Altered regulation of iron transport and storage in Parkinson’s disease

J Neural Transm (2006) [Suppl] 71: 201–204

# Springer-Verlag 2006

Altered regulation of iron transport and storage in Parkinson’s disease

E. C. Hirsch

INSERM, UMR679, Experimental Neurology and Therapeutics, H^oopital de la Salp^eetri
eere; Universitee Pierre & Marie Curie – Paris 6, Paris, France

SummaryParkinson’s disease (PD) is characterized by the death of dopa- minergic neurons in the substantia nigra. This neuronal degeneration is associated with a strong microglial activation and iron accumulation in the affected brain structures. The increased iron content may result from an increased iron penetration into the brain parenchyma due to a higher expression of lactoferrin and lactoferrin receptors at the level of the blood vessels and dopaminergic neurons in the substantia nigra in PD. Iron may also accumulate in microglial cells after phagocytosis of dopaminergic neurons. These effects may be reinforced by a lack of up-regulation of the iron storage protein ferritin, as suggested by an absence of change in iron regulatory protein 1 (IRP-1) control of ferritin mRNA translation in PD.

Thus, a dysregulation of the labile iron pool may participate in the degen- erative process affecting dopaminergic neurons in PD.

Introduction

Parkinson’s disease (PD) is characterized by a preferential vulnerability of dopaminergic neurons in the substantia nigra. Yet evidence suggests that the loss of these neurons is heterogeneous across different catecholaminergic cell groups (Hirsch et al., 1988). Despite the recent identifica- tion of genes involved in inherited forms of the disease, the exact mechanism by which dopaminergic neurons degen- erate in idiopathic Parkinson’s disease is still poorly un- derstood. Several molecular and cellular alterations may, however, contribute to cellular dysfunction and ultimately cell death. Among these factors, protein accumulation, pro- teasome dysfunction, mitochondrial complex-1 deficiency, and oxidative stress are believed to participate in the cas- cade of events leading to neuronal death. In line with this, the research groups headed by Youdim and Riederer were among the first to report an increase in the content of iron (III) and total iron in the postmortem substantia nigra of

patients with PD (Sofic et al., 1988). These results, which were confirmed by several other studies, raised several questions, which have been addressed during the last fifteen years: is there a dysregulation of iron entry into the sub- stantia nigra ? Is there a dysregulation of iron storage in the substantia nigra ? Where is iron increased within the sub- stantia nigra ? What is the consequence of increased iron content in the parkinsonian substantia nigra ?

Altered iron penetration into the parkinsonian nigrostriatal pathway

The mechanism by which iron accumulates in the substan- tia nigra pars compacta in Parkinson’s disease is poorly understood. A possible pathway for iron to penetrate into brain parenchyma involves the binding of differic trans- ferrin to a high affinity receptor (Aisen, 1992). To address this question, we performed a quantitative analysis of iodo- transferrin binding in the mesencephalon of patients with PD and matched control subjects (Faucheux et al., 1993).

We found a low level of binding of transferrin in the sub- stantia nigra pars compacta of healthy control subjects and no change in PD. These data suggest that transferrin and its receptor are unlikely to be involved in the increased iron content in the substantia nigra of patients with PD. Never- theless, this does not exclude a retrograde transport of iron taken up in the dopaminergic terminals at the striatal level.

In human control subjects, iodotransferrin binding was highest in the putamen and caudate nucleus and lowest in the globus pallidus (Faucheux et al., 1995). In parkinsonian patients, mean density values of transferrin binding were increased in the putamen and caudate nucleus as compared to control subjects. These data suggest transferrin recep- tors may be located in the putamen and caudate nucleus and iron could be retrogradely transported to perikarya of

Correspondence: E. C. Hirsch, INSERM U679 – Experimental Neurology and Therapeutics, H^oopital de la Salp^eetri
eere, 47 Boulevard de l’H^oopital, 75651 Paris Cedex 13, France

e-mail: hirsch@ccr.jussieu.fr

melanized dopaminergic neurons (Arvidson, 1994). Fur- thermore, despite the small number of patients studied in this report, PD patients with the highest density of trans- ferrin binding were those in whom the severity of the dis- ease was the highest, in agreement with data published by Riederer et al. (1989). Yet, using film autoradiography, it is difficult to precisely localize transferrin binding sites. Thus, a higher density at the level of blood vessels or glial cells could also explain the increased binding found in PD.

