Iron-sulfur enzyme mediated mitochondrial superoxide toxicity in experimental Parkinson's disease

Mitochondrial oxidative stress is thought to be an important pathological mediator of neuronal death in Parkinson's disease. However, the precise mechanism by which mitochondrial oxidative stress mediates the death of dopaminergic neurons of the substantia nigra remains unclear. We tested the idea that neuronal damage in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease results, in part, from superoxide radical toxicity via inactivation of an iron-sulfur (Fe-S) protein, mitochondrial aconitase. Administration of MPTP in mice resulted in inactivation of mitochondrial aconitase, but not fumarase in the substantia nigra. MPTP treatment mobilized an early mitochondrial pool of iron detectable by bleomycin chelation that coincided with mitochondrial aconitase inactivation. MPTP-induced mitochondrial aconitase inactivation, iron accumulation and dopamine depletion were significantly attenuated in transgenic mice overexpressing mitochondrial Sod2 and exacerbated in partial deficient Sod2 mice. These results suggest that mitochondrial aconitase may be an important early source of mitochondrial iron accumulation in experimental Parkinson's disease, and that superoxide radical toxicity manifested by oxidative inactivation of mitochondrial aconitase may play a pathogenic role in Parkinson's disease.     

Time Course Changes in the Dopaminergic Nigrostriatal System Following Transection of the Medial Forebrain Bundle: Detection of Oxidatively Modified Proteins in Substantia Nigra

Abstract: We studied the time course of oxidatively modified proteins in the nigrostriatal dopaminergic system following transection of the medial forebrain bundle by quantifying the number of carbonyl groups coupled to striatal and nigral protein homogenates, an index of metal-catalyzed oxidations. We found a striking effect of axotomy on the number of oxidatively modified proteins in the substantia nigra but not in the striatum within the first 5 days postlesion. This effect was correlated with the neurochemical activity of the dopaminergic and serotoninergic systems in the substantia nigra, which suggests a role of dopamine- and serotonin-derived radical oxygen species in the oxidative stress detected in this brain area. We then searched for the type of cell death in the substantia nigra following axotomy. The fragmentation pattern obtained by agarose gel electrophoresis of DNA isolated from nigral tissue was indicative of cell death being entirely necrotic. In fact, no evidence of apoptosis was detected at any postlesion time as revealed by TdT-mediated dUTP-biotin nick end-labeling (TUNEL) staining. The course of necrotic cell death in the substantia nigra coincided with the maximal levels of oxidatively modified proteins in the substantia nigra, suggesting a link between oxidative stress and nerve cell death and also coinciding with the neurochemical activity of both dopaminergic and serotoninergic systems.     

Inflammation induces mitochondrial dysfunction and dopaminergic neurodegeneration in the nigrostriatal system

Evidence suggests that chronic inflammation, mitochondrial dysfunction, and oxidative stress play significant and perhaps synergistic roles in Parkinson's disease (PD), where the primary pathology is significant loss of the dopaminergic neurons in the substantia nigra. The use of anti-inflammatory drugs for PD treatment has been proposed, and inhibition of cyclo-oxygenase-2 (COX-2) or activation of peroxisome proliferator-activated receptor gamma (PPAR-gamma) yields neuroprotection in MPTP-induced PD. Lipopolysaccharide (LPS) induces inflammation-driven dopaminergic neurodegeneration. We tested the hypothesis that celecoxib (Celebrex, COX-2 inhibitor) or pioglitazone (Actos, PPAR-gamma agonist) will reduce the LPS-induced inflammatory response, spare mitochondrial bioenergetics, and improve nigral dopaminergic neuronal survival. Rats were treated with vehicle, celecoxib, or pioglitazone and were intrastriatally injected with LPS. Inflammation, mitochondrial dysfunction, oxidative stress, decreased dopamine, and nigral dopaminergic neuronal loss were observed post-LPS. Celecoxib and pioglitazone provided neuroprotective properties by decreasing inflammation and restoring mitochondrial function. Pioglitazone also attenuated oxidative stress and partially restored striatal dopamine as well as demonstrated dopaminergic neuroprotection and reduced nigral microglial activation. In summary, intrastriatal LPS served as a model for inflammation-induced dopaminergic neurodegeneration, anti-inflammatory drugs provided protective properties, and pioglitazone or celecoxib may have therapeutic potential for the treatment of neuro-inflammation and PD.     

