxic peptides obtained during their proteolysis (polyglutamine diseases). A relatively abundant example is Huntington’s Chorea [15]. Besides such gain of toxic function, some autosomal diseases are characterized by a marked quantitative loss of a single protein. The most abundant inherited disease of this ‘loss of function’ type is Friedreich’s ataxia (FRDA), which exhibits a genetic defect leading to a pronounced decrease of the mitochondrial iron-binding protein frataxin (FXN) [16]. While missense alleles exist, most patients do not show any changes of the amino 4

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acid sequence. The phenotype is thus suggested to be caused solely by an alteration of protein quantity and to be mediated mainly by the resulting disturbance of irondependent metabolism within the mitochondria.

Despite the fact that many genes have been linked to the common diseases

AD, PD and ALS, approximately 90% of cases are sporadic, with pathogenic mechanisms being less well understood thus far. A common feature of the above mentioned inherited and sporadic disorders are altered biochemical markers indicating oxidative stress, accompanied by disturbed iron homeostasis. Iron overload might contribute to oxidative stress through the Fenton reaction. The roles of iron and oxidative stress in neurodegeneration have been discussed before, but many issues are yet not fully understood [17-23]. The analysis of expression and regulation of proteins involved in iron metabolism, however, may contribute to an understanding of the disturbed iron homeostasis in neurodegeneration. In this review we will summarize the recent data regarding dysregulation of such genes and of metabolic pathways of iron in FRDA, which exemplifies the pathogenic role of an inherited decrease of a single mitochondrial iron-protein, and in AD, PD and ALS, where the contribution of disturbed iron homeostasis and of a dysregulation of ironrelated genes is more speculative. We will shortly discuss technical problems to specifically detect iron overload and the controversy regarding the impact of total versus labile iron in neurodegeneration and suggest some opportunities and strategies to substantiate the debate of iron-mediated oxidative stress in neurodegeneration.

Pathways of iron transport and processing within the cell 5


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Figure 1 provides an overview about the major molecular players of cellular iron trafficking. The main iron binding proteins are transferrin and ferritin. Transferrin receptor (TfR) is present at the surface membrane of all cells and tissues and has high affinity for binding iron-loaded transferrin (Tf), mediating endocytosis of iron. Ferritin is the intracellular iron storage protein composed of 24 subunits, which can be either light chains (L) or heavy chains (H), which assemble in varying proportions to form the mature ferritin protein. The assembled ferritin protein can bind several thousand iron atoms per molecule. The two ferritin chains are functionally different. The H-ferritin is more active in iron turnover and is considered as a major cytoprotectant, because it catalyzes the oxidation of ferrous (Fe 2+) to ferric iron (Fe3+) without forming oxygen radicals. H-ferritin thus lowers the pool of labile Fe2+, which can cause tissue damage via the Fenton reaction. The ferric iron is stored within ferritin as an essentially inert metal. The L-chain is associated with long term iron

storage [24]. In tissues with pronounced iron storage, such as liver and spleen, L-rich ferritins are found, while the proportion of H-chains is increased in metabolically

active tissues such as the brain. Only 0.05% of the brain’s iron seems to belong to the cytosolic labile iron pool, while the rest is bound to proteins, at least 90% to ferritin [25]. TfR and ferritin expression are controlled via the iron responsive element/ iron regulatory protein (IRE/ IRP) system.

The IRE/IRP system includes IRP1 and IRP2, which are cytosolic RNA-binding proteins that control iron homeostasis. These proteins exert their regulatory function by binding to the conserved stem-loop-stem RNA structures (IREs), located in the 5’or 3’- untranslated mRNA regions (UTRs) of proteins involved in iron transport and metabolism [20]. TfR mRNA for example contains five distinct IREs, located in the 3’untranslated region (UTR), while ferritin expression is controlled through a single IRE 6


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located in the 5’-UTR of H- and L-chain, thus allowing an antipodal regulation of iron import and storage as a reflection of the available cytosolic iron pool.

The mRNAs regulated in this way include those encoding proteins for iron

acquisition (TfR1, divalent metal transporter 1), storage (H- and L-chains of ferritin), utilization (erythroid 5-aminolevulinic amino acid synthase (ALAS2), mitochondrial acconitase) and export (ferroportin) [26]. Both IRPs inhibit the initiation of mRNA translation when they bind to the 5’-UTR-IREs, while binding to the 3’-UTR-IREs normally leads to protection from mRNA degradation. The binding activities of IRP1 and IRP2 are regulated by the cellular iron pool. Under conditions of high iron levels, an iron-sulphur cluster (4Fe-4S) assembles in IRP1, preventing it from binding to IREs; in conditions of low iron levels, IRP1 undergoes conformational change allowing IRE binding. In contrast to IRP1, IRP2 does not contain an iron sulphur cluster (ISC) but it is regulated by iron-dependent ubiquitination and degradation.

Once internalized by receptor mediated endocytosis, Tf-iron is found in the

endosome, where it is released from Tf upon acidification. Upon reduction to the ferrous form (Fe2+), the metal is transported from the endosome to the cytoplasm primarily by divalent metal transporter 1 (DMT1) [27]. The acquisition of iron by mitochondria of mammals requires mitochondrial transporters termed mitoferrins (Mfrn1 and Mfrn2), which import the metal into the organelle [28]. These solute carriers are located in the inner mitochondrial membrane. The yeast homologues Mrs3p and Mrs4p are required for Fe import into yeast mitochondria [29], and the poor growth of deletion mutants under restricted availability of iron could be corrected by the mammalian mitoferrins. While Mfrn1 is essential for the high levels of mitochondrial heme synthesis during erythropoiesis, the ubiquitous expression of Mfrn2 suggests a primary role of this solute carrier in other cell types. A targeted 7


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Mfrn1 deletion in mice revealed a phenotype when the deletion was induced in adult haematopoietic tissues (anaemia) showing again it was essential for red blood cell haemoglobinization. Deletion of mitoferrin 1 in adult hepatocytes showed no phenotype under normal conditions. Under conditions of a diet-enforced increase in porphyrin synthesis, animals with a hepatocyte specific knockout were less able to incorporate Fe into mature heme, leading to protoporphyria, cholestasis and cirrhosis [30].