Studies at the cellular level are the only means of deter- mining the exact contribution of transferrin receptors to the increased iron content seen in the parkinsonian substantia nigra. To address this issue, we measured the density of transferrin receptors on perikarya of melanized neurons using micro-autoradiography. The mean transferrin recep- tor density on melanized perikarya was decreased by 50%

in the ventral part of the substantia nigra where dopami- nergic neuronal loss is the most severe. These data are thus in apparent contradiction with our results obtained micro- scopically. Yet the absence of decrease observed macro- scopically is very likely explained by the presence of transferrin receptors on blood vessels, astrocytes and reac- tive microglial cells, since the density of these receptors is known to increase in PD (Sofic et al., 1991; Faucheux et al., 1997). In sum, it is unlikely that transferrin receptors are involved in the increased iron content found in melanized dopaminergic neurons in PD.

Lactoferrin receptors could represent another way of iron entry into neurons. Indeed, lactoferrin is another protein binding iron that is also involved in iron transport into the brain via specific receptors called lactoferrin receptors (Mazurier et al., 1989). Using immunohistochemistry, we demonstrated the presence of lactoferrin receptors at the level of blood vessels and nigral dopaminergic neurons (Faucheux et al., 1995). This suggests that these receptors may be involved in iron penetration into brain parenchyma from the blood circulation and from the parenchyma to the cytoplasm of melanized neurons. Moreover, in the substan- tia nigra, we found lactoferrin receptor immunoreactivity to be increased on both melanized dopaminergic neurons and microvasculature in patients with PD as compared to healthy control subjects (Faucheux et al., 1995). Interest- ingly, this increase was highest in the most severely affected dopaminergic cell groups, suggesting a relationship be- tween lactoferrin receptor increase and dopaminergic de- generation. The involvement of lactoferrin and its receptor is further supported by an increase in lactoferrin staining within nigral neurons in PD cases. Thus, the concomitant increase of lactoferrin and its receptor on dopaminergic neurons in PD may be responsible for the excessive ac-

cumulation of iron in vulnerable neuronal populations.

This concept is further supported by the fact that lacto- ferrin expression is increased in MPTP-intoxicated mice (Fillebeen et al., 2001). Yet, the involvement of lactoferrin and its receptor is not selective for neuronal degeneration in PD but merely represents a common pathway of neuro- nal degeneration, as similar changes have been described in several neurodegenerative disorders including Alzheimer’s disease (AD), Down syndrome, Pick’s disease, sporadic amyotrophic lateral sclerosis, and amyotrophic lateral scler- osis =parkinsonism-dementia complex of Guam (Kawamata et al., 1993; Leveugle et al., 1994; Osmand et al., 1991;

Rebeck et al., 1995). The identification of lactoferrin changes in other neurodegenerative disorders raises the question as to whether these changes play a primary role in the neurodegenerative process or are merely a conse- quence of neuronal degeneration.

The exact mechanism by which iron concentration increases in the substantia nigra of patients with PD is not fully understood. Yet the data reviewed here suggest that it is unlikely that transferrin and its receptor participate in this increase in iron content. Other iron transporters such as lactoferrin and its receptor may therefore be particularly relevant to this increased iron concentration. Further stud- ies on the expression level of lactoferrin and =or its receptor are now needed to determine whether these changes are really involved in neuronal degeneration. Likewise, studies on other iron transport proteins such as DMT1 and ferro- portin are also needed. Furthermore, a better knowledge of the regulation of iron transport and buffering mechanisms may also help to address this question.

Altered regulation of iron homeostasis in Parkinson’s disease

Well regulated iron homeostasis is necessary for cell survi- val because this metal is a co-factor of numerous biochem- ical reactions. In contrast, an increased iron concentration is deleterious given the ability of iron to react with hy- drogen peroxide and catalyze the formation of the highly reactive hydroxyl radical. Thus, intracellular iron concen- trations are controlled by ferritin and transferrin, the level of which depends on both transcriptional control and a post-transcriptional regulatory system (Harrison and Arosio, 1996; Hentze and K €uunh, 1996). Two cytoplasmic proteins, iron regulatory proteins 1 and 2 (IRP1 and 2) are involved in the control of ferritin synthesis by binding to a stem-loop structure located in the 5

untranslated region of ferritin mRNA and known as the iron responsive element (IRE).

