Coexistence of zinc and iron augmented oxidative injuries in the nigrostriatal dopaminergic system of SD rats

Clinical studies have demonstrated an excess of transition metals, including zinc and iron, in the substantia nigra (SN) of Parkinson's patients. In the present study, the neurotoxic effect of zinc was investigated using iron as a positive control. Addition of zinc or iron to brain homogenates increased lipid peroxidation. Zinc was less potent than iron in inducing lipid peroxidation. Coincubation with desferrioxamine prevented zinc- and iron-induced lipid peroxidation. Furthermore, glutathione (GSH), S-nitroso-N-acetylpenicillamine, or melatonin inhibited zinc-induced lipid peroxidation. The oxidative effect of zinc was further investigated in anesthetized rats. Seven days after intranigral infusion of zinc, lipid peroxidation was elevated in the infused SN, and dopamine content and tyrosine hydroxylase-positive axons were decreased in the ipsilateral striatum. Zinc was less potent than iron in inducing neurodegeneration in vivo. L-Buthionine-[S,R]-sulfoximine pretreatment (i.c.v.), which depletes cellular GSH levels, enhanced zinc-induced oxidative injuries in the nigrostriatal dopaminergic system. Moreover, simultaneous infusion of zinc and iron appeared to augment oxidative injuries in rat brain. Taken together, our results demonstrate that intranigral infusion of zinc caused degeneration of the nigrostriatal dopaminergic system in rat brain. Furthermore, coexistence of zinc and iron augmented oxidative injuries in rat brain. These findings indicate that both zinc and iron contribute to the etiology of Parkinsonism.     

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 Parkinson's disease. Because, as a transitional metal, iron promotes free radical formation, the role of iron in the degeneration of the nigrostriatal dopamine neurons in Parkinson's 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 micrograms of Fe3+ in 5 microliters) 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 micrograms of Fe3+ resulted in a substantial selective decrease of striatal dopamine (95%), 3,4-dihydroxyphenylacetic acid (82%), and homovanillic 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 ipsilateral 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 neurodegeneration in Parkinson's disease.     

Lipid peroxidation-mediated oxidative stress and dopamine neuronal apoptosis in the substantia nigra during development.

The cellular pathways underlying naturally occurring neuronal apoptosis in the rat substantia nigra (SN) during the perinatal period remain largely unknown. Determining the mediators of this process in development may shed light on causes of premature neuronal death in adult neurodegenerative disorders, including the loss of dopamine neurons in Parkinson's disease. In the present study, we investigated whether lipid peroxidation-mediated oxidative stress mediates developmental death of nigral neurons by (1) establishing the profile of lipid peroxidation and other oxidative stress markers throughout the postnatal period both in the SN and striatum, and (2) examining whether the inhibitor of lipid peroxidation, alpha-tocopherol, protects these neurons from death. In addition to monitoring, the level of lipid peroxidation throughout development, we also measured the activities of three antioxidant enzymes, namely superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx). We have shown that lipid peroxidation and SOD activity progressively increased from postnatal day (PND) 3 to PND 42 in both SN and striatum. During this period, GPx activity remained stable, while catalase activity transiently increased at PND 8 only in the SN. Furthermore, alpha-tocopherol treatment from embryonic day 18 to PND 2 did not reduce the number of apoptotic neurons at PND 3. These results do not support the hypothesis that lipid peroxidation-mediated oxidative stress is the major mediator of nigral dopamine neuronal apoptosis during the perinatal period.     

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

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 Parkinson's disease (PD) [one with Alzheimer's 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.     

Neuroprotective effects of iron chelator Desferal on dopaminergic neurons in the substantia nigra of rats with iron-overload

The aim of the present study was to investigate whether the iron chelator Desferal prevents the degeneration of dopaminergic neurons in the substantia nigra (SN) induced by iron-overload in rats. Using fast cyclic voltammetry, tyrosine hydroxylase (TH) immunohistochemistry, Perls' iron staining, and high-performance liquid chromatography-electrochemical detection, we measured the degeneration of dopaminergic neurons and increased iron content in the SN of rats overloaded with iron dextran and assessed the effects of treatment with Desferal. The results showed that iron dextran overload increased the iron content in the SN, decreased dopamine release and content, and reduced the numbers of TH-immunoreactive neurons. Treatment with Desferal prevented the increased iron content in the SN. As a result, dopamine release and content remained at almost normal levels, while the numbers of TH-immunoreactive neurons remained at control values. This study suggests that the iron chelator Desferal is neuroprotective against iron-overload, so iron chelators that can cross the blood-brain barrier may have the potential to treat cases where abnormal iron accumulation in the brain is associated with the degenerative processes, as in Parkinson's disease.     

