M. B. H. Youdim

Iron, brain ageing and neurodegenerative disorders

There is increasing evidence that iron is involved in the mechanisms that underlie many neurodegenerative diseases. Conditions such as neuroferritinopathy and Friedreich ataxia are associated with mutations in genes that encode proteins that are involved in iron metabolism, and as the brain ages, iron accumulates in regions that are affected by Alzheimers disease and Parkinsons disease. High concentrations of reactive iron can increase oxidative-stress induced neuronal vulnerability, and iron accumulation might increase the toxicity of environmental or endogenous toxins. By studying the accumulation and cellular distribution of iron during ageing, we should be able to increase our understanding of these neurodegenerative disorders and develop new therapeutic strategies.

Increased iron (III) and total iron content in post mortem substantia nigra of parkinsonian brain.

Significant differences in the content of iron (III) and total iron were found in post mortem substantia nigra of Parkinsons disease. There was an increase of 176% in the levels of total iron and 255% of iron (III) in the substantia nigra of the parkinsonian patients compared to age matched controls. In the cortex (Brodmann area 21), hippocampus, putamen, and globus pallidus there was no significant difference in the levels of iron (III) and total iron. Thus the changes in total iron, iron (III) and the iron (II)/iron (III) ratio in the parkinsonian substantia nigra are likely to be involved in the pathophysiology and treatment of this disorder.

Monoamine oxidase: isoforms and inhibitors in Parkinson's disease and depressive illness

A few years after the foundation of the British Pharmacological Society, monoamine oxidase (MAO) was recognized as an enzyme of crucial interest to pharmacologists because it catalyzed the major inactivation pathway for the catecholamine neurotransmitters, noradrenaline, adrenaline and dopamine (and, later, 5‐hydroxytryptamine, as well). Within the next decade, the therapeutic value of inhibitors of MAO in the treatment of depressive illness was established. Although this first clinical use exposed serious side effects, pharmacological interest in, and investigation of, MAO continued, resulting in the characterization of two isoforms, MAO‐A and ‐B, and isoform‐selective inhibitors. Selective inhibitors of MAO‐B have found a therapeutic role in the treatment of Parkinsons disease and further developments have provided reversible inhibitors of MAO‐A, which offer antidepressant activity without the serious side effects of the earlier inhibitors. Clinical observation and subsequent pharmacological analysis have also generated the concept of neuroprotection, reflecting the possibility of slowing, halting and maybe reversing, neurodegeneration in Parkinsons or Alzheimers diseases. Increased levels of oxidative stress in the brain may be critical for the initiation and progress of neurodegeneration and selective inhibition of brain MAO could contribute importantly to lowering such stress. There are complex interactions between free iron levels in brain and MAO, which may have practical outcomes for depressive disorders. These aspects of MAO and its inhibition and some indication of how this important area of pharmacology and therapeutics might develop in the future are summarized in this review.

The iron chelator desferrioxamine (Desferal) retards 6-hydroxydopamine-induced degeneration of nigrostriatal dopamine neurons.

Abstract: A selective increase in content of iron in the pars compacta of the substantia nigra has been implicated in the biochemical pathology of Parkinsons disease. Iron is thought to induce oxidative stress by liberation of oxygen free radicals from H2O2. Because 6‐hydroxydopamine (6‐OHDA) is thought to induce nigrostriatal dopaminergic neuronal lesions via metal‐catalyzed free radical formation, the effect of the iron chelator desferrioxamine was investigated on 6‐OHDA‐induced dopaminergic neuron degeneration in the rat. Intracerebroventricular injection of 6‐OHDA (250 μg) caused a 88, 79, and 70% reduction in striatal tissue content of dopamine (DA), 3,4‐dihydroxyphenylacetic acid, and homovanillic acid (HVA), respectively, and a 2.5‐fold increase in DA release as indicated by the HVA/DA ratio. Prior injection of desferrioxamine (130 ng i.c.v.) resulted in a significant protection (<60%) against the 6‐OHDA‐induced reduction in striatal DA content and a normalization of DA release. Dopaminergic‐reiated behavioral responses, such as spontaneous movements in a novel environment and rearing, were significantly impaired in the 6‐OHDA‐treated group. By contrast, the desferrioxamine‐pretreated rats exhibited almost normal behavioral responses. The ability of iron chelators to retard dopaminergic neurodegeneration in the substantia nigra may indicate a new therapeutic strategy in the treatment of Parkinsons disease.

