Desferal

Abnormality of iron in tissues, either its increase or decrease, is associated with some of the most devastating diseases in humans. Until recently, very little attention was paid to iron metabolism in the brain.

Iron Deficiency

Iron deficiency in the brain is thought to cause cognitive and learning impairment in infants and young children.1-3 Animal studies on iron-deficient young rats (28-80 days old) showed that the rats have 30-40% lower brain iron. These animals exhibit learning deficits in Y-water maze and Morris water tank. If iron deficiency is induced in 10-- day-old rats, iron supplementation does not restore brain iron or learning deficits, even after long-term treatment. This picture is mimicked in children with nutritional iron deficiency. The neurochemical basis of this defect may be related to down-regulation (subsensitivity) of striatal dopamine D2 receptors and increased striatal and ventral tegmentum endogenous opiate peptides (dynorphin B and enkephalins). Furthermore, we showed degeneration of C1 and C2 areas of hippocampus as a consequence of iron and transferrin deficiency in these regions.4 The hippocampus is prominent because its cholinergic neurodegeneration is associated with cognitive impairment in Alzheimer's disease. Iron supplementation does not necessarily correct cognitive impairment because deficiency occurs during a critical state of brain development and neural differentiation and appears to have longterm consequences.3

Excess Brain Iron

Iron and transferrin distribution in the brain are unequal and do not show similar patterns. Whereas the highest concentrations of iron are found in globus pallidus, substantia nigra, red nucleus, detate gyrus, thalamus, and putamen, transferrin concentrations are relatively low in these regions. These brain regions are associated with the most prominent neurodegenerative diseases (Parkinson's, Alzheimer's, Huntington chorea, amyotropic lateral sclerosis). The highest transferrin concentration in the brain is present in the hippocampus and cortical region, however, which have relatively low iron content as compared with the other regions. Increase of iron in specific brain regions is thought to have a pivotal role in neurodegeneration and many neurodegenerative diseases show accumulation of iron at the neuronal sites that degenerate.5-7

There have been many hypotheses regarding the cause of Parkinson's disease (PD) and other neurodegenerative diseases. These hypotheses include genetic aberrations, the presence of exogenously and endogenously derived neurotoxins similar to either 6-- hydroxydopamine (6-OHDA) or 1-methyl-4-phenyl- 1,2,3,6-- tetrahydropyridine (MPTP), viral infection, apoptosis, inflammatory immunology processes and reactive oxygen species (ROS), and induced oxidative stress (OS),8-11 and have been confirmed by cDNA microarray gene expression studies. OS has received much publicity because the chemical pathology of PD points to selective biochemical changes solely in the sunstantia nigra pars compacta (SNPC), one prominent aspect of which is accumulation of iron in reactive microglia and dopamine neurons in this region. The pathologic changes include proliferation of reactive microglia, accumulated iron, activation of proinflammatory redox transcription factor, nuclear factor kappa B (NF-kB) (Figure 1), induction of transcription of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL1), and IL6, which can induce promotion of ROS production or lack of ROS disposal. Theoretically, this can lead to accumulation of ROS near or in the neurons. Such ROS have the ability to cause membrane lipid peroxidation, which in turn results in fluidity of the neuron membrane and consequent death of the neuron. Indeed, others and we have reported on several occasions that the evidence for OS includes increases of monoamine oxidase B, iron, ironbinding melanin, lipid peroxidation, superoxide dismutase, lipofiucin, and ubiquitin. There are also concomitant decreases of mitochondrial complex I, calcium-binding protein (calbindin 24), and reduced glutathione (GSH). These data support, but do not necessarily confirm that there are OS in the SNPC. Nevertheless, these findings are supported by animal studies in which 6-OHDA and MPTP have been used to develop the model of PD. Both these neurotoxins owe their neurotoxicity to their ability to induce OS via significant production of ROS, and both neurotoxins deplete cellular GSH in the striatum and release iron from its binding site on ferritin. Furthermore, similar to the observation in PD, both neurotoxins induce accumulation of iron in the SNPC. How the iron accumulates in the brain is unclear because serum iron has no access to adult brain. Iron, more than any other divalent metal, is involved with production of ROS because of its ability to react with hydrogen peroxide to produce the most reactive ROS, the hydroxyl radical, via the Fenton chemistry. This can occur if hydrogen peroxide disposition is compromised; this seems to occur in a number of neurodegenerative diseases. It is the free chelatable iron that is thought to be most dangerous for cell viability. It has the ability to deplete reduced GSH, the rate-limiting cofactor for glutathione peroxidase, which is the enzyme responsible for disposition of hydrogen peroxide in the brain. Although many attempts have been made to isolate an MPTP-like substance, they have not met with success. However, our recent results clearly support the notion that 6-OHDA can be formed in vivo. We showed that in the presence of iron and hydrogen peroxide, dopamine can be converted to 6-OHDA, as identified by mass-spectrometer and high performance liquid chromatography analysis. These findings support the recent observation for the presence of 6-OHDA in the urine of those with PD.

