Experimental Approaches To Brain Iron Loading

The neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA), induce many of the pathological changes that occur in PD and are widely used as animal models of this disease. MPTP induces nigral degeneration in several species including rat, mouse, dog, cat, and monkey (reviewed in [151]). Essentially, MPTP will cross the BBB to be converted by monoamine oxidase B in glial cells to the active form, 1-methyl-4-phenylpyridinium (MPP+) (Figure 23). MPP+ will accumulate in dopaminergic cells prior to its entry into mitochondria, via an energy-dependent mechanism. MPP+ will inhibit mitochondrial activity leading to decreases of ATP levels, thereby inducing cell death. MPTP induces changes in iron metabolism, up-regulating the expression of both transferrin and lactoferrin receptors, as well as increasing free iron content in substantia nigra. Oxidative stress occurs which is associated with cellular changes of both GSH-GSSG ratio and cytoprotective enzymes. The loss of GSH may reflect the inactivation of glutathione perox-idase [152]. Primates treated with MPTP develop motor disturbances resembling those seen in idiopathic PD, including bradykinesia, rigidity and postural abnormalities.

6-Hydroxydopamine, a hydroxylated analogue of dopamine, is unable to cross the BBB, such that intracerebral administration is necessary to destroy nigral dopaminergic neurons in the striatum, the SN or the ascending medial forebrain

OhP

1

•O-

MPP^

• Lipid peroxidation

• Protein peroxidation

• DNA damages

Activation of kinases, proteases endonucleases

Gsh Transporter Bbb

Activation of kinases, proteases endonucleases

Figure 23. Hypothetical mechanism of MPTP toxicity. MPTP injected peripherally, crosses the BBB and is transformed by glial monoamine oxidase (MAO) into the active compound MPP+. Once inside the cells, MPP+ will inhibit the respiratory chain, cause oxidative stress, both triggering cell death. DAT = dopamine transporter. (Reproduced with permission from [151]).

Figure 23. Hypothetical mechanism of MPTP toxicity. MPTP injected peripherally, crosses the BBB and is transformed by glial monoamine oxidase (MAO) into the active compound MPP+. Once inside the cells, MPP+ will inhibit the respiratory chain, cause oxidative stress, both triggering cell death. DAT = dopamine transporter. (Reproduced with permission from [151]).

bundle (reviewed in [151]) (Figure 24). The iron content of SN and striatum is increased, which will participate in Fenton chemistry to generate reactive oxygen species to exacerbate the lesion. The importance of iron in the exacerbation of this lesion can be verified by the facts that iron-deficient rats are resistant to 6-OHDA-induced damage [152,153], and iron chelating agents can prevent 6-OHDA-induced deleterious effects [154].

It has been difficult to engender an animal model which shows selective accumulation of iron in specific brain regions. In normal rats the blood-brain barrier will normally restrict iron uptake. However, the orally administered compound 3,5,5-trimethyl hexanoyl ferrocene, will rapidly traverse the BBB, its iron moiety being concealed between the two aromatic rings, releasing and increasing iron content of various brain regions, including SN, cerebellum and cerebral cortex, by approximately 50%, after four weeks of administration. This animal model has been utilized to test the ability of a range of chelators to reduce brain iron content [96].

Mechanism Parkinson Disease

Figure 24. Hypothetical mechanism of 6-OHDA toxicity. This is induced by (a) reactive oxygen species generated by intra- or extracellular auto-oxidation, (b) hydrogen peroxide formation induced by MAO activity, or (c) direct inhibition of the mitochondrial respiratory chain. (Reproduced with permission from [151]).

Figure 24. Hypothetical mechanism of 6-OHDA toxicity. This is induced by (a) reactive oxygen species generated by intra- or extracellular auto-oxidation, (b) hydrogen peroxide formation induced by MAO activity, or (c) direct inhibition of the mitochondrial respiratory chain. (Reproduced with permission from [151]).

