alpha-synuclein and 3-nitrotyrosine

Lipid-protein adducts are formed during oxidative and nitrative stress conditions associated with increasing lipid and protein oxidation and nitration. The focus of this review is the analysis of interactions between oxidative-modified lipids and proteins and how lipid nitration can modulate lipid-protein adducts formation. For this, two biologically-relevant models will be analysed: a) human low density lipoprotein, whose oxidation is involved in the early steps of atherogenesis, and b) alpha-synuclein/lipid membranes system, where lipid-protein adducts are being associated with the develop of Parkinson disease and other synucleinopathies.

Topics: alpha-Synuclein; Humans; Lipid Metabolism; Lipids; Lipoproteins, LDL; Nitrates; Oxidative Stress; Proteins; Reactive Nitrogen Species; Tyrosine

Tyrosine nitration of proteins is one of the most important oxidative post-translational modifications in vivo. A major obstacle for its biochemical and physiological studies is the lack of efficient and chemoselective protein tyrosine nitration reagents. Herein, we report a generalizable strategy for light-controlled protein tyrosine nitration by employing biocompatible dinitroimidazole reagents. Upon 390 nm irradiation, dinitroimidazoles efficiently convert tyrosine residues into 3-nitrotyrosine residues in peptides and proteins with fast kinetics and high chemoselectivity under neutral aqueous buffer conditions. The incorporation of 3-nitrotyrosine residues enhances the thermostability of lasso peptide natural products and endows murine tumor necrosis factor-α with strong immunogenicity to break self-tolerance. The light-controlled time resolution of this method allows the investigation of the impact of tyrosine nitration on the self-assembly behavior of α-synuclein.

Topics: alpha-Synuclein; Animals; Light; Mice; Nitrates; Oxidation-Reduction; Tumor Necrosis Factor-alpha; Tyrosine

Tyrosine nitration is a post-translational protein modification relevant to various pathophysiological processes. Chemical nitration procedures have been used to generate and study nitrated proteins, but these methods regularly lead to modifications at other amino acid residues. A novel strategy employs a genetic code modification that allows incorporation of 3-nitrotyrosine (3-NT) during ribosomal protein synthesis to generate a recombinant protein with defined 3-NT-sites, in the absence of other post-translational modifications. This approach was applied to study the generation and stability of the 3-NT moiety in recombinant proteins produced in E.coli. Nitrated alpha-synuclein (ASYN) was selected as exemplary protein, relevant in Parkinson's disease (PD). A procedure was established to obtain pure tyrosine-modified ASYN in mg amounts. However, a rapid (t

Topics: alpha-Synuclein; Cloning, Molecular; Escherichia coli; Escherichia coli Proteins; Gene Expression; Genetic Vectors; Green Fluorescent Proteins; Humans; Metabolic Networks and Pathways; Oxidation-Reduction; Protein Engineering; Recombinant Proteins; Tyrosine

Current pharmacological management of Parkinson disease (PD) does not provide for disease modification, but addresses only symptomatic features. Here, we explore a new approach to neuroprotection based on the use of 2-pentadecyl-2-oxazoline (PEA-OXA), the oxazoline derivative of the fatty acid amide signaling molecule palmitoylethanolamide (PEA), in an experimental model of PD. Daily oral treatment with PEA-OXA (10 mg/kg) significantly reduced behavioral impairments and neuronal cell degeneration of the dopaminergic tract induced by four intraperitoneal injections of the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on 8-week-old male C57 mice. Moreover, PEA-OXA treatment prevented dopamine depletion, increased tyrosine hydroxylase and dopamine transporter activities, and decreased α-synuclein aggregation in neurons. PEA-OXA treatment also diminished nuclear factor-κB traslocation, cyclooxygenase-2, and inducible nitric oxide synthase expression and through upregulation of the nuclear factor E2-related factor 2 pathway, induced activation of Mn-superoxide dismutase and heme oxygenase-1. Further, PEA-OXA modulated microglia and astrocyte activation and preserved microtubule-associated protein-2 alterations. In conclusion, pharmacological activation of nuclear factor E2-related factor 2 pathways with PEA-OXA may be effective in the future therapy of PD.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; alpha-Synuclein; Animals; Astrocytes; Behavior, Animal; Cyclooxygenase 2; Cytokines; Disease Models, Animal; DNA Damage; Dopamine Plasma Membrane Transport Proteins; Inflammation; Mice, Inbred C57BL; Microglia; NF-E2-Related Factor 2; Nitric Oxide Synthase Type II; Nitrosative Stress; Oxazoles; Oxidative Stress; Parkinson Disease; Poly Adenosine Diphosphate Ribose; Transcription Factor RelA; Tyrosine; Tyrosine 3-Monooxygenase