The two main iron-dependent biosynthetic pathways are housed in mitochondria: which are the synthesis of Iron sulfur clusters (ISC) [31, 32] prosthetic groups on enzymes both inside and outside these organelles, and the synthesis of heme [33],

which is needed in huge amounts in specialized cells (erythrocytes), but also in various redox-enzymes in all cells. Mitoferrin-1 is thought to interact with the protein Abcb10 to enhance mitochondrial iron uptake and that these two proteins can form oligomeric complexes with ferrochelatase, an enzyme of the heme biosynthesis pathway [34]. ISCs are formed within mitochondria and can be assembled on mitochondrial apotproteins, such as IscU in mammals. Mitochondrial ISCs are also exported to the cytosol where a cytosolic complex of proteins delivers the ISC to both cytosolic and nuclear proteins. IscU and other proteins of the ISC biogenesis complexes were shown to interact physically with the mitochondrial iron protein frataxin (FXN) (25). For review on iron traffic and processing in mitochondria and

cytosol see [35].

Iron accumulates in many organs as a function of age [36] and is also

associated with the pathology of many age-related diseases. In this review we will summarise the findings about the expression of iron-related genes in FRDA, AD, PD and ALS, all of which have been associated with disturbance in iron homeostasis. 8 This article is protected by copyright. All rights reserved.

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Friedreich’s ataxia: an inherited neurodegenerative disease caused by loss of an iron-binding protein

FRDA is an autosomal recessive disease characterized by progressive degeneration in the central and peripheral nervous system, cardiomyopathy and increased risk of diabetes mellitus (for review see [37]). The neuropathology of FRDA involves spinal cord, peripheral nerves and cerebellum [38]. The disease most often results from decreased expression of the mitochondrial protein FXN, caused by a GAA repeat expansion within the first intron of the gene localized on chromosome 13. The functions of FXN are still not completely known and several new functions that have not been well-analysed to-date, can be hypothesized from recent literature data, including an involvement in redox-signaling or nitric oxide signaling [39]. However, the pathogenesis of the disease has been linked early to a disturbed ISC biogenesis [40]. The impairment of this biosynthetic pathway has been nicely supported by studies in yeast cells, exhibiting a loss of the FXN homolog Yfh1 (yeast frataxin

homolog 1) [41]. FXN is thought to act as an iron donor for mitochondrial iron consuming reactions, most prominantly ISC synthesis, but perhaps heme synthesis as well. The disturbance of the ISC pathway, together with a diminished heme biogenesis in Yfh1/FXN deficient cells [42] are thought to rely on a direct interaction of Yhf1/FXN with the ISC scaffold proteins [43] and with the enzyme ferrochelatase [44]. ISC- and heme- dependent enzymes inside and outside mitochondria will be affected in parallel. Inside mitochondria , complexes of the mitochondrial electron transport chain (ETC) may be targets of both types of metabolic alterations, since they contain ISCs and heme, required for electron transport. Moreover, ETC complexes and the ISC-containing mitochondrial enzyme aconitase may be affected 9 This article is protected by copyright. All rights reserved.

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by FXN-deficiency on a third level, i.e. oxidative stress. The resulting impairment of oxidative energy metabolism may be especially relevant in neuronal cell bodies, axons and synapses with their high demand of energy required to establish plasma membrane potential and to perform neurotransmission.

While even normal physiological ETC activity leads to the production of

reactive oxygen species (ROS), mainly superoxide and the strong oxidant hydrogen peroxide, mitochondrial iron deposits in FRDA tissues may enhance oxidative stress. A notable finding in both yeast and human cells with frataxin deficiency is the occurrence of iron deposits. These deposits (along with impaired ETC) may promote the generation of highly reactive hydroxyl radicals via the Fenton reaction, able to further destabilize ETC complexes I, II and III [45]. Mitochondrial ETC enzyme deficiency (complexes I, II and III) together with aconitase deficiency was reported early in the history of analyzing FXN functions [40] using endomyocardial biopsies from two independent FRDA patients with hypertrophic cardiomyopathy, having a homozygous trinucleotide expansion of the FXN gene.

The mechanism of mitochondrial iron load was partly deciphered in a study

investigating the expression of iron metabolism genes using a muscle creatine kinase (MCK) conditional FXN knock out mouse model of FRDA developed earlier [35, 46, 47]. Besides downregulation of FXN by the primary gene defect, Huang and colleagues identified dysregulation on the mRNA and protein level for a number of proteins of iron metabolism in heart muscle tissue of young FRDA mice [35, 47], although the authors did not provide an explanation how differential gene regulation was achieved. Nevertheless, these results were highly interesting, since they suggested a combination of previously unknown pathways of iron accumulation in diseased heart muscle mitochondria. 10 This article is protected by copyright. All rights reserved.

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First of all, decreased availability of the downregulated iron-sulphur-cluster

scaffold proteins, which are the primary acceptors of Yfh1-bound [43] and mammalian FXN-bound iron [48], seemed to contribute to decreased Fe utilization, complemented by a corresponding decrease of cysteine desulphurase Nfs1, an essential enzyme for the acquisition of sulphur prior to ISC synthesis on the scaffold protein [35, 47]. Besides ferrochelatase, another suggested acceptor of FXN-bound iron, four other enzymes of the heme synthesis pathway were downregulated. These results suggested that the disturbance of Fe canalization into biosynthetic pathways within the mitochondria had a broader mechanistic basis than previously thought.

Moreover, a network of transporters regulating iron trafficking was altered:

TfR1 and the mitochondrial iron transporter Mfrn2 were upregulated and ferroportin1, a protein exporting Fe, was downregulated. Together, these dysregulations may explain higher total cellular uptake of Fe, which is concentrated via Mfrn2 in the mitochondria. The observed increase of heme oxygenase-I, a cytoplasmic enzyme of heme catabolism, and the decrease of ferritin L- and H-chains may contribute to free iron available for mitochondrial import, which in the face of impaired iron consuming processes of ISC and heme synthesis might result in increased iron accumulation.

FRDA can thus serve as a well-defined example of an inherited disease, which

includes a specific pattern of neurodegeneration and is caused by metabolic shifts and oxidative stress elicited solely by a severe decline of a single mitochondrial iron protein, but very likely assisted by secondary dysregulation of other genes involved in Fe metabolism and trafficking.