Peripheral iron dextran induced degeneration of dopaminergic neurons in rat substantia nigra

Iron accumulation is considered to be involved in the pathogenesis of Parkinson's disease. To demonstrate the relationship between peripheral iron overload and dopaminergic neuron loss in rat substantia nigra (SN), in the present study we used fast cyclic voltammetry, tyrosine hydroxylase (TH) immunohistochemistry, Perls' iron staining, and high performance liquid chromatography-electrochemical detection to study the degeneration of dopaminergic neurons and increased iron content in the SN of iron dextran overloaded animals. The findings showed that peripheral iron dextran overload increased the iron staining positive cells and reduced the number of TH-immunoreactive neurons in the SN. As a result, dopamine release and content, as well as its metabolites contents were decreased in caudate putamen. Even more dramatic changes were found in chronic overload group. These results suggest that peripheral iron dextran can increase the iron level in the SN, where excessive iron causes the degeneration of dopaminergic neurons. The chronic iron overload may be more destructive to dopaminergic neurons than the acute iron overload.     

Neuroprotection by iron chelator against proteasome inhibitor-induced nigral degeneration

The cause of the neurodegenerative process in Parkinson's disease (PD) remains unclear, but evidence suggests that failure of the ubiquitin-proteasome system may play a major role in the pathogenesis of the disease. Iron is believed to be a key contributor to PD pathology by inducing aggregation of alpha-synuclein and by generating oxidative stress. Our present studies have shown that micro-injection of the proteasome inhibitor lactacystin into the substantia nigra (SN) of C57BL/6 mice causes significant loss of dopaminergic cells and induces intracellular inclusion body formation. We have also found that co-injection of the iron chelator desferrioxamine not only attenuates the lactacystin-induced dopamine neuron loss, but also reduces the presence of ubiquitin-positive intracellular inclusions in the SN, whereas use of iron-deficient diet has no such protective effects. These results may support that iron plays a key role in proteasome inhibitor-induced nigral pathology and that reducing iron reactivity may prevent dopaminergic neuron degeneration and reduce abnormal protein aggregation.     

MAO-B elevation in mouse brain astrocytes results in Parkinson's pathology

Age-related increases in monoamine oxidase B (MAO-B) may contribute to neurodegeneration associated with Parkinson's disease (PD). The MAO-B inhibitor deprenyl, a long-standing antiparkinsonian therapy, is currently used clinically in concert with the dopamine precursor L-DOPA. Clinical studies suggesting that deprenyl treatment alone is not protective against PD associated mortality were targeted to symptomatic patients. However, dopamine loss is at least 60% by the time PD is symptomatically detectable, therefore lack of effect of MAO-B inhibition in these patients does not negate a role for MAO-B in pre-symptomatic dopaminergic loss. In order to directly evaluate the role of age-related elevations in astroglial MAO-B in the early initiation or progression of PD, we created genetically engineered transgenic mice in which MAO-B levels could be specifically induced within astroglia in adult animals. Elevated astrocytic MAO-B mimicking age related increase resulted in specific, selective and progressive loss of dopaminergic neurons in the substantia nigra (SN), the same subset of neurons primarily impacted in the human condition. This was accompanied by other PD-related alterations including selective decreases in mitochondrial complex I activity and increased mitochondrial oxidative stress. Along with a global astrogliosis, we observed local microglial activation within the SN. These pathologies correlated with decreased locomotor activity. Importantly, these events occurred even in the absence of the PD-inducing neurotoxin MPTP. Our data demonstrates that elevation of murine astrocytic MAO-B by itself can induce several phenotypes of PD, signifying that MAO-B could be directly involved in multiple aspects of disease neuropathology. Mechanistically this may involve increases in membrane permeant H(2)O(2) which can oxidize dopamine within dopaminergic neurons to dopaminochrome which, via interaction with mitochondrial complex I, can result in increased mitochondrial superoxide. Our inducible astrocytic MAO-B transgenic provides a novel model for exploring pathways involved in initiation and progression of several key features associated with PD pathology and for therapeutic drug testing.     