In our early study [96] intraperitoneal injection of either bidendate (e.g., deferiprone), or the hexadendate (desferoxamine = DFO) iron chelators (Figure 25) to ferrocene-loaded rats, reduced excess iron from a variety of brain regions, with no apparent adverse effect on brain iron metabolism. Dopamine metabolism was adversely affected by both groups of chelators in this study. Tridendates (e.g., desferrithiocein) were also efficient at removing iron from various brain regions in the ferrocene-loaded brain and most importantly did not interfere with dopamine metabolism [97].

Both 6-OHDA and MPTP increase the iron content of the substantia nigra pars compacta (SNPC) in rats and monkeys [155-159] and in mice [160,161]. Various factors are implicated in iron accumulation, which include up-regulation of DMT1 [162], down regulation of transferrin receptors, loss of IRP-2 activity, and iron release from ferritin [163].

Many compounds have been evaluated in these two animal models for their iron-chelating properties and antiinflammatory action. In the MPTP model, des-feral [161,162] and green tea catechin ((-)-epigallactocatechin-3-gallate) all show iron chelating properties and, as such, are potent neuroprotective agents against

(a) Deferiprone

(a) Deferiprone

Deferiprone Parkinson

(b) Desferrithiocin and ICL670

OH OH

OH OH

(c) Desferoxamine

Figure 25. Iron chelators, past and future therapeutic agents for the removal of iron from neurodegenerative diseases where excessive iron accumulation occurs. (a) Bidendates: deferiprone; (b) tridendates: desferrithiocein and ICL670; (c) hexadendates: desferriox-

MPTP. DFO administration either by intracranial pretreatment or intraperi-toneally prevented 6-OHDA-induced changes in dopaminagic neurons [162]. Another iron chelator, the lipophilic VK-28, (5-[4-hydroxyethyl) piperazine-1-ylmethyl]-quiniline-8-ol) (Figure 26), traverses the blood-brain barrier to prevent 6-OHDA-induced malondialdehyde (MDA) production, without altering dopamine metabolism [163].

IRP-1 knockout mice show misregulation of iron metabolism in the kidney and brown fat, the two tissues in which the endogenous expression of IRP-1 exceeds that of IRP-2 [164]. IRP-2 knockout mice, have dysregulated iron metabolism associated with cytosolic iron accumulation (possibly as ferritin) in neuronal axons, particularly striatum, which in normal circumstances

VK-28

Epigallactocatechin-3-gallate

Figure 26. Iron chelators of possible therapeutic use: (-)-epigallactocatechin-3-gallate and VK-28.

Epigallactocatechin-3-gallate

Figure 26. Iron chelators of possible therapeutic use: (-)-epigallactocatechin-3-gallate and VK-28.

has high IRP-2 content. Degradation of ferritin occurs in lysosomes, which could lead to increased release of free iron and oxidative damage. These animals exhibit movement disorders which include ataxia, tremor and bradykinesia. Mice that are homozygous for a targeted deletion of IRP-2 and heterozygous for a targeted deletion of IRP-1 (IRP + /-IRP2-/-) develop severe neurodegeneration, characterized by widespread axonopathy and vacuolisation in several brain regions, including SN. Ultimately neuronal cell bodies degenerate in the SN activating microglia. Mice show gait and motor impairment when axonopathy is pronounced [165].

There is considerable evidence for abnormal metal homeostasis in AD. The presence of the 5'-UTR in the APP transcript may indicate that APP is a metalloprotein. Iron chelation has been proposed as a possible therapeutic approach for the treatment of AD. Indeed, as already stated earlier, APP was selectively down-regulated in response to iron chelation [98]. Chelation may also prevent iron-induced inflammation as well as Ap aggregation. The therapeutic advantages of various chelators, such as clioquinol (a copper chelator), desferrioxamine (an iron chelator) as well as polyphenols, e.g., epigallactocatechin-3-gallate (EGCG) and curcumin (a constituent of turmeric) [166], should be assessed in suitable animal models, for as yet, there are no details of changes in iron metabolism in any of these transgenic AD animal models.

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