Neuromelanin associated redox-active iron is increased in the substantia nigra of patients with Parkinson's disease

Degeneration of dopaminergic neurones during Parkinson's disease is most extensive in the subpopulation of melanized-neurones located in the substantia nigra pars compacta. Neuromelanin is a dark pigment produced in the dopaminergic neurones of the human substantia nigra and has the ability to bind a variety of metal ions, especially iron. Post-mortem analyses of the human brain have established that oxidative stress and iron content are enhanced in association with neuronal death. As redox-active iron (free Fe2+ form) and other transition metals have the ability to generate highly reactive hydroxyl radicals by a catalytic process, we investigated the redox activity of neuromelanin (NM)-aggregates in a group of parkinsonian patients, who presented a statistically significant reduction (- 70%) in the number of melanized-neurones and an increased non-heme (Fe3+) iron content as compared with a group of matched-control subjects. The level of redox activity detected in neuromelanin-aggregates was significantly increased (+ 69%) in parkinsonian patients and was highest in patients with the most severe neuronal loss. This change was not observed in tissue in the immediate vicinity of melanized-neurones. A possible consequence of an overloading of neuromelanin with redox-active elements is an increased contribution to oxidative stress and intraneuronal damage in patients with Parkinson's disease.     

Modifications of the iron-neuromelanin system in Parkinson's disease

Parkinson's disease is a common neurodegenerative disorder with a mainly sporadic aetiology, although a number of monogenic familiar forms are known. Most of the motor symptoms are due to selective depletion of dopaminergic, neuromelanin-containing neurones of the substantia nigra pars compacta. Neuromelanin is the dark insoluble macromolecule that confers the black (substantia nigra) or grey (locus coeruleus) colour to monoaminergic basal ganglia. In particular, nigral neurones are pigmented because of the accumulation of by-products of oxidative metabolism of the neurotransmitter dopamine. The occurrence of dopamine (and all the enzymatic machinery required for dopamine synthesis, re-uptake and disposal) and neuromelanin, and a large amount of iron ions that interact with them, makes dopaminergic nigral neurones peculiarly susceptible to oxidative stress conditions that, in turn, may become amplified by the iron-neuromelanin system itself. In this mini-review we describe biophysical evidence for iron-neuromelanin modifications that support this hypothesis. Furthermore, we discuss the formation of the covalent linkage between alpha-synuclein and neuromelanin from the early stages of the disease.     

Curcumin treatment alleviates the effects of glutathione depletion in vitro and in vivo: therapeutic implications for Parkinson's disease explained via in silico studies

Oxidative stress has been implicated in the degeneration of dopaminergic neurons in the substantia nigra (SN) of Parkinson's disease (PD) patients. An important biochemical feature of presymptomatic PD is a significant depletion of the thiol antioxidant glutathione (GSH) in these neurons resulting in oxidative stress, mitochondrial dysfunction, and ultimately cell death. We have earlier demonstrated that curcumin, a natural polyphenol obtained from turmeric, protects against peroxynitrite-mediated mitochondrial dysfunction both in vitro and in vivo. Here we report that treatment of dopaminergic neuronal cells and mice with curcumin restores depletion of GSH levels, protects against protein oxidation, and preserves mitochondrial complex I activity which normally is impaired due to GSH loss. Using systems biology and dynamic modeling we have explained the mechanism of curcumin action in a model of mitochondrial dysfunction linked to GSH metabolism that corroborates the major findings of our experimental work. These data suggest that curcumin has potential therapeutic value for neurodegenerative diseases involving GSH depletion-mediated oxidative stress.     