Sporadic neurodegenerative diseases. 11 This article is protected by copyright. All rights reserved.

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Alzheimer’s disease (AD) is the most common form of dementia characterized by progressive loss of neurons in the cerebral cortex and formation of extracellular proteinaceous deposits referred to as senile plaques, which contain aggregated amyloid-β peptide (Aβ). They are accompanied by intracellular neurofibrillary tangles (NFTs) made of aggregates of the microtubule-associated protein tau. The 40-42 amino acid Aβ-peptides, which are the main component of the senile plaques,

accumulate during the progression of AD and result from the cleavage of the βamyloid precursor protein (APP), an ubiquitously expressed type I transmembrane receptor and metal binding protein [49, 50]. A leading hypothesis for AD aetiology suggests that neurodegeneration in AD is primarily caused by Aβ- accumulation and all other disease processes, such as tau pathology, result from the imbalance between Aβ production and clearance [51].

The involvement of iron in the pathogenesis of AD was suggested by

descriptive post mortem and sometimes in vivo imaging studies. Increased iron and ferritin levels were observed in the cerebral cortex of AD patients [52]. An immunohistochemical study showed higher iron accumulation as well as higher expression of both, transferrin and ferritin predominantly in oligodendrocytes [53]. In Alzheimer’s brain tissue, a homogeneous distribution of transferrin was observed around the senile plaques as well as strong ferritin immunoreaction in senile plaques and along cerebral cortical and hippocampal grey matter blood vessels. A significantly increased iron concentration was measured in Alzheimer’s disease globus pallidus and frontal cortex of human post-mortem brains. Significantly elevated levels of transferrin were measured in the frontal cortex of Alzheimer disease brains as compared to elderly controls [54]. A magnetic resonance imaging


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study reported elevated ferritin-iron levels in the basal ganglia (caudate, putamen and globus pallidus) as compared to age matched controls [55].

Based on the previous findings about a disturbance in metal homeostasis in

AD, Lovell and colleagues [56] used microparticle induced X-ray emission analysis and found elevated levels of copper, iron and zinc in the insoluble Aβ-plaques of AD patients. This finding is of particular interest, because these transition metals are suggested to induce a spontaneous self-aggregation and oligomerization of Aß and subsequent amyloidosis [57]. Understanding the mechanism of disturbed transition metal homeostasis might suggest potential therapeutic targets that might prevent increased transition metal concentration and therefore reduce Aβ-aggregation. Such a target could be DMT1, which was found to be required for the generation of Aβ [58]. In this study, two isoforms of DMT1, either containing an iron responsive element (IRE) or not, were found to colocalize with Aβ in the senile plaques of postmortem AD brains and APP/PS1 (presenilin-1) transgenic mouse brains. These mice are characterized by enhanced levels of Aß-peptides in their brains and rapid accumulation of amyloid plaques during aging, accompanied by a gradual decrease of various learning skills, which indicate memory impairment. Furthermore, DMT1 silencing in SH-SY5Y cells, overexpressing the APP Swedish mutant (APPsw) reduced APP mRNA and protein expression, as well as the Aβ 42 peptide level [58], which suggests the involvement of DMT1 in the pathogenesis of AD. Aβ is generated from APP through sequential cleavages, first by β-secretase

and then by γ-secretase (for review see [59]). The mRNA of APP was found to contain an IRE in its 5’-untranslated region and its translation was extremely

dependent on the intracellular iron concentration i.e. iron chelators inhibited the APP translation while iron influx reversed the inhibition [60]. A positive regulation of APP 13


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by Fe seems to fit well with the recently observed negative impact of DMT1 silencing on APP expression. In addition, the APP 5’-UTR-IRE was found to be more selective

for iron than copper and was unresponsive to zinc [49]. If Aß-pathology should indeed be the central aspect of Alzheimer’s disease, these findings would support a role for iron in the expression of APP and in the pathogenesis of AD. The 5’-UTR of

APP was shown to interact specifically with IRP1 in human neuroblastoma and hepatoma cells [60]. Interestingly, IRP2 appeared to be localized to neurofibrillary tangles and neurofibrillary lesions in AD brains [61]. Since IRP2 normally undergoes iron-dependent degradation in the proteasome, its accumulation might be due to proteolytic abnormalities [58]. However, dysregulation in the binding of IRPs to IRE was found to occur in AD brains [62].

In recent years, some reports used spectroscopic methods to quantify iron

concentrations directly in tissue samples. Mössbauer spectroscopy of a relevant number of post-mortem hippocampus samples, derived from AD patients (N = 10) and elderly controls (N = 10), revealed only a moderate (1.47-fold) increase of total iron in the AD samples, which was within the error range of the method, while ELISAs confirmed a previously suggested significant increase of both, H- chain ferritin (3.9fold) and L-chain ferritin (3.2-fold) in AD [25]. No analysis of the small labile iron pool, has been undertaken in AD brains. As mentioned by Galazka-Friedman and colleagues [25], even extremely low concentrations of labile iron may be sufficient to generate toxic ROS species and induce neurodegeneration. A focused analysis of the non-ferritin iron pool will be necessary to further evaluate a potential role of ironmediated oxidative stress in neurodegeneration. Although not providing a final proof for a pathogenetic role of iron overload, the observations depicted above connect AD with iron imbalance. Moreover, a few recent observations and experimental results 14 This article is protected by copyright. All rights reserved.

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connect DMT1 divalent transition metal transporter with Aß-deposits and the regulation of APP by iron. Parkinson’s disease (PD) is characterized by degeneration of dopaminergic neurons (DN) in the substantia nigra pars compacta (SNpc) and accumulation of αsynuclein in the proteinaceous cytoplasmic inclusions (Lewy bodies) in the remaining DN. The main clinical features of PD include tremor at rest, rigidity, akinesia and postural instability [63]. In various studies, iron was reported to accumulate at the sites of neuronal death in PD brain. In particular, increased iron was found in microglia, astrocytes, oligodendrocytes and melanin containing DN of the substantia nigra (SN), the main site of neuronal degeneration.