Employing the 6-OHDA model of PD, we have examined several compounds as neuroprotective antioxidants. These include the iron chelator desferal, vitamin E, the radical scavenger lipoic acid, the nonreceptor-binding glutamate antagonist flupritine, and the dopamine D1-D2 agonist apomorphine. The first three prevent the neurotoxic action of 6-OHDA in the rat, whereas flupritine and apomorphine have been shown to be potent antioxidants with iron-chelating properties. In cell culture studies using PC12 cells, both latter drugs prevent 6-OHDA and hydrogen peroxide-induced PC12 cell death. We are now examining their in vivo neuroprotective properties. Thus, nonsteroid antiinflammatory drugs such as aspirin, thalidomide, supidimide and COX2 inhibitors, antioxidants, and noncompetitive glutamate antagonists devoid of NMDA receptor-blocking action, appear to be natural candidates as neuroprotective drugs in PD and possibly in other neurodegenerative diseases, including Alzheimer's disease. Unfortunately, iron chelators, including desferal or many other antioxidants and COX2 inhibitors, do not cross the blood-brain barrier (BBB). Iron chelators devoid of side effects and capable of crossing the BBB are being sought. Vitamin E and lipoic acid have been shown to be useful in Alzheimer's disease and in diabetes neuropathy, respectively. Antiinflammatory drugs such as thalidomide and its derivatives have been shown to be neuroprotective in animal studies. These neuroprotective drugs are thought to produce their effects by blocking cytotoxic cytokines such as TNF-alpha production because iron induces, and iron chelators inhibit the proinflammatory transcription factor NF-kB activation. Iron is therefore thought to play a pivotal role as an activator of NF-kB (Figures 1 and 2).6

Discussion

Hernandez: Regarding the potential toxicity of iron, what is your opinion about its role in the etiology of neurodegenerative diseases?

Youdim: Iron is toxic and can produce free radicals so it presents a controversy. I want to come back to the copper question because in a 1989 paper on brains of Parkinsonians, we showed an increase of iron but low levels of copper. There is a relation between copper and iron such that if you make rats copper-deficient, iron increases, and ifyou make them iron-deficient, copper increases. We don't actually understand what the copper is doing, and I find your data is fascinating in that it may have some protective effect. I don't know how exactly it will be linked, maybe to some very important protein that is protecting the neurons. That was very nice data.

Hernandez: In your iron deficiency model, in which region did you see reduction of D-2 receptors? And did you also see cellular death induced by iron load?

Youdim: We concentrated on the hippocampal area. The transferring ferritin and transferring receptor were in the C-1, C-2, C-3, and C-4 areas of the hippocampus in nutritional iron deficiency. Regarding the D-2 receptor, we've seen it decrease in the striatum as well as in the pituitary.

Hernandez: Right, in that area, is there also cell death?