3-Nitrotyrosine (Nit) belongs to products of oxidative stress and could probably influence conformation of neurodegenerative proteins. Syntheses of peptides require availability of suitable synthon for introduction of Nit residue. Common phenolic protection groups are more acid labile, when they are attached to Nit residue. We have found that Fmoc-Nit(Bn)-OH is a good building block for syntheses of Nit containing peptides by Fmoc/tBu strategy. Interestingly, the peptides containing multiple Nit residues can be available solely by use of Fmoc-Nit(Bn)-OH synthon. Bn is removed rapidly with ca 80 % trifluoroacetic acid in dark. The cleavage of Bn from Fmoc-Nit(Bn)-OH proceeds via pseudo-first order mechanism with activation barrier 32 kcal mol(-1) and rate k = 15.3 s(-1) at 20 °C. This rate is more than 2,000,000 times faster than that for cleavage of benzyl from Tyr(Bn).

Topics: alpha-Synuclein; Amino Acid Sequence; Fluorenes; Hydrolysis; Oxidative Stress; Peptides; Prions; Trifluoroacetic Acid; Tyrosine

α-Synuclein (α-Syn), one of the most abundant proteins in the CNS, is known to be a major player in the neurodegeneration observed in Parkinson's disease. We currently report that transient focal ischemia upregulates α-Syn protein expression and nuclear translocation in neurons of the adult rodent brain. We further show that knockdown or knock-out of α-Syn significantly decreases the infarction and promotes better neurological recovery in rodents subjected to focal ischemia. Furthermore, α-Syn knockdown significantly reduced postischemic induction of phospho-Drp1, 3-nitrotyrosine, cleaved caspase-3, and LC-3 II/I, indicating its role in modulating mitochondrial fragmentation, oxidative stress, apoptosis, and autophagy, which are known to mediate poststroke neuronal death. Transient focal ischemia also significantly upregulated serine-129 (S129) phosphorylation (pα-Syn) of α-Syn and nuclear translocation of pα-Syn. Furthermore, knock-out mice that lack PLK2 (the predominant kinase that mediates S129 phosphorylation) showed better functional recovery and smaller infarcts when subjected to transient focal ischemia, indicating a detrimental role of S129 phosphorylation of α-Syn. In conclusion, our studies indicate that α-Syn is a potential therapeutic target to minimize poststroke brain damage.. Abnormal aggregation of α-synuclein (α-Syn) has been known to cause Parkinson's disease and other chronic synucleinopathies. However, even though α-Syn is linked to pathophysiological mechanisms similar to those that produce acute neurodenegerative disorders, such as stroke, the role of α-Syn in such disorder is not clear. We presently studied whether α-Syn mediates poststroke brain damage and more importantly whether preventing α-Syn expression is neuroprotective and leads to better physiological and functional outcome after stroke. Our study indicates that α-Syn is a potential therapeutic target for stroke therapy.

Topics: alpha-Synuclein; Animals; Brain Infarction; Brain Ischemia; Caspase 3; Death-Associated Protein Kinases; Disease Models, Animal; Gene Expression Regulation; Male; Mice; Mice, Transgenic; Microtubule-Associated Proteins; Motor Activity; PC12 Cells; Protein Serine-Threonine Kinases; Rats; Rats, Inbred SHR; RNA, Small Interfering; Stroke; Tyrosine

Nitrosative stress, where nitrosylation of tyrosine (Tyr) leading to 3-nitrotyrosine proteins or free 3-nitrotyrosine is the most prominent change, has been proposed as a pathogenic mechanism in Parkinson's disease (PD). Levels of 3-nitrotyrosine proteins in serum and cerebrospinal fluid (CSF) of patients with PD have not been studied. Nitrosative stress-induced protein changes in serum and CSF were analyzed in patients with PD (n=54) and controls (n=40). Herein, we demonstrate the presence of nitrosative stress in serum and CSF of patients with early PD leading to selective increase of 3-nitrotyrosine proteins other than nitroalbumin, without free 3-nitrotyrosine (Hoehn-Yahr stage 1, p<0.05; stage 2, p<0.01). Among 3-nitrotyrosine proteins, nitro-α-synuclein (N-αSyn) was detected in serum, not CSF, and the sites of Tyr nitrosylation were observed to be modified in patients with early PD. Thus, the intensity of nitrosylation of Tyr125/136 residues is enhanced (stage 1, p<0.05; stage 2, p<0.01), and that of the Tyr39 site is reduced (stage 1, p<0.05), and the ratio between both parameters (α-synuclein with nitrosylated tyrosines 125 and 136 [N-αSyn-Tyr125/136]:α-synuclein with nitrosylated tyrosine 39 [N-αSyn-Tyr39] ratio) is significantly higher in patients with early PD (p<0.01). These observations lead to the hypothesis that evaluating nitrosative stress through enhanced levels of 3-nitrotyrosine proteins in serum and CSF without changes in nitroalbumin, together with the profile of tyrosine nitrosylation of serum αSyn characterized by dominant nitrosylation of Tyr125/136, could serve for the diagnosis of sporadic PD.