When the concentration of cellular iron is low, IRP binds

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to IRE, thus inhibiting translation of ferritin. In contrast, if iron concentration increases, IRP does not bind to IRE, allowing translation of ferritin mRNA. Similarly, IRP binds to the 3

non-coding region of the transferrin receptor mRNA and, when cellular iron levels are low, it stabilizes the transferrin receptor message by preventing its endonu- clease cleavage. In contrast, when the iron level increases, this stabilization of the transferrin receptor mRNA is re- duced, leading to a decreased transferrin receptor expres- sion and consequently a reduced iron penetration in the cell. Given the high iron concentration observed in the substantia nigra of patients in PD, one may thus expect IRP to be bound to the iron responsive element of ferritin mRNA in the substantia nigra. To test this hypothesis we performed electrophoretic mobility shift assays on post- mortem samples from patients with PD and control sub- jects (Faucheux et al., 2002). We observed no change in the binding activity of IRP to an IRE-ferritin mRNA probe, indicating the absence of any substantial change in post- transcriptional regulation in the substantia nigra in PD. In agreement with these data, we found no change in ferritin mRNA transcript, thus confirming our previous results and other studies performed both in parkinsonian patients and MPTP-intoxicated monkeys (Dexter et al., 1991; Mann et al., 1994; Connor et al., 1995; Goto et al., 1996).

Furthermore, using the supershift technique, we showed that one of the IRPs, IRP1 but not IRP2, bound to ferritin mRNA in these samples. This indicates that the increased iron content found in the substantia nigra of patients with PD is not-associated with an up-regulation of ferritin at the translational level. The reason for such an absence of reg- ulation is not known but may be explained by several non- exclusive mechanisms. First, a cellular dysfunction asso- ciated with the disease may prevent ferritin up-regulation.

In line with this, the mRNA coding for tyrosine hydroxy- lase has also been shown to be decreased in the surviving dopaminergic neurons in the substantia nigra of patients with PD (Javoy-Agid et al., 1990). Alternatively, it has been reported that nitric oxide increases the binding activ- ity of IRP1 to IRE-ferritin mRNA and blocks ferritin mRNA translation (Mulero and Brock, 1999). In line with this, astroglial and microglial cells expressing the inducible form of nitric synthase have been found in the vicinity of dopaminergic neurons in the substantia nigra of patients with PD and animal models of the disease (Hunot et al., 1996, 1999; Liberatore et al., 1999; Dehmer et al., 2000).

Thus, the production of nitric oxide could interfere with the binding of IRP1 to IRE and may contribute to a deficiency in ferritin subunit synthesis in the substantia nigra of patients with PD.

Another explanation for the absence of ferritin up-regu- lation could be related to the fact that the pool of iron resulting in an increased concentration may not be located in the cytoplasm where ferritin mRNA is localized. Indeed, intra-cellular analysis of iron has shown that it is also local- ized on the melanin granules (Good et al., 1992; Jellinger et al., 1992) and in Lewy bodies (Hirsch et al., 1991).

Thus, neuromelanin may be involved in intra-neuronal iron homeostasis as a result of its strong chelating ability for iron (Zecca et al., 2001). In line with this, the level of redox activity detected in neuromelanin aggregates was signifi- cantly increased in parkinsonian patients and was highest in patients with the most severe neuronal loss (Faucheux et al., 2003). A possible consequence of an overloading of neuromelanin with redox element may thus be an increased contribution to oxidative stress and intra-neuronal damage in patients with PD. The increase in iron concentration may also take place in non-dopaminergic neurons. Indeed, his- tochemical identification of iron within the parkinsonian substantia nigra evidenced strong staining in microglial cells. Thus, a part of the iron contributing to its increase concentration as measured on brain homogenates may be localized in microglial cells. This may result in an indirect involvement in the neurodegenerative process by inducing glial activation and neuroinflammatory processes.

Conclusion

In summary, while it has been well established that iron concentration increases in the substantia nigra of patients with PD, its exact role in neurodegeneration has yet to be determined. The mechanisms potentially involved in such an increase include the possible release of iron from neu- romelanin aggregates and an increased penetration by lac- toferrin and its receptor. Furthermore, the absence of ferritin and transferrin regulation, possibly due to the pre- sence of nitric oxide, may facilitate iron entry into dopa- minergic neurons. The consequence of this increase may be oxidative stress, protein alteration including alpha-synu- clein aggregation and ultimately cell death. Thus, therapies aimed at regulating iron concentration within dopaminergic neurons may be useful in helping to reduce neuronal de- generation in PD.

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