Semichronic inhibition of glutathione reductase promotes oxidative damage to proteins and induces both transcription and translation of tyrosine hydroxylase in the nigrostriatal system

We have evaluated the effect of N,N-bis (2-chloroethyl)-N-nitrosourea (BCNU), an inhibitor of glutathione reductase (GR), on the oxidative status along with the integrity of the nigrostriatal dopaminergic system of the rat. The oxidative status was studied by the quantification of carbonyl groups coupled to protein homogenates. Moreover, the specific oxidations in glial fibrillary acidic protein (GFAP) and neurofilament-200 (NF-200) were also measured. The results show that oxidative damage in proteins in the nigrostriatal system is confined to the striatum. Specific carbonyl groups coupled to native NF-200 and GFAP were also increased. These changes were accompanied by reactive astrocytosis in striatum but not in substantia nigra. In substantia nigra, decreased levels of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were observed following BCNU treatment. In contrast, DA levels were increased in the striatum along with an overall decrease in the ratios of DA metabolites to DA. We also studied the mRNA levels for tyrosine hydroxylase (TH) and the dopamine transporter (DAT) by in situ hybridization. TH mRNA but not DAT mRNA was significantly induced in substantia nigra following BCNU treatment, which was consistent with significant elevations in TH enzyme amount and activity and unchanged DA uptake in striatum. All these results support the DA free radical hypothesis and the key role of the striatal glutathione system in protecting the striatal system against oxidative stress.     

铁与帕金森病关系研究进展

大量研究证实帕金森患者黑质铁异常沉积,脑内铁代谢紊乱,铁促进氧化应激是神经细胞损伤的重要病理机制。本文综述了铁代谢异常与帕金森病关系的研究概况,指出从中药中寻找金属螯合活性成分治疗PD是极其诱人的研究方向。     

Increased Protein Oxidation in Human Substantia Nigra Pars Compacta in Comparison with Basal Ganglia and Prefrontal Cortex Measured with an Improved Dinitrophenylhydrazine Assay

Abstract: The dopaminergic phenotype of neurons in human substantia nigra deteriorates during normal aging, and loss of these neurons is prominent in Parkinson's disease. These degenerative processes are hypothesized to involve oxidative stress. To compare oxidative stress in the nigra and related regions, we measured carbonyl modifications of soluble proteins in postmortem samples of substantia nigra, basal ganglia, and prefrontal cortex from neurologically normal subjects, using an improved 2,4-dinitrophenylhydrazine assay. The protein carbonyl content was found to be about twofold higher in substantia nigra pars compacta than in the other regions. To further analyze this oxidative damage, the distribution of carbonyl groups on soluble proteins was determined by western immunoblot analysis. This method revealed that carbonyl content of the major proteins in each region was linearly dependent on molecular weight. This distribution raises the possibility that protein carbonyl content is controlled by a size-dependent mechanism in vivo. Our results suggest that oxidative stress is elevated in human substantia nigra pars compacta in comparison with other regions and that oxidative damage is higher within the dopaminergic neurons. Elevated oxidative damage may contribute to the degeneration of nigral dopaminergic neurons in aging and in Parkinson's disease.     

Is there a rationale for neuroprotection against dopamine toxicity in Parkinson's disease?

Parkinson's disease is a progressive neurological disease caused by rather selective degeneration of the dopaminergic neurons in the substantia nigra. Though subject to intensive research, the etiology of this nigral loss is still undetermined and treatment is basically symptomatic. The current major hypothesis is that nigral neuronal death in PD is due to excessive oxidative stress generated by auto and enzymatic oxidation of the endogenous neurotransmitter dopamine (DA), the formation of neuromelanin (NM) and the presence of a high concentration of iron. In this review article although we concisely describe the effects of NM and iron on neuronal survival, we mainly focus on the molecular mechanisms of DA-induced apoptosis. DA exerts its toxic effects through its oxidative metabolites either in vitro or in vivo The oxidative metabolites then activate a very intricate web of signals, which culminate in cell death. The signal transduction pathways and genes, which are associated with DA toxicity are described in detail.     