Topics: Albumins; alpha-Synuclein; Biomarkers; Humans; Mass Spectrometry; Oxidation-Reduction; Oxidative Stress; Parkinson Disease; Tyrosine

Vitamin A has been characterized as a potential neurotoxin, because ingestion of such vitamin - or its derivatives, the retinoids - at moderate to high doses elicits a myriad of deleterious effects, from acute intoxication involving head-ache, confusion, and 'pseudo tumor cerebri' to chronic, and perhaps irreversible, abnormalities, including irritability, anxiety, depression, and suicide ideation. Nevertheless, it still remains to be found the mechanism by which vitamin A induces cognitive decline. Based on the fact that vitamin A at clinical doses is a potent pro-oxidant agent to the central nervous system, we performed the present work to analyze whether a cotreatment with L-NAME at 30 mg/kg (four times a week) was able to prevent (or minimize) the biochemical and/or behavioral disturbances resulting from a 28-day daily supplementation with retinol palmitate at doses from 1000 to 9000 IU/kg/day. Then, we investigated mitochondrial function, redox parameters, and the levels of proteins potentially involved in neurodegenerative events, as for instance α-synuclein and receptor for advanced glycation endproducts. Besides, monoamine oxidase enzyme activity was quantified in this work. We observed that L-NAME cotreatment was not completely effective in preventing the redox disturbances induced by vitamin A supplementation. Moreover, L-NAME cotreatment did not affect the behavioral deficits elicited by vitamin A supplementation. We conclude that other parameters rather than NO levels or its derivatives, as for example ONOO(-), take a more important role in mediating the negative effects triggered by vitamin A supplementation.

Topics: alpha-Synuclein; Animals; Anxiety Disorders; Behavior, Animal; Central Nervous System; Dietary Supplements; Diterpenes; Electron Transport; Glutathione Transferase; Heat-Shock Proteins; Illness Behavior; Locomotion; Male; Manganese; Mitochondria; Monoamine Oxidase; Neurodegenerative Diseases; NG-Nitroarginine Methyl Ester; Nitric Oxide; Oxidation-Reduction; Rats; Rats, Wistar; Receptor for Advanced Glycation End Products; Receptors, Dopamine; Receptors, Immunologic; Retinyl Esters; Superoxide Dismutase; Superoxides; Tyrosine; Vitamin A

The diagnosis of Parkinson's disease rests on motor signs of advanced central dopamine deficiency. There is an urgent need for disease biomarkers. Clinicopathological evidence suggests that α-synuclein aggregation, the pathological signature of Parkinson's disease, can be detected in gastrointestinal tract neurons in Parkinson's disease. We studied whether we could demonstrate α-synuclein pathology in specimens from unprepped flexible sigmoidoscopy of the distal sigmoid colon in early subjects with Parkinson's disease. We also looked for 3-nitrotyrosine, a marker of oxidative stress. Ten subjects with early Parkinson's disease not treated with dopaminergic agents (7 men; median age, 58.5 years; median disease duration, 1.5 years) underwent unprepped flexible sigmoidoscopy with biopsy of the distal sigmoid colon. Immunohistochemistry studies for α-synuclein and 3-nitrotyrosine were performed on biopsy specimens and control specimens from a tissue repository (23 healthy subjects and 23 subjects with inflammatory bowel disease). Nine of 10 Parkinson's disease samples were adequate for study. All showed staining for α-synuclein in nerve fibers in colonic submucosa. No control sample showed this pattern. A few showed light α-synuclein staining in round cells. 3-Nitrotyrosine staining was seen in 87% of Parkinson's disease cases but was not specific for Parkinson's disease. This study suggests a pattern of α-synuclein staining in Parkinson's disease that was distinct from healthy subjects and those with inflammatory bowel disease. The absence of this pattern in subjects with inflammatory bowel disease suggests it is not a sequel of inflammation or oxidative stress. 3-Nitrotyrosine immunostaining was common in all groups studied, suggesting oxidative stress in the colonic submucosa.