A detailed study examining H and L- ferritins by quantitative immunoassay in

selected brain regions (frontal cortex, caudate, putamen, SN and globus pallidus) revealed a normal age-related increase of both isoforms in brains of elderly persons, H-ferritin being the predominant type in the brain [24]. The predominance of H-ferritin was recently confirmed by others [25] and is in accordance with a main function of brain ferritin in iron turnover and detoxification, rather than long term storage. The concentrations of H-ferritin (normalized to wet weight) were highest in putamen, caudate and SN. The normal age-dependent increase of H-ferritin in these regions was not observed to the same extent in PD brains. In all brain regions analyzed, the concentration of L-ferritin was also significantly lower as compared to elderly controls, a result which was recently confirmed for the SN by others [25]. Similarly, Connor and colleagues observed again the lack of an age-dependent increase of both ferritins in AD brains, suggesting a lowered capacity of PD and AD brains to bind iron and perhaps withdraw it from toxic Fenton reactions. The authors suggest that a


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deficiency in the sequestration of iron in a chemically inactive form in aging brains may favour neurodegeneration.

At the same time TfR was found in very low densities in SN and globus

pallidus in normal brains, which does not correspond to high iron concentration in SN, meaning that transferrin internalization is not the mechanism leading to iron accumulation in this area [64]. In contrast, increased lactoferrin (Lf) receptor immunoreactivity was found in neurons of the SNpc where 50-80% of DA neuronal loss occurs (60). Lf may be responsible for the excessive accumulation of iron in dopaminergic neurons in PD, since the main lactoferrin functions are binding and transportation of iron. However, another study describes experiments which suggest that PD’s ferritin is more heavily loaded with iron in comparison to age-matched controls [65]. In addition, H-ferritin overexpression within dopaminergic SN neurons successfully sequestered excess iron and prevented neuronal cell loss induced by the Parkinson-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrapyradine (MPTP) in mice [66].

Besides the ferritin iron load, increased neuromelanin iron load was also

observed in PD, both of which have been associated with the increased iron load [6769]. Neuromelanin is thought to be the intracellular by-product of the oxidative polymerisation of dopamine [70] with an important metal chelating/sequestering role [71].

Using immunohistochemical approaches, a general correlation was discovered

between the level of DMT1 and the ferrous iron distribution in the basal ganglia of a monkey [57], where regions such as caudate nucleus, putamen and SN showed dense staining of DMT1 and large numbers of ferrous granules. The DMT1 (+IRE 16 This article is protected by copyright. All rights reserved.

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isoform) expression was shown to be increased in the substantia nigra of PD patients. In the MPTP-induced PD mouse model expression of DMT1 was accompanied by iron accumulation, oxidative stress, and dopaminergic cell loss. However mice with a mutation in DMT1, which impairs iron transport activity, were much less susceptable to MPTP-induced dopaminergic cell death, suggesting the involvement of DMT1 in the pathogenesis of PD [72]. Similarly to APP in AD, a putative IRE in the 5’-UTR of α-synuclein mRNA was

reported, suggesting post-transcriptional regulation of α-synuclein protein expression in response to cellular iron [73]. Moreover, α- synuclein was suggested to bind free

iron, which in turn greatly accelerates its aggregation (for review: [74]). While enhanced total iron levels in SN of PD brains had been reported early [75], some reports failed to confirm this, including a recent Mössbauer spectroscopy study, which compared the SN from 17 PD patients with that of 29 controls. The observed mean iron concentrations were identical (177ng/mg wet weight) in both groups [25],

which argues against a role of general iron overload in SN neurodegeneration, as long as local inhomogeneity within the SN and potential differences between glia and dopaminergic neurons are neglected. Mössbauer spectroscopy neither revealed any ferrous iron (Fe2+) in the SN of PD patients, nor in the SN of controls, but the sensitivity of the technique was limited. Most interestingly, atomic absorption spectroscopy, designed to measure iron only in particles below 10 kDa [76] revealed a 2.4-fold increase of the concentration of non-ferritin iron in the SN of PD versus control brains [25]. Although the measured concentrations represented only 0.05% of the total iron in the SN of PD brains, the authors suggest that the fraction of Fe 2+ in this pool may play a role for neurodegeneration via oxidative stress.


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Although the relatively rare autosomal PD subtypes with early age of onset do

not currently add significant information regarding the role of iron in PD pathophysiology, it is interesting to compare some of them with Friedreich’s ataxia, because these diseases have in common a prominent role of mitochondria, i.e. of the organelles harbouring essential branches of iron-metabolism.

Besides autosomal inheritance, PARK-2, -6 and -7 share biochemical features

of a mitochondrial dysfunction or at least enhanced sensitivity towards mitochondrial noxes with Friedreich’s ataxia. The most common mutations causing autosomal recessive parkinsonism (PARK-2) are located in the parkin gene, which encodes an ubiquitin E3-ligase, involved in ubiquitin-labeling of proteins for proteasomal degradation. Although knockout-mice surprisingly did not exhibit a major behavioural phenotype, it could be shown in various models that parkin protects neurons against a variety of cellular stresses, including the prevention of mitochondrial swelling, cytochrome c release and subsequent mitochondria-dependent cell death [77]. The second-most common cause of autosomal recessive parkinsonism (PARK-6) are mutations in the gene PINK-1 (phosphatase and tensin homologe induced kinase 1). The localization of this kinase is still debated and it seems to occur in cytoplasm and mitochondria [78]. Moreover, series of potential targets have been suggested, as it is the case for parkin, leaving the question largely unresolved, which targets may play a dominating role in neurodegeneration. However, it seems that the mitochondriaprotective protein parkin is among the targets of the kinase PINK-1 and knockoutphenotypes in Drosophila are similar for both proteins, i.e. shorter lifespan, male infertility and motor deficiency. These and several other hints suggested a participation of both proteins in the same neuroprotective, mitochondria-based pathways (for review see [79]). Although again knockout mice did not show a major 18 This article is protected by copyright. All rights reserved.

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phenotype, e.g. no nigrostriatal degeneration, the role of PINK-1 for regular mitochondrial function was supported by respiration-deficits in the striatum of knockout-mice and under conditions of oxidative stress in the cortex [80]. More specifically, a decreased activity of NADH:ubichinon oxidoreductase (complex I of the ETC) was observed in PINK-1 knockout-models of mouse and Drosophila [81],

leading to defective synaptic function, probably via ATP deficiency. Autosomal recessive parkinsonism (PARK-7) can also be caused by mutations of the gene DJ-1, which seems to have a protective function against oxidative stress [82, 83], including the induction of antioxidative defences [84, 85] and protection against apoptosis by interaction with a modulator of the mitochondrial apoptotic pathway [86].