The herbicide paraquat induces dopaminergic nigral apoptosis through sustained activation of the JNK pathway

Environmental exposure to the oxidant-producing herbicide paraquat has been implicated as a risk factor in Parkinson's disease. Although intraperitoneal paraquat injections in mice cause a selective loss of dopaminergic neurons in the substantia nigra pars compacta, the exact mechanism involved is still poorly understood. Our data show that paraquat induces the sequential phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun and the activation of caspase-3 and sequential neuronal death both in vitro and in vivo. These effects are diminished by the specific JNK inhibitor SP600125 and the antioxidant manganese(III) tetrakis (4-benzoic acid) porphyrin in vitro. Furthermore, JNK pathway inhibitor CEP-11004 effectively blocks paraquat-induced dopaminergic neuronal death in vivo. These results suggest that the JNK signaling cascade is a direct activator of the paraquat-mediated nigral dopaminergic neuronal apoptotic machinery and provides a molecular linkage between oxidative stress and neuronal apoptosis.     

Neuroprotective Effects of Alpha Lipoic Acid on Haloperidol-Induced Oxidative Stress in the Rat Brain

Haloperidol is an antipsychotic drug that exerts its' antipsychotic effects by inhibiting dopaminergic neurons. Although the exact pathophysiology of haloperidol extrapyramidal symptoms are not known, the role of reactive oxygen species in inducing oxidative stress has been proposed as one of the mechanisms of prolonged haloperidol-induced neurotoxicity. In the present study, we evaluate the protective effect of alpha lipoic acid against haloperidol-induced oxidative stress in the rat brain. Sprague Dawley rats were divided into control, alpha lipoic acid alone (100 mg/kg p.o for 21 days), haloperidol alone (2 mg/kg i.p for 21 days), and haloperidol with alpha lipoic acid groups (for 21 days). Haloperidol treatment significantly decreased levels of the brain antioxidant enzymes super oxide dismutase and glutathione peroxidase and concurrent treatment with alpha lipoic acid significantly reversed the oxidative effects of haloperidol. Histopathological changes revealed significant haloperidol-induced damage in the cerebral cortex, internal capsule, and substantia nigra. Alpha lipoic acid significantly reduced this damage and there were very little neuronal atrophy. Areas of angiogenesis were also seen in the alpha lipoic acid-treated group. In conclusion, the study proves that alpha lipoic acid treatment significantly reduces haloperidol-induced neuronal damage.     

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.     

A dibenzoylmethane derivative protects dopaminergic neurons against both oxidative stress and endoplasmic reticulum stress

The enhancement of intracellular stresses such as oxidative stress and endoplasmic reticulum (ER) stress has been implicated in several neurodegenerative disorders including Parkinson's disease (PD). During a search for compounds that regulate ER stress, a dibenzoylmethane (DBM) derivative 14-26 (2,2'-dimethoxydibenzoylmethane) was identified as a novel neuroprotective agent. Analysis in SH-SY5Y cells and in PC12 cells revealed that the regulation of ER stress by 14-26 was associated with its anti-oxidative property. 14-26 prevented the production of reactive oxygen species (ROS) when the cells were exposed to oxidants such as hydrogen peroxide and 6-hydroxydopamine (6-OHDA) or an ER stressor brefeldin A (BFA). 14-26 also prevented ROS-induced damage in both the ER and the mitochondria, including the protein carbonylation in the microsome and the reduction of the mitochondrial membrane potential. Further examination disclosed the presence of the iron-chelating activity in 14-26. In vivo, 14-26 suppressed both oxidative stress and ER stress and prevented neuronal death in the substantia nigra pars compacta (SNpc) after injection of 6-OHDA in mice. These results suggest that 14-26 is an antioxidant that protects dopaminergic neurons against both oxidative stress and ER stress and could be a therapeutic candidate for the treatment of PD.     