Increased levels of ubiquitin in the 6-OHDA-lesioned striatum of rats

Multiple genetic deficits have linked impaired ubiquitin-conjugation pathways to various forms of familiar Parkinson's disease. We therefore examined the possible role of 6-hydroxydopamine, a dopaminergic neurotoxin used in Parkinson's disease experimental models, in causing protein degradation and its association with the ubiquitin proteasome system. Using unilaterally 6-hydroxydopamine-denervated rats and mass spectrometry profiling directly on brain tissue sections, we here report for the first time an increased level of unconjugated ubiquitin specifically in the dorsal striatum of the dopamine depleted hemisphere. No similar changes were found in the intact hemisphere or in the ventral striatum of the dopamine depleted hemisphere. The lesioning of the dopamine innervation to the striatum was confirmed by a strongly reduced dopamine transporter binding in the striatum, indicating an abundant loss of dopamine neurons. These results suggest that denervation of dopamine neurons per se is implicated in the regulation of ubiquitin pathways, at least in a classical animal model of Parkinson's disease. This study adds additional information regarding the involvement of the ubiquitin-proteasome system in Parkinson's disease.     

Inhibitors of Mitochondrial Respiration, Iron (II), and Hydroxyl Radical Evoke Release and Extracellular Hydrolysis of Glutathione in Rat Striatum and Substantia Nigra

In this investigation, microdialysis has been used to study the effects of 1-methyl-4-phenylpyridinium (MPP+), an inhibitor of mitochondrial complex I and alpha-ketoglutarate dehydrogenase and the active metabolite of the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), on extracellular concentrations of glutathione (GSH) and cysteine (CySH) in the rat striatum and substantia nigra (SN). During perfusion of a neurotoxic concentration of MPP+ (2.5 mM) into the rat striatum or SN, extracellular concentrations of GSH and CySH remain at basal levels (both approximately 2 microM). However, when the perfusion is discontinued, a massive but transient release of GSH occurs, peaking at 5,000% of basal levels in the striatum and 2,000% of basal levels in the SN. The release of GSH is followed by a slightly delayed and smaller elevation of extracellular concentrations of CySH that can be blocked by the gamma-glutamyl transpeptidase (gamma-GT) inhibitor acivicin. Low-molecular-weight iron and extracellular hydroxyl radical (OH*) have been implicated as participants in the mechanism underlying the dopaminergic neurotoxicity of MPTP/MPP+. During perfusion of Fe2+ (OH*) into the rat striatum and SN, extracellular levels of GSH also remain at basal levels. When perfusions of Fe2+ are discontinued, a massive transient release of GSH occurs followed by a delayed, small, but progressive elevation of extracellular CySH level that again can be blocked by acivicin. Previous investigators have noted that extracellular concentrations of the excitatory/excitotoxic amino acid glutamate increase dramatically when perfusions of neurotoxic concentrations of MPP+ are discontinued. This observation and the fact that MPTP/MPP+ causes the loss of nigrostriatal GSH without corresponding increases of glutathione disulfide (GSSG) and the results of the present investigation suggest that the release and gamma-GT/dipeptidase-mediated hydrolysis of GSH to glutamate, glycine, and CySH may be important factors involved with the degeneration of dopamine neurons. It is interesting that a very early event in the pathogenesis of Parkinson's disease is a massive loss of GSH in the SN pars compacta that is not accompanied by corresponding increases of GSSG levels. Based on the results of this and prior investigations, a new hypothesis is proposed that might contribute to an understanding of the mechanisms that underlie the degeneration of dopamine neurons evoked by MPTP/MPP+, other agents that impair neuronal energy metabolism, and Parkinson's disease.     

Toxic Effects of Iron for Cultured Mesencephalic Dopaminergic Neurons Derived from Rat Embryonic Brains