Although these suggested mitochondrial involvements in neurodegeneration fit

with a discussed mitochondrial (ETC complex-I) dysfunction in sporadic PD in the SN and other tissues [87-90], and with the well-known potency of the complex-I inhibitor MPTP to induce parkinsonism in humans [91] and mice, it does not link directly ironmetabolism within the mitochondria to neurodegeneration, as it is the case in Friedreich’s ataxia. However, it is thinkable that mitochondrial dysfunction (decreased complex-I

activity,

respiration,

membrane

potential

and

ATP

production)

accompanied by enhanced mitochondrial ROS production, either due to loss or mutation of mitochondria-protective proteins (parkin, PINK-1) or by the loss of an important mitochondrial iron-acceptor (FXN), may have similar consequences in neurons. As depicted earlier, ETC dysfunction accompanied by signs for oxidative stress (aconitase deficiency) have also been reported in patients suffering from Friedreich’s ataxia. Inhibition of energy metabolism, of support with metabolic intermediates from the citric acid cycle and enhanced apoptotic sensitivity may thus be mechanistic overlaps between Friedreich’s ataxia and some types of monogenic 19


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parkinsonism. At least, mutations in FXN, parkin, PINK-1 and DJ-1 support the notion that

mitochondrial

dysfunction

and

oxidative

stress

can

participate

in

neurodegeneration, while leaving open the question of regional specificity of neuron loss.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by selective loss of motor neurons in the spinal cord, brainstem and cortex, and occurrence of Bunina bodies, skein-like inclusions (SLI), Lewy body- like inclusions (LBLI) and basophilic inclusions in the remaining anterior horn cells of the spinal cord.

Increased calcium and iron levels in the cytoplasm of cervical spinal cords

from sporadic ALS patients had been reported in a study using Laser Microprobe Mass Spectrometry [92], which was in line with T2-weighed MRI scans, which may be interpreted in terms of increased iron load [93]. A large recent study of serum samples from ALS patients (N = 694) and healthy controls (N = 297) revealed higher serum ferritin levels and higher iron saturation of serum transferrin in the ALS group. Even more important, the survival time of the group with high level ferritin was 33% shorter (p ≤ 0.01) than that of the low level ferritin group [94]. A similar negative effect of high serum ferritin levels was recently reported in a study comparing 92 ALS patients and 92 age, sex and body mass index matched controls. Disease progression, according to the ALS functional rating scale, was positively correlated with serum ferritin levels [95]. From the clinical point of view there is another recent hint, which points towards a possible relation between brain iron overload and ALS. A mutation of an orphan mitochondrial protein, c19orf12, has been found to explain a 20 This article is protected by copyright. All rights reserved.

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fraction of rare neurodegenerative diseases (NBIA, neurodegeneration with brain iron accumulation) showing iron accumulation in the basal ganglia [96]. While

histopathological characterization exhibited an overlap of NBIA with more common neurodegenerative diseases, i.e. tau and tangle pathology and Lewy bodies, two families have been recently identified, in which the C19orf12 mutation caused a phenotype mimicking ALS in three individuals [97].

Increased iron levels in spinal cord motor neurons were also reported recently

[98, 99] in two ALS mouse models of different disease kinetics, harboring SOD1 mutations known to cause familial ALS. One of these mutations (G93A) causes a rapid degeneration of spinal motor neurons leading to death within three months, while the other (G37R) causes a much slower disease progression in mice. Even in the extremely rapid disease (G93A), iron dysregulation was strong enough to observe a measurable iron overload within the life-time of the mice. All these findings imply a disturbed iron homeostasis in ALS. A pathogenic impact of this dysregulation was further supported by the partially protective effects of iron-chelators in cell culture and mouse models of ALS. In recent years, various iron-chelators were shown to prolong the survival of transgenic mice [98, 100] and rats [101] carrying the G93A mutation of SOD1. Iron-chelators not only attenuated iron levels, but also reduced oxidative stress markers in the tissue and attenuated neuronal loss, astroglial and microglial activation.

Together with the elevated iron-levels in ALS, an increase in TfR1 protein

expression in the spinal cords of SOD1-G93A mice [98] was reported. Similarly, in our recent study an increase in TfR1 gene expression was observed in cells harboring the same mutation [102]. TfR1 was found to accumulate in Bunina bodies and basophilic inclusions from the lumbar spinal cords of ALS patients [103], which 21


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suggests an involvement of TfR1 in Bunina body formation. Elevated serum ferritin levels were found in ALS patients, being a reflection of the general increase in the amount of iron stored in these patients [104], as well as an upregulation of H- and Lferritin in SOD1-G93A mice just prior to the end-stage of the disease has been reported [105]. In our study, increased levels of ferritin mRNA were confirmed in the SOD1-G93A cell model. Altogether, these findings support the idea of increased neuronal iron uptake and overload in ALS. The gene solute carrier family 11 (SLC11A2), encoding DMT1, a carrier necessary for the release of iron from endocytotic vesicles, was associated with ALS disease duration [106]. We found significant upregulation of DMT1 mRNA expression in SOD1-G93A cells, suggesting increased release of iron into the cytoplasm, which is in line with the findings about iron overload in ALS.

Although iron dysregulation causes several human diseases, including

neurodegenerative disorders (FRDA, NBIA), a surprising deficit of knowledge exists regarding the basics of the interplay between cytoplasmic and mitochondrial iron. Nothing is known about the normal, physiological role of mitoferrins 1 and 2 for mitochondrial iron uptake in neurons and glial cells. Our ALS cell culture model suggested a general upregulation of these solute carriers and of the primary intramitochondrial Fe-acceptor FXN in cells harboring the SOD1-mutation G93A [102].