Youdim: No. In fact iron is there, but we do not see any cell death. Where we may see cell death is in the hippocampus; we've seen changes with iron deficiency that we have not completely analyzed. But we did not notice any cell death in the striatum, only the dopamine D-2 receptor was down in number.

Hernandez: You mentioned in the iron deficiency table there is a 5HD-2 decrease also.

Youdim: No change. 5HD metabolism and 5HD, as well as noradrenaline, were not changed at all. And this is what surprised us. I think part of the thing that you saw was that the liver iron can be reduced by approximately 90%, whereas the maximum brain level of iron is 35-40%. And we now know that there is a functional pool in which you can reduce the level of iron to a certain extent and the enzymes will not change. You need a much more severe deficiency before you see changes in brain enzymes. I think this shows how the brain protects itself; it probably has much more protective capacity than the liver or the heart, which we see by the way these enzyme changes occur. Now, one of the oddities is that although nutritional iron deficiency appears to be detrimental to the cognitive aspects that I described, I mentioned earlier that if you make the rats iron-deficient and reduce the brain level of iron approximately 15%, you will not be able to induce 6-hydroxydopamine-induced neurodegeneration, 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP), or choanate. Somehow, iron deficiency can also be protective. Actually, we believe that iron level is so tightly controlled that either deficiency or excess can lead to a pathologic condition, especially in the brain.

Hernandez: Have you seen any damage to glial cells?

Youdim: One of the reasons we did the nutritional iron deficiency study and looked at 6-hydroxidopamine-induced neurodegeneration is that if you give 6-hydroxidopamine, MPTP, or choanate, which is what we used for hippocampal damage, you see tremendous proliferation of reactive micro-glia, which are pouring out all their superoxide, nitrate-oxide and, peroxidants. This is exactly what you see in Parkinson's and Alzheimer's disease. If you look at the area of the nucleus bacillus or if you look at the substantia in Parkinson's, there is proliferation of the reactive micro-glia that we think is associated with the inflammatory process. Now, if you make the rats iron-deficient and then you give choanate or 6-hydroxydopamine, not only is the rat protected, but also you do not see any reactive micro-glia. We therefore think that this iron really does have a fundamental role, a pivotal role, though not necessarily a primary role. We think something else may be affecting iron metabolism that leads to a cascade of events.

Garry: In any of your experiments, are you able to measure free iron versus bound iron?

Youdim: Yes, it is crucial. We know that it is the chelatable iron that is dangerous because we've done all the work with animal models. I've been showing you data such that if we pretreat the animals with the iron chelators like desferrioxamine or apomorphine, or some of the new compounds I showed you on a list, you protect the animal from MPTP and 6-hydroxydopamine because it releases iron. We believe it's in a free form.

Invited Comment

Parkinson's disease is a clear example of how knowledge about a particular pathology of the nervous system starts with a precise and accurate description of the symptomatology, and how step-by-step truth emerges in spite of many misinterpretations. James Parkinson would be amazed by the amount of research and variety of therapeutic strategies generated for what he termed shaking palsy. That first step, the precise description of the disease by Parkinson was enriched by others such as Jean-Martin Charcot and Armand Trousseau. Later, other researches described the lesions in the substantia nigra and showed its relevance for the disease.

One hundred fifty years after Parkinson's description came L-dihydroxyphenylalanine (L-DOPA), the first pharmacologic treatment that had a significant impact on the lives of the patients. The dopamine precursor and the discovery of the dopaminergic pathway explained the relationship between neural loss in the substantia nigra compacta and dopamine depletion in the striatum. L-DOPA does not cure Parkinson's disease, and its significant side effects led to other drugs such as decarboxylation inhibitors, dopamine agonists, anticholinergics, and, more recently monoamine oxidase-B inhibitors. Other treatments now include transplants of adrenal or neural cells, electrical stimulation of the subthalamic nucleus or globus pallidus, thalamotomy, and, perhaps soon gene therapy. They have met with variable results. With regard to transplantation, initial optimism has tempered it is now believed that transplants are useful in some cases but the patients must be carefully screened; more research is needed to establish the most important features related to successful treatment. Neurosurgical techniques reduce akinesia and rigidity and generally improve gait and speech disturbances of the patients. Gene therapy offers a novel way for treating the disease not only symptomatically but also as a neuroprotective and neuroregenerative therapy.