Iron, a transition metal possibly involved in the pathogenesis of Parkinson's disease, was tested for its toxic effects toward cultures of dissociated rat mesencephalic cells. When cultures were switched for 24 h to serum-free conditions, the effective concentrations of ferrous iron (Fe2+) producing a loss of 50% of dopaminergic neurons, as quantified by tyrosine hydroxylase (TH) immunocytochemistry, TH mRNA in situ hybridization, and measurement of TH activity, were on the order of 200 microM. High-affinity dopamine (DA) uptake, which reflects integrity and function of dopaminergic nerve terminals, was impaired at significantly lower concentrations (EC50 = 67 microM). Toxic effects were not restricted to dopaminergic neurons inasmuch as trypan blue dye exclusion index and gamma-aminobutyric acid uptake, two parameters used to assess survival of other types of cells present in these cultures, were also affected. Protection against iron cytotoxicity was afforded by desferrioxamine and apotransferrin, two ferric iron-chelating agents. Normal supplementation of the culture medium by serum proteins during treatment was also effective, presumably via nonspecific sequestration. Potential interactions with DA were also investigated. Fe2+ at subtoxic concentrations and desferrioxamine in the absence of exogenous iron added to the cultures failed to potentiate or reduce DA cytotoxicity for mesencephalic cells, respectively. Transferrin, the glycoprotein responsible for intracellular delivery of iron, was ineffective in initiating selective cytotoxic effects toward dopaminergic neurons preloaded with DA. Altogether, these results suggest (a) that ferrous iron is a potent neurotoxin for dopaminergic neurons as well as for other cell types in dissociated mesencephalic cultures, acting likely via autoxidation into its ferric form, and (b) that the presence of intra- and extracellular DA is not required for the observed toxic effects.     

Protection of midbrain dopaminergic neurons by the end-product of purine metabolism uric acid: potentiation by low-level depolarization

High plasma levels of the end product of purine metabolism uric acid (UA) predict a reduced risk of developing Parkinson's disease suggesting that UA may operate as a protective factor for midbrain dopaminergic neurons. Consistent with this view, UA exerted partial but long-term protection in a culture model in which these neurons die spontaneously. The rescued neurons were functional as they accumulated dopamine, efficiently. The use of the fluorescent probe dihydrorhodamine-123 revealed that UA operated by an antioxidant mechanism. The iron chelating agent desferrioxamine, the H₂O₂ scavenger enzyme catalase and the inhibitor of lipid peroxidation Trolox mimicked the effects of UA, suggesting that UA neutralized reactive oxygen species produced via a Fenton-type chemical reaction. UA was, however, not significantly accumulated into neurons, which indicates that the antioxidant effect occurred probably extracellularly. Structure – activity relationships among purine derivatives revealed that the antioxidant properties of UA resulted from the presence of a 8-one substituent in its chemical structure. Of interest, the stimulation of L-type Ca²⁺ channels by high K⁺-induced depolarization and the ensuing activation of extracellular signal-regulated kinases 1/2 strongly improved the neuroprotective effect of UA whereas the depolarizing signal alone had no effect. In summary, our data indicate that UA may interfere directly with the disease's pathomechanism.     

The Iron Chelator Desferrioxamine (Desferal) Retards 6-Hydroxydopamine-Induced Degeneration of Nigrostriatal Dopamine Neurons

A selective increase in content of iron in the pars compacta of the substantia nigra has been implicated in the biochemical pathology of Parkinson's 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 micrograms) 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 (approximately 60%) against the 6-OHDA-induced reduction in striatal DA content and a normalization of DA release. Dopaminergic-related 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 Parkinson's disease.     

Neuromelanin can protect against iron-mediated oxidative damage in system modeling iron overload of brain aging and Parkinson's disease

In Parkinson's disease (PD), dopamine neurons containing neuromelanin selectively degenerate. Neuromelanin binds iron and accumulates in aging. Iron accumulates in reactive form during aging, PD, and is involved in neurodegeneration. It is not clear how the interaction of neuromelanin and iron can be protective or toxic by modulating redox processes. Here, we investigated the interaction of neuromelanin from human substantia nigra with iron in the presence of ascorbic acid, dopamine, and hydrogen peroxide. We observed that neuromelanin blocks hydroxyl radical production by Fenton's reaction, in a dose-dependent manner. Neuromelanin also inhibited the iron-mediated oxidation of ascorbic acid, thus sparing this major antioxidant molecule in brain. The protective effect of neuromelanin on ascorbate oxidation occurs even in conditions of iron overload into neuromelanin. The blockade of iron into a stable iron–neuromelanin complex prevents dopamine oxidation, inhibiting the formation of neurotoxic dopamine quinones. The above processes occur intraneuronally in aging and PD, thus showing that neuromelanin is neuroprotective. The iron–neuromelanin complex is completely decomposed by hydrogen peroxide and its degradation rate increases with the amount of iron bound to neuromelanin. This occurs in PD when extraneuronal iron–neuromelanin is phagocytosed by microglia and iron–neuromelanin degradation releases reactive/toxic iron.     