The existing literature supports the idea of oxidative stress in ALS. Our cell

culture model revealed that the mRNA levels of iron-related genes (TfR1, Ferritin, DMT1, IscU, FXN, Mfrn2, IRP1) may further increase when the cytoplasmic ROS levels rise in response to treatment with retinoic acid [102]. This observation suggests the involvement of ROS in the dysregulation of iron related genes in the 22 This article is protected by copyright. All rights reserved.

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mutant cells. Critically it is not known yet what comes first in ALS, iron dysregulation or oxidative stress. Does iron via the Fenton reaction trigger oxidative stress, a scenario for which solely the unknown labile iron pool is responsible ? Or is oxidative stress from other sources merely accompanied by altered gene expression leading to a secondary overload of iron, which is then safely stored in proteins ? A central question is, whether or not an increase of stored iron in the neurons really leads to an increase of labile iron, which can catalyze the production of hydroxyl radicals. The molecular mechanism of neurodegeneration in ALS is complex. We suggest that ROS-dependent transcriptional regulation may affect the machinery for mitochondrial iron import (Mfrn-2) and utilization (FXN, IscU), since these mRNAS were among those, which were upregulated under conditions of enhanced ROS levels (retinoic acid treatment), as monitored by the ROS sensitive dye DCF [102].

Inherited SOD1 mutations (G93A), modeled in mice and rats, were shown to

exhibit increased ROS production, which may be explained e.g. by monomers of SOD1 with incomplete metal loading, which become sources of ROS [107] or indirectly by a disturbance of the mitochondrial electron transport chain (ETC) in brain and spinal cord [108]. In the mouse model, this ROS overproduction leads to enhanced oxidative damage in older animals, which is most pronounced in the central nervous system, but also occurs in skeletal muscle and liver [109]. The interplay between disturbed iron homeostasis and other potential sources of oxidative stress in these models (e.g. mutant SOD1 itself or ETC), represents a completely unresolved issue. Therefore, It remains unclear, up to what extend these models exemplify the mechanisms of oxidative stress, which can be observed in patients with sporadic ALS, according to molecular markers of oxidative lipid and protein damage in plasma and urine [110-112]. 23 This article is protected by copyright. All rights reserved.

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Controversies about the role of iron in neurodegeneration

There is an ongoing debate as to whether or not iron deposits play a significant role in triggering or accelerating neurodegeneration. Alternatively, iron deposits may just reflect

a

disturbed

metabolism

as

a

secondary

event

accompanying

neurodegeneration. Moreover, not all studies support iron accumulation at all and several potential artifacts of quantitative estimation from MRI scans, spectroscopic measureents and other methods have been discussed. If iron deposits occur early during pathogenesis in the relevant brain regions, this circumstance would greatly support a significant role in pathogenesis. However, longitudinal MRI studies were rare and a direct comparison between ex vivo MRI data and histological iron stains or

biochemical iron measurement are possible only post mortem, i.e. usually in a very late stage of the disease.

At least, it can be stated that an increased iron content has been found using

various spectroscopic and other techniques in the SN of PD brains by most studies undertaken [for review of the research history see [113]. For ALS, some of the larger MRI-studies did either not perform any attempt to correlate hypo-intensities with biochemically measured iron concentrations in the same region, or performed this detailed analysis only with an extremely small fraction of their cases post mortem. In a recent study, Ignjatovic and colleagues were able to correlate MRI hypo-intensities in the precentral gyrus, i.e. in the primary motorcortex, with ALS, suggesting these scans to be suitable non-invasive biomarkers, but did not perform an independent iron determination post mortem [114]. The cortical hypo-intensities measured recently by Kwan and colleagues in a relatively large ALS cohort, were shown only in two 24 This article is protected by copyright. All rights reserved.

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histologically examined cases to correspond to local iron-deposits in the motor cortex [115]. Moreover, histological stains suggested major iron-accumulation mainly in microglia, but not in neurons. Such observations make it difficult to assume a direct iron-based (e.g. oxidative) neuronal damage and feed the speculation that iron deposits in the motor cortex may reflect the final destruction of already degenerating neurons by activated microglia. Other authors have put into question the significance of MRI imaging as a measure of iron in ALS-motorcortex at all, because different MRI techniques, assumed to reflect the iron-content of brain tissue, did not yield consistent results [116].

In AD, the situation is even more complex, due to the wide distribution of Aß-

pathology in the affected brains, making the localization of iron-deposits a less significant feature regarding the controversy of an active involvement of the metal in the pathogenesis. At least, MRI-based studies suggested iron-accumulation in AD brains to include relevant regions, such as hippocampus and cortex [117, 118] By comparison of histological stains with ex vivo MRI data in the entorhinal cortex , i.e. in the most early affected brain region in AD, it could be shown that MRI and histology both found iron accumulation, thus supporting an active role of iron and also the usefulness of MRI to measure it [119]. However, iron and amyloid plaques themselves, even if iron-free at the sensitivity- level of histology, were able to induce hypo-intensities in MRI images, thus demonstrating a limitation to quantify the metal in AD by imaging techniques. Alterations in MRI scans can also be elicited by several other metals [120]. Generally, the non-invasive MRI techniques lack an absolute specificity for iron, which makes precise longitudinal studies impossible.

But even more specific techniques, requiring post mortem tissue samples,

revealed their limitations in the past. Technical difficulties and inconsistency between 25 This article is protected by copyright. All rights reserved.

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studies to determine a potential iron overload with various spectroscopic and other methods have been extensively discussed by Friedman and colleagues for the example of SN in PD [25, 121]. In their review of the history of iron-research in PD, the authors state that sometimes no change in iron-content or even a moderate decrease was observed in the SN of PD-cases, although the opposite was observed in most studies. More important, the mean values for iron per gram wet weight of the SN varied in the majority of studies by a factor of 4.5. If all studies were taken into account, the range of measured iron-concentrations differed 10-fold between the minimal and maximal values ever published. This pointed to severe differences between the methods used and several technical problems have been discussed, such as leakage of iron from the sample, if the method required a destruction of the protein shell of the metal or a thinkable inhomogeneous distribution of iron within the SN (e.g. pars compacta vs. pars reticulata), playing a role for very small tissue samples [25, 122]. The authors favour Mössbauer spectroscopy, which does not require pretreatment, is able to assess iron in a volume comparable to the whole human SN, distinguishes Fe2+ (involved in the Fenton reaction) and Fe3+ and various forms of protein-bound iron. As mentioned above, the authors did not reveal any significant difference in total iron-content between PD and controls using this method, and were unable to detect any Fe2+, although the limited sensitivity of the method would have allowed the presence of small amounts of divalent iron capable to participate in the Fenton reaction. Most interesting, the authors discuss a dominating role of the changed composition of the ferritin-shell in PD. Since a decline

of L-ferritin was shown to occur in the SN of PD patients, this decline may result in an enhanced leakage of iron from ferritin to become part of the labile iron pool.