Semichronic Inhibition of Glutathione Reductase Promotes Oxidative Damage to Proteins and Induces both Transcription and Translation of Tyrosine Hydroxylase in the Nigrostriatal System

We have evaluated the effect of N,N-bis (2-chloroethyl)-N-nitrosourea (BCNU), an inhibitor of glutathione reductase (GR), on the oxidative status along with the integrity of the nigrostriatal dopaminergic system of the rat. The oxidative status was studied by the quantification of carbonyl groups coupled to protein homogenates. Moreover, the specific oxidations in glial fibrillary acidic protein (GFAP) and neurofilament-200 (NF-200) were also measured. The results show that oxidative damage in proteins in the nigrostriatal system is confined to the striatum. Specific carbonyl groups coupled to native NF-200 and GFAP were also increased. These changes were accompanied by reactive astrocytosis in striatum but not in substantia nigra. In substantia nigra, decreased levels of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were observed following BCNU treatment. In contrast, DA levels were increased in the striatum along with an overall decrease in the ratios of DA metabolites to DA. We also studied the mRNA levels for tyrosine hydroxylase (TH) and the dopamine transporter (DAT) by in situ hybridization. TH mRNA but not DAT mRNA was significantly induced in substantia nigra following BCNU treatment, which was consistent with significant elevations in TH enzyme amount and activity and unchanged DA uptake in striatum. All these results support the DA free radical hypothesis and the key role of the striatal glutathione system in protecting the striatal system against oxidative stress.     

Antioxidant Effects of SelegilIne in Oxidative Stress Induced by Iron Neonatal Treatment in Rats

Increased levels of iron in specific brain regions have been reported in neurodegenerative disorders. It has been postulated that iron exerts its deleterious effects on the nervous system by inducing oxidative damage. In a previous study, we have shown that iron administered during a particular period of the neonatal life induces oxidative damage in brain regions in adult rats. The aim of the present study was to evaluate the possible protective effect of selegiline, a monoamino-oxidase B (MAO-B) inhibitor used in pharmacotherapy of Parkinson’s disease, against iron-induced oxidative stress in the brain. Results have shown that selegiline (1.0 and 10.0 mg/kg), when administered early in life was able to protect the substantia nigra as well as the hippocampus against iron-induced oxidative stress, without affecting striatum. When selegiline (10.0 mg/kg) was administered in the adult life to iron-treated rats, oxidative stress was reduced only in the substantia nigra.     

A rat model of human FENIB (familial encephalopathy with neuroserpin inclusion bodies)

FENIB (familial encephalopathy with neuroserpin inclusion bodies) is caused by intracellular accumulation/polymerization of mutant neuroserpins in the endoplasmic reticulum (ER). Transgenic rats overexpressing megsin (Tg meg), a newly identified serine protease inhibitor (serpin), demonstrated intraneuronal periodic-acid Schiff (PAS)-positive inclusions distributed throughout deeper layers of cerebral cortex, CA1 of the hippocampus, and substantia nigra. Hippocampal extracts from Tg meg rats showed increased expression of ER stress proteins, and activation of caspases-12 and -3, associated with decreased neuronal density. Enhanced ER stress was also observed in dopaminergic neurons in the substantia nigra, in parallel with decreased neuronal viability and motor coordination. In each case, PAS-positive inclusions were also positive for megsin. These data suggest that overexpression of megsin results in ER stress, eventuating in the formation of PAS-positive inclusions. Tg meg rats provide a novel model of FENIB, where accumulation of serpins in the ER induces selective dysfunction/loss of specific neuronal populations.     