Nevertheless, the development of effective treatments depends on expanding our knowledge about the etiology of the disease. Neurosurgical interventions can control symptoms but do not cure. As with other diseases, prevention would be the best strategy, but this is impossible without knowing its causes. Dr. Youdim is clearly heading in that direction: finding the triggers and the mayor players in Parkinson's disease that will eventually lead to more effective therapies and possibly to prevention by use of neuroprotective molecules.

A patient suffering from Parkinson's disease called it a cruel disease, a disease that does not allow the body to follow the commands from the brain.1 With continued research and dedication, we can achieve the goal of less people held hostage by this cruel disease.

1. Youdim MB, Ben-Shachar D, Yehuda S. Putative biological mechanisms of the effect of iron deficiency on brain biochemistry and behavior. Am J Clin Nutr 1989;50:607-17

2. Yehuda S, Youdim MB. Brain iron: a lesson from animal models. Am J Clin Nutr 1989;50:618-29

3. Youdim MBH, Yehuda S. The neurochemical basis of cognitive deficits induced by brain iron deficiency: involvement of dopamine-opiate system. Cell Mol Biol 2000;46:491-500

4. Shoham S, Youdim MBH. Iron involvement in neural damage and microgliosis in models of neurodegenerative diseases. Cell Mol Biol 2000;46:743-60

5. Youdim MBH, Riederer P Iron in the brain, normal and pathological. In: Adelman G, Smith BH, eds. Elsevier's encyclopedia of neuroscience. Amsterdam: Elsevier Science, 1999:984-7

6. Youdim MB, Grunblatt E, Mandel S. The pivotal role of iron in NF-kappa B activation and nigrostriatal dopaminergic neurodegeneration. Prospects for neuroprotection in Parkinson's disease with iron chelators. Ann N Y Acad Sci 1999;890:7-25

7. Samson FE, Nelson SR. The aging brain, metals and oxygen free radicals. Cell Mol Biol 2000;46:699-707

8. Gerlach M, Ben-Shachar D, Riederer P, Youdim MBH. Altered brain metabolism of iron as a cause of neurodegenerative diseases? J Neurochem 1994;63:793-807

9. Jenner P Oxidative mechanisms in nigral cell death in Parkinson's disease. Mov Disord 1998;13:24-- 34

10. Youdim MB, Ben-Shachar D, Riederer P The possible role of iron in the etiopathology of Parkinson's disease. Mov Disord 1993;8:1-12

11. Grunblatt E, Mandel S, Maor G, Youdim MBH. Gene expression analysis in MPTP model of Parkinson's disease and neuroprotection by apomorphine using cDNA microarray. J Neurochem 2001;77:1-13

1. Tivol M. Experiment in hope. Washington Post Magazine, October 17, 1993:14-9, 423

Mousa B.H. Youdim, Ph.D., M.Sc.

Dr. Youdim is Faculty of Medicine with Technion, Eve Topf and U.S. National Parkinson Foundation Centers of Excellence for Research in Neurodegenerative Diseases, Haifa, Israel.

Magda Giordano, Ph.D., and Camilo Rios, Ph.D.

Dr. Giordano is with the Center for Neurobiology, National Autonomous University of Mexico, Campus Juriquilla, Queretaro, Qro. 76230 Mexico. Dr. Rios is with the Department of Neurochemistry, National Institute of Neurology and Neurosurgery, Mexico City, D.F., 14269 Mexico.

Copyright International Life Sciences Institute and Nutrition Foundation Aug 2001
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