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At this point, the unresolved debate about the iron storage capacity of

neuromelanin comes into play. If significant amounts of iron may bind loosely to this pigment, neuromelanin may be a good source to feed the labile iron pool and keep the deleterious Fenton reaction running. An increased content of neuromelaninassociated iron was demonstrated in the SN of PD brains [123]. However, as already stated by Friedman and colleagues [113] the neuromelanin content per se cannot

explain neurodegeneration, because melanin-containing neurons outside the SN are not affected. If playing a role at all, a high proportion of neuromelanin-bound iron may only be a basic condition, enhancing ROS-production in such dopaminergic neurons, in which an as yet unknown specific change occured. An at least thinkable specific condition may be a lowered capacity of the main iron-store ferritin, due to structural changes. However, iron-independent specific conditions are also imaginable and have been discussed, such as enhanced mitochondrial ROS-generation by alterations of the ETC, e.g. elicited by age-dependent somatic mitochondrial DNA deletions, which expand clonally within dopaminergic neurons of the SN [124, 125] A further alternative would be an age-dependent decline of antioxidative defense within the SN [126, 127] , which may per se sufficiently explain the observed oxidative tissue damage [128-131]. All these mechanisms of oxidative stress may be favored by the availability of labile iron, but a difference in the content of this metal between control and PD brains is not necessary to explain neurodegeneration in the latter. In conlusion, it should be kept in mind that labile iron may participate as a basic cofactor in oxidative stress and neurodegeneration, even in the absence of any measurable quantitative difference between affected individuals and controls. At least it should be consensus that the measurement of total iron does not allow any final conclusions regarding a role of the metal for oxidative stress.


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As already mentioned above, another indirect support of an active role of

chelatable iron for neurodegeneration comes from several studies, which demonstrated beneficial effects of chelator-treatment in transgenic mice, which represent severe forms of inherited neurodegenerative disorders. In the SOD1-G93A ALS mouse model, the iron-chelators VK-28 andf M30 both inhibited ROSgeneration, microglia activation and astrocyte activation in the spinal cords [98].

Moreover the chelating compound M30 led to later onset of disease and prolonged survival in the same model [100]. The classical iron-chelator deferoxamine was shown to inhibit hippocampal tau phosphorylation, to reduce amyloidogenic processing of the amyloid precursor protein (APP), and to reduce Aß-deposition and memory loss in transgenic Alzheimer mice (APP/PS1) fed with high-dose iron [132, 133]. Correspondingly, the iron-chelator M30 also exhibited protecting effects in otherwise untreated APP/PS1 mice by lowering cerebral iron concentrations, lowering Aß-levels and plaque formation, decreasing APP- and tau-phosphorylation and improving learning skills in a broad variety of behavioral essays [134]. Hints for a limited rescuing activity of M30 towards dopaminergic neurons were obtained in the mouse model of MPTP-induced parkinsonism [135]. However, it is difficult to attribute all these effects to the process of iron-chelation, since M30 is a hybrid molecule, which also possesses properties of an antioxidant and a monoamine-oxidase B (MAO-B)- inhibitor moiety, which both could be suspected to play a role in these models. Finally, a more specific support of an active role of iron in dopaminergic neurodegeneration came from a mouse mode, where stereotactic injections of a proteasome-inhibitor elicited dopaminergic degeneration. If inhibitor injections into wild type mice were compared with injections into mice overexpressing human Hferritin, a neuroprotective effect of enhanced H-ferritin levels was observed, suggesting a role for non-ferritin bound iron in the process of neurodegeneration 28 This article is protected by copyright. All rights reserved.

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[136]. Although H-ferritin was used in this transgenic model, the message of a neuroprotective role of enhanced storage of iron in ferritin-shells may fit with the discussed role of a lowered iron-storage capacity of ferritin in the SN of PD brains (see above).

Opportunities and strategies to move the field forward

Post mortem analyses: It should be a consensus to use methods for determination

of the total iron load of a tissue sample of patients or experimental animals that are as free of artifacts as possible. As discussed by Friedman and colleagues, an uncontrolled leakage of iron by a variable degree of denaturation of iron-binding proteins (e.g. by storage or preparation) should be avoided. It should also be a consensus that the total iron concentration can hardly be discussed as a feature predicting oxidative stress-based neurodegeneration. To match this goal more closely, it is necessary to find tools for a visualization and reliable quantitative estimation of the pool of labile iron, either by development and use of sufficiently specific and sensitive iron-chelating fluorescent dyes [137-139] which eventually can target also mitochondria [140], or by using the fraction of iron, which is not bound to high molecular weight particles as a surrogate marker of the labile iron pool. Ultrafiltration excluding ferritin- and transferrin-bound iron and any particles above 10

kDa, followed by atomic absorption spectroscopy, was successfully used to demonstrate an increase of this ‘low molecular weight iron pool’ in the SN of PD patients [76]. The fact that further homogenization in a glass homogenizer after ultrafiltration, i.e. the destruction of membranes, organelles and artificial lipid vesicles, was necessary to discover the difference between PD and controls in this “low 29 This article is protected by copyright. All rights reserved.

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molecular weight iron”, suggests that iron-binding mechanisms of low-affinity may mask differences in the redox-reactive iron pool of tissue homogenates even in carefully planned experiments, depending on the precise technique of tissue preparation used. While protein denaturation has to be avoided in order to preserve the major stores of redox-inactive iron, the remaining iron pool must be made accessible as completely as possible to spectroscopic or fluorescent detection. In most studies, labile iron has not been the focus, but it should be the focus in order to substantiate the long controversy about iron and oxidative stress. Another important issue to substantiate this debate would be any attempt to find longitudinal correlations between ‘labile iron’ and ROS or ‘labile iron’ and disease progression in genetic mouse models for PD, ALS and AD and in MTPT-induced parkinsonism in the mouse. A refinement of fluorescent techniques may help to better define the relevant cell types and to separately assess mitochondria.