Celastrol protects against MPTP- and 3-nitropropionic acid-induced neurotoxicity

Oxidative stress and inflammation are implicated in neurodegenerative diseases including Parkinson's disease (PD) and Huntington's disease (HD). Celastrol is a potent anti-inflammatory and antioxidant compound extracted from a perennial creeping plant belonging to the Celastraceae family. Celastrol is known to prevent the production of proinflammatory cytokines, inducible nitric oxide synthase and lipid peroxidation. Mice were treated with celastrol before and after injections of MPTP, a dopaminergic neurotoxin, which produces a model of PD. A 48% loss of dopaminergic neurons induced by MPTP in the substantia nigra pars compacta was significantly attenuated by celastrol treatment. Moreover, celastrol treatment significantly reduced the depletion in dopamine concentration induced by MPTP. Similarly, celastrol significantly decreased the striatal lesion volume induced by 3-nitropropionic acid, a neurotoxin used to model HD in rats. Celastrol induced heat shock protein 70 within dopaminergic neurons and decreased tumor necrosis factor-alpha and nuclear factor kappa B immunostainings as well as astrogliosis. Celastrol is therefore a promising neuroprotective agent for the treatment of PD and HD.     

The antioxidant activity of haptoglobin towards haemoglobin-stimulated lipid peroxidation

Haemoglobin stimulates the peroxidation of lipids in two discernable phases. The first phase is inhibited by binding haemoglobin to the protein haptoglobin. The second phase is stimulated by complexable iron released from the haemoglobin molecule during the process of lipid peroxidation. This latter peroxidation is inhibitable by transferrin and the iron chelator desferrioxamine. Heat-denatured haemoglobin and haemin both stimulated lipid peroxidation but this is not inhibitable by haptoglobin. It is suggested that the haptoglobins play an important antioxidant role in vivo by preventing iron-stimulated formation of oxygen radicals.     

Stimulation and inhibition of iron-dependent lipid peroxidation by desferrioxamine

Peroxidation of rat brain synaptosomes was assessed by the formation of thiobarbituric acid reactive products in either 50 mM potassium phosphate buffer (pH 7.4) or pH adjusted saline. In phosphate, addition of Fe2+ resulted in a dose-related increase in lipid peroxidation. In saline, stimulation of lipid peroxidation by Fe2+ was maximal at 30 uM, and was less at concentrations of 100 uM and above. Whereas desferrioxamine caused a dose-related inhibition of iron-dependent lipid peroxidation in phosphate, it stimulated lipid peroxidation with Fe2+ by as much as 7-fold in saline. The effects of desferrioxamine depended upon the oxidation state of iron, and the concentration of desferrioxamine and lipid. The results suggest that lipid and desferrioxamine compete for available iron. The data are consistent with the hypothesis that either phosphate or desferrioxamine may stimulate iron-dependent lipid peroxidation under certain circumstances by favoring formation of Fe2+/Fe3+ ratios.     

Paraquat-Induced Free Radical Reaction in Mouse Brain Microsomes

Paraquat has been implicated as an environmental toxin which may induce the syndrome of Parkinson's disease after exposure to this agent. However, the biochemical mechanism by which paraquat causes cell death and neurodegeneration has not been extensively studied. Paraquat was rapidly taken up by nerve terminals isolated from mouse cerebral cortices. It induced lipid peroxidation in a concentration dependent manner in the presence of NADPH and ferrous ion. The maximal stimulation effect was obtained at a paraquat concentration around 100 M and the K_mvalue for paraquat was 46.7 M. The lipid peroxidation required microsomal enzymes. Antioxidants, such as superoxide dismutase, catalase and promethazine significantly inhibited paraquat-induced lipid peroxidation. Due to its structural similarity to the pyridinium compound MPP⁺(N-methyl-4-phenyl pyridium ion), it may be taken up by dopamine neurons and cause lipid peroxidation and cell death resulting in the manifestation of Parkinsonian syndrome.     

Oxygen free radicals and Parkinson's disease

The involvement of oxygen radicals in the pathogenesis of Parkinson's disease has been suggested for some time. This article reviews the evidence supporting the involvement of oxygen radicals in the disease process in the brain. This includes a discussion of iron, lipid peroxidation, peroxidase, catalase, superoxide dismutase, and glutathione levels in the brain. In addition, various theories of induction of Parkinson's disease are discussed in relation to the possible involvement of oxygen radicals. These theories include the environmental toxin theory, the dopamine turnover theory, and the cerebral blood flow theory.