Mechanism of 6-hydroxydopamine neurotoxicity

The catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) has recently been found to be formed endogenously in patients suffering from Parkinsons disease. In this article, we highlight the latest findings on the biochemical mechanism of 6-OHDA toxicity. 6-OHDA has two ways of action: it easily forms free radicals and it is a potent inhibitor of the mitochondrial respiratory chain complexes I and IV. The inhibition of respiratory enzymes by 6-OHDA is reversible and insensitive towards radical scavengers and iron chelators with the exception of desferrioxamine. We conclude that free radicals are not involved in the interaction between 6-OHDA and the respiratory chain and that the two mechanisms are biochemically independent, although they may act synergistically in vivo.

Iron‐Melanin Complex in Substantia Nigra of Parkinsonian Brains: An X‐Ray Microanalysis

Abstract: Using energy‐dispersive x‐ray analysis on an electron microscope working in the scanning transmission electron microscopy mode equipped with a microanalysis system, we studied the subcellular distribution of trace elements in neuromelanin‐containing neurons of the substantia nigra zona compacta (SNZC) of three cases of idiopathic Parkinsons disease (PD) [one with Alzheimers disease (AD)] and of three controls, in Lewy bodies of SNZC, and in synthetic dopamine‐melanin chemically charged or uncharged with Fe. Weak but significant Fe peaks similar to those of a synthetic melanin‐Fe3+ complex were seen only in intraneuronal highly electron‐dense neuromelanin granules of SNZC cells of PD brains, with the highest levels in a case of PD plus AD. whereas a synthetic melanin‐Fe2+ complex showed much lower iron peaks, indicating that neuromelanin has higher affinity for Fe3+ than for Fe2+. No detectable Fe was seen in nonmelanized cytoplasm of SNZC neurons and in the adjacent neuropil in both PD and controls, in Lewy bodies in SNZC neurons in PD, and in synthetic dopamine‐melanin uncharged with iron. These findings, demonstrating for the first time a neuromelanin‐iron complex in dopaminergic SNZC neurons in PD, support the assumption that an iron‐melanin interaction contributes significantly to dopaminergic neurodegeneration in PD and PD plus AD.

MPTP mechanisms of neurotoxicity and their implications for Parkinson's disease

Systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) gives rise to motor deficits in humans and other primates which closely resemble those seen in patients with Parkinsons disease. These deficits are associated with a relatively selective loss of cells in the pars compacta of the substantia nigra and severe reductions in the concentrations of dopamine, noradrenaline and serotonin in the striatum. Similarly, in mice of various different strains the administration of MPTP also induces a marked loss of dopaminergic cells with severe depletion of biogenic amines, but higher doses of MPTP are required to produce these effects in mice than in primates. This review summarises advances made in understanding the biochemical events which underlie the remarkable neurotoxic action of MPTP. Major steps in the expression of neurotoxicity involve the conversion of MPTP to the toxic agent 1-methyl-4-phenylpyridinium ion (MPP+) by type B monoamine oxidase (MAO-B) in the glia, specific uptake of MPP+ into the nigro-striatal dopaminergic neurones, the intraneuronal accumulation of MPP+, and the neurotoxic action of MPP+. This is exerted mainly through the inhibition of the enzymes of the respiratory chain (Complex I), the disturbance of Ca2+ homeostasis, and possibly by the formation of free radicals. The relevance of the MPTP model to idiopathic Parkinsons disease is discussed.

Ironing iron out in Parkinson's disease and other neurodegenerative diseases with iron chelators: A lesson from 6-hydroxydopamine and iron chelators,desferal and VK-28