Last but not least, much basic research is still required to understand the

import of iron into mitochondria. These organelles at the same time house important pathways of iron-metabolism and provide most of the H2O2, which labile divalent iron would need to participate in oxidative stress. In this connection it would be of interest to assess the total and labile iron pool of mitochondrial preparations from the affected tissues of neurodegenerative mouse models. It is yet unclear, if the ubiquitously expressed iron transporter of the inner mitochondrial membrane (Mfrn-2), or the most important mitochondrial iron-importer of erythrocyte-precursors (Mfrn-1) or both play any role for iron-uptake into neuronal or glial mitochondria. A first step to identify the relevant transporters would be the analysis of mitochondrial iron load, mitochondrial iron-metabolism (heme synthesis, ISC biogenesis) and histology of brains from mice with corresponding neuronal or glial knockouts. 30 This article is protected by copyright. All rights reserved.

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Studies in vivo: For MRI-based estimation of total iron load in vivo, a standard method has to be established to allow better comparison between different studies. Meanwhile it seems to be consensus that R2* relaxometry yields the most reliable results and is associated with tolerable variation among institutions [141]. The use of this technique in longitudinal studies, which correlate a progression of the MRI-score (hypo-intensities) with disease progression should be extended, even though an unequivocal longitudinal correlation with accumulating iron is not possible. However, the application of MRI techniques in neurodegenerative mouse models may allow to follow disease progression, as e.g. demonstrated in AD mice [142]. Especially in AD models, it may be difficult to decide, if MRI scans mainly detect iron-associated amyloid plaques or also iron-free amyloid plaques above a certain size. However, mouse models would allow a longitudinal comparison of MRI data in a defined brain region with the biochemically determined iron load of the mice.

Finally, a further application of chelating agents in neurodegenerative mouse

models, especially of non-hybrid molecules without other functional moieties besides the iron-chelating one, may help to further underpin a deleterious role of iron in these models.

Acknowledgements: We thank Jerry Kaplan (University of Utah, USA) for

critical reading and helpful comments. The work was supported by grants from the Christa Lorenz Foundation and the CBBS. MH, EK and CM contributed equally to the manuscript writing/correction, and generation of the figure.


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Figure legend:

Figure 1:

Scheme representing the main routes of cellular iron-trafficking and -metabolism. In most cells transferrin-bound iron is imported through the plasma membrane via transferrin-receptor mediated endocytosis, leading to iron loaded complexes in endosomes. Following liberation of iron from these complexes, the metal is transported as Fe2+ via divalent metal transporter 1 (DMT1) from the endosomes into the cytoplasm and either stored mainly in a ferritin-bound form or imported into mitochondria via mitoferrins. Inside mitochondria, iron is channeled into two main biosynthetic pathways, i.e. biosynthesis of iron sulfur clusters (ISC) and heme. ISC biogenesis occurs on the surface of iron sulfur cluster scaffold proteins (IscU), which thus need to receive iron, while ferrochelatase is the iron-accepting enzyme in heme biosynthesis, which chelates iron with the protoporphyrin IX. The protein frataxin (FXN) is another important acceptor of mitochondrial iron, which certainly plays a role for correct ISC biogenesis and most likely for correct heme synthesis. It cooperates with IscU and ferrochelatase.


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Abbreviations

AD

Alzheimer’s disease

ALAS2

5-aminolevulinic amino acid synthase

ALS

Amyotrophic lateral sclerosis

ALS2

alsin gene

APP

amyloid precursor protein

APT13A2

ATPase Typ 13A2

C9ORF72

chromosome 9 open reading frame 72

C19ORF12

chromosome 19 open reading frame 12

DMT1

divalent metal transporter 1

DN

dopaminergic neuron

ETC

electron transport chain

FRDA

Friedreich’s ataxia

FTDL-U

frontotemporal lobar degeneration with ubiquitin inclusions

FUS

fused in sarcoma (gene relevant in Parkinson’s disease)

FXN

frataxin

IRE

iron responsive element

IRP

iron regulatory protein

ISC

iron sulfur cluster

IscU

iron sulfur cluster scaffold protein

Lf

lactoferrin

LRRK2

leucine-rich repeat kinase 2

Mfrn-1 (2)

mitoferrin 1 (2)

MPTP

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin

NBIA

neurodegeneration with brain iron accumulation 33


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Parkinson’s disease

PINK-1

phosphatase and tensin homologe induced kinase 1

PS-1

presenelin 1

ROS

reactive oxygen species

SETX

senataxin

SN

substantia nigra

SNCA

alpha-synuclein gene

SOD1

superoxide dismutase 1

TDP-43

transcription response DNA-binding protein 43

Tf

transferrin

TfR

transferrin receptor

UTR

untranslated mRNA region

VAPB

vesicle-associated membrane protein-associated protein B/C

Yfh1

yeast frataxin homolog 1

Accepted Article

PD


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Schenck JF. Magnetic resonance imaging of brain iron. J Neurol Sci 2003; 207: 99-

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120 102

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133 Guo C, Wang T, Zheng W, Shan ZY, Teng WP, Wang ZY. Intranasal deferoxamine reverses iron-induced memory deficits and inhibits amyloidogenic APP processing in a transgenic mouse model of Alzheimer's disease. Neurobiol Aging 2013; 34: 562-75 134 Kupershmidt L, Amit T, Bar-Am O, Youdim MB, Weinreb O. The novel multi-target iron chelating-radical scavenging compound M30 possesses beneficial effects on major hallmarks of Alzheimer's disease. Antioxid Redox Signal 2012; 17: 860-77 135 Youdim MB. M30, a brain permeable multitarget neurorestorative drug in post nigrostriatal dopamine neuron lesion of parkinsonism animal models. Parkinsonism Relat Disord 2012; 18 Suppl 1: S151-4 136 Zhu W, Li X, Xie W, Luo F, Kaur D, Andersen JK, Jankovic J, Le W. Genetic iron chelation protects against proteasome inhibition-induced dopamine neuron degeneration. Neurobiol Dis 2010; 37: 307-13

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