Therapeutic Vitamin A Doses Increase the Levels of Markers of Oxidative Insult in <Emphasis Type="Italic">Substantia Nigra</Emphasis> and Decrease Locomotory and Exploratory Activity in Rats after Acute and Chronic Supplementation

Vitamin A is known to regulate some central nervous system (CNS)-associated functions. Vitamin A at high doses has been demonstrated to be beneficial in the treatment of some diseases, for instance acute promyelocytic leukemia. However, vitamin A and its naturally occurring metabolites (retinoids) are known to alter neuronal function, inducing behavioral disorders. Here we provide an evidence to indicate that vitamin A supplementation, at both therapeutic and excessive doses, induces oxidative stress in the rat substantia nigra. Vitamin A supplementation induced lipid peroxidation, protein carbonylation, and oxidation of protein thiol groups, as well as change in catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activity. Surprisingly, locomotory and exploratory activity of rats were decreased after acute and chronic vitamin A supplementation. Therefore, we may conclude from our results that vitamin A supplementation is prooxidant to the rat substantia nigra and effective in altering behavior.     

Intranigral iron infusion in the rat

Iron is known to induce lipid perocidation and recent evidence indicates that both iron and lipid peroxidation are elevated in the substantia nigra in Parkinson's disease (PD). To test whether excess intranigral iron induces lipid peroxidation, we infused an iron citrate solution (0.63 nmol in 0.25 μL) into the rat substantia nigra and measured nigral thiobarbituric acid reactive products at 1-h, 1-d, 1-wk, and 1-mo postinfusion. In a separate group of iron-infused animals, histologic analysis within the substantia nigra through 1-mo postinfusion was accomplished by thionine- and iron-staining, with concurrent assessment of striatal neurochemical markers. Concentrations of nigral thiobarbituric acid reactive products were significantly elevated at 1 h and 1 d in iron-infused animals compared to vehicle-infused and unoperated animals, with a return to control values by 1 wk. Similarly, striatal dopamine turnover was acutely elevated, suggesting damage to dopaminergic neurons, which was confirmed histologically. Although iron-staining within the iron diffusionary area was increased through the postinfusion month, there was an apparent progression of the cellular character of staining from predominantly neuronal to reactive glial and finally to oligodendroglial by 1 mo postinfusion. this progression of cellular iron-staining may indicate a shifting of infused iron to a more bound unreactive form, thus explaining only an acute elevation in lipid peroxidation through 1 d following intranigral iron infusion. The data indicate that damage to nigral neurons induced by iron infusion is transciently associated with a marker of oxidative damage and supports the possibility that iron-induced oxidative stress contributes to the pathogenesis of PD.     

Dynorphin immunocytochemistry in the rat central nervous system

The distribution of dynorphin in the central nervous system was investigated in rats pretreated with relatively high doses (300–400 μg) of colchicine administered intracerebroventricularly. To circumvent the problems of antibody cross-reactivity, antisera were generated against different portions as well as the full dynorphin molecule (i.e., residues 1–13, 7–17, or 1–17). For comparison, antisera to [Leu]enkephalin (residues 1–5) were also utilized. Dynorphin was found to be widely distributed throughout the neuraxis. Immunoreactive neuronal perikarya exist in hypothalamic magnocellular nuclei, periaqueductal gray, scattered reticular formation sites, and other brain stem nuclei, as well as in spinal cord. Additionally, dynorphin-positive fibers or terminals occur in the cerebral cortex, olfactory bulb, nucleus accumbens, caudate-putamen, globus pallidus, hypothalamus, substantia nigra, periaqueductal gray, many brain stem sties, and the spinal cord. In many areas studied, dynorphin and enkephalin appeared to form parallel but probably separate anatomical systems. The results suggest that dynorphin occurs in neuronal systems that are immunocytochemically distinct from those containing other opioid peptides.