Abstract: In Parkinsons disease (PD) and its neurotoxin‐induced models, 6‐hydroxydopamine (6‐OHDA) and N‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), significant accumulation of iron occurs in the substantia nigra pars compacta. The iron is thought to be in a labile pool, unbound to ferritin, and is thought to have a pivotal role to induce oxidative stress‐dependent neurodegeneration of dopamine neurons via Fenton chemistry. The consequence of this is its interaction with H2O2 to generate the most reactive radical oxygen species, the hydroxyl radical. This scenario is supported by studies in both human and neurotoxin‐induced parkinsonism showing that disposition of H2O2 is compromised via depletion of glutathione (GSH), the rate‐limiting cofactor of glutathione peroxide, the major enzyme source to dispose H2O2 as water in the brain. Further, radical scavengers have been shown to prevent the neurotoxic action of the above neurotoxins and depletion of GSH. However, our group was the first to demonstrate that the prototype iron chelator, desferal, is a potent neuroprotective agent in the 6‐OHDA model. We have extended these studies and examined the neuroprotective effect of intracerebraventricular (ICV) pretreatment with the prototype iron chelator, desferal (1.3, 13, 134 mg), on ICV induced 6‐OHDA (250 μg) lesion of striatal dopamine neurons. Desferal alone at the doses studied did not affect striatal tyrosine hydroxylase (TH) activity or dopamine (DA) metabolism. All three pretreatment (30 min) doses of desferal prevented the fall in striatal and frontal cortex DA, dihydroxyphenylacetic acid, and homovalinic acid, as well as the left and right striatum TH activity and DA turnover resulting from 6‐OHDA lesion of dopaminergic neurons. A concentration bell‐shaped neuroprotective effect of desferal was observed in the striatum, with 13 μg being the most effective. Neither desferal nor 6‐OHDA affected striatal serotonin, 5‐hydroxyindole acetic acid, or noradrenaline. Desferal also protected against 6‐OHDA‐induced deficit in locomotor activity, rearing, and exploratory behavior (sniffing) in a novel environment. Since the lowest neuroprotective dose (1.3 μg) of desferal was 200 times less than 6‐OHDA, its neuroprotective activity may not be attributed to interference with the neurotoxin activity, but rather iron chelation. These studies led us to develop novel brain‐permeable iron chelators, the VK‐28 series, with iron chelating and neuroprotective activity similar to desferal for ironing iron out from PD and other neurodegenerative diseases, such as Alzheimers disease, Friedreichs ataxia, and Huntingtons disease.

Neuroprotection by a novel brain permeable iron chelator, VK-28, against 6-hydroxydopamine lession in rats

Significant increase in iron occurs in the substantia nigra pars compacta of Parkinsonian subjects, and in 6-hydroxydopamine (6-OHDA) treated rats and monkeys. This increase in iron has been attributed to its release from ferritin and is associated with the generation of reactive oxygen species and the onset of oxidative stress-induced neurodegeneration. Several iron chelators with hydroxyquinoline backbone were synthesized and their ability to inhibit basal as well as iron-induced mitochondrial lipid peroxidation was examined. The neuroprotective potential of the brain permeable iron chelator, VK-28 (5-[4-(2-hydroxyethyl) piperazine-1-ylmethyl]-quinoline-8-ol), injected either intraventricularly (ICV) or intraperitoneally (IP), to 6-OHDA lesioned rats was investigated. VK-28 inhibited both basal and Fe/ascorbate induced mitochondrial membrane lipid peroxidation, with an IC(50) (12.7 microM) value comparable to that of the prototype iron chelator, desferal, which does not cross the blood brain barrier. At an ICV pretreatment dose as low as 1 microg, VK-28 was able to completely protect against ICV 6-OHDA (250 microg) induced striatal dopaminergic lesion, as measured by dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) levels. IP injection of rats with VK-28 (1 and 5 mg/kg) daily for 10 and 7 days, respectively, demonstrated significant neuroprotection against ICV 6-OHDA at the higher dose, with 68% protection against loss of dopamine at 5mg/kg dosage of VK-28. The present study is the first to show neuroprotection with a brain permeable iron chelator. The latter can have implications for the treatment of Parkinsons disease and other neurodegenerative diseases (Alzheimers disease, Friedreich ataxia, aceruloplasminemia, Hallervorden Spatz syndrome) where abnormal iron accumulation in the brain is thought to be associated with the degenerative processes.

Intranigral Iron Injection Induces Behavioral and Biochemical “Parkinsonism” in Rats

Elevated iron concentrations in the substantia nigra (SN) pars compacta have been implicated in the development of idiopathic Parkinsons disease. Because, as a transitional metal, iron promotes free radical formation, the role of iron in the degeneration of the nigrostriatal dopamine neurons in Parkinsons disease has received much attention. This study further investigates the cytotoxic effects of iron in the SN. Various concentrations of FeCl3 (1, 5, and 50 μg of Fe3+ in 5 μl) were unilaterally injected into the SN of adult rats. The two lower doses of iron had no effect on striatal dopamine levels or on the behavioral responses of the rats. However, injection of 50 μg of Fe3+ resulted in a substantial selective decrease of striaial dopamine (95%), 3,4‐dihydroxyphenylacetic acid (82%), and homo‐vanillic acid (45%), without any change in norepinephrine concentration. Dopamine‐related behavioral responses, such as spontaneous movements in a novel space and rearing, were significantly impaired, whereas amphetamine administration induced ipsilatcral rotation in the iron‐treated rats. The present study indicates that the nigrostriatal dopamine neurons are susceptible to the presence of ionic iron and thus supports the assumption that iron initiates dopaminergic neu‐rodegeneration in Parkinsons disease.