Topics: Aged; alpha-Synuclein; Biomarkers; Colon; Enteric Nervous System; Female; Humans; Male; Middle Aged; Neurons; Oxidative Stress; Parkinson Disease; Substantia Nigra; Tyrosine

Lifetimes of fluorescent states and their fluorescence intensities are strictly coupled and very sensitive to the environment of the fluorophores. The advantage of measuring lifetimes, next to intensities, comes from the fact that it can reveal heterogeneity and dynamic properties of this environment. In this way lifetime analysis can be used to characterize static and dynamic conformational properties and heterogeneity of fluorescent groups in different areas of a protein and as a function of time for an evolving protein. The phenomena that determine the lifetime of a label are its intrinsic properties, dynamic quenching by neighboring groups, exposure to the solvent, as well as Förster resonance energy transfer (FRET) between different groups. The basic principles of these fluorescence phenomena can be found extensively described in the excellent book of Lakowicz (Principles of fluorescence spectroscopy, 3rd edn. Springer, New York, 2006). The fluorescent groups involved are either natural amino acid side chains like tryptophan (Trp) or tyrosine (Tyr), or fluorescent labels covalently engineered into the protein. Even a single fluorescent group can show indications of heterogeneity in the local environment. If several natural fluorescent groups are present, the properties of the individual groups can be separated using site-directed mutagenesis, and additivity of their contributions can be analyzed (Engelborghs, Spectrochim Acta A Mol Biomol Spectrosc 57(11):2255-2270, 2001). If no fluorescent group is naturally present, site-directed mutagenesis can be used to introduce either a fluorescent amino acid or a cysteine allowing chemical labeling.

Topics: Algorithms; alpha-Synuclein; Amino Acid Substitution; Anisotropy; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Naphthalenesulfonates; Protein Conformation; Spectrometry, Fluorescence; Staining and Labeling; Tryptophan; Tyrosine

Although previous studies have demonstrated the involvement of nitrated α-synuclein in neurodegenerative disorders (synucleinopathies), the effects of nitrated α-synuclein and the molecular mechanisms underlying its toxicity are still unclear. In the present study, nitrated α-synuclein with four 3-nitrotyrosines (Tyr(39), Tyr(125), Tyr(133), and Tyr(136)) was obtained non-enzymatically by incubation with nitrite. The nitrated protein existed as a mixture of monomers, dimers, and polymers in solution. The nitrated α-synuclein could induce cell death in a time- and concentration-dependent manner when SH-SY5Y cells (a human neuroblastoma cell line) were incubated with the dimers and polymers. Treatment with anti-integrin α5β1 antibody partially rescued the SH-SY5Y cells from the cell death. Dot blotting and immunoprecipitation revealed that the nitrated protein bound to integrin on the cell membranes. Level of nitric oxide (NO) and calcium-independent inducible NO synthase (iNOS) activity increased during the initial stages of the treatment. The expression of phosphorylated focal adhesion kinase (FAK) decreased in the cells. Subsequently, an increase in caspase 3 activity was observed in SH-SY5Y cells. Our results demonstrate that activation of iNOS and inhibition of FAK may both be responsible for the cell death induced by nitrated α-synuclein. These data suggest that the cytotoxicity of nitrated α-synuclein is mediated via an integrin-iNOS/-FAK signaling pathway, and that the nitration of α-synuclein plays a role in neuronal degeneration.

Topics: alpha-Synuclein; Antibodies; Apoptosis; Calcium; Caspase 3; Cell Line, Tumor; Focal Adhesion Protein-Tyrosine Kinases; Humans; Immunoprecipitation; Integrin alpha5beta1; Nitric Oxide; Nitric Oxide Synthase Type II; Nitrites; Polymerization; Recombinant Proteins; Signal Transduction; Tyrosine

Parkinson's disease (PD) is the second most common neurodegenerative disorder of aging. The pathological hallmark of PD is neuronal inclusions termed Lewy bodies whose main component is alpha-synuclein protein. The finding of these Lewy bodies in the intestinal enteric nerves led to the hypothesis that the intestine might be an early site of PD disease in response to an environmental toxin or pathogen. One potential mechanism for environmental toxin(s) and proinflammatory luminal products to gain access to mucosal neuronal tissue and promote oxidative stress is compromised intestinal barrier integrity. However, the role of intestinal permeability in PD has never been tested. We hypothesized that PD subjects might exhibit increased intestinal permeability to proinflammatory bacterial products in the intestine. To test our hypothesis we evaluated intestinal permeability in subjects newly diagnosed with PD and compared their values to healthy subjects. In addition, we obtained intestinal biopsies from both groups and used immunohistochemistry to assess bacterial translocation, nitrotyrosine (oxidative stress), and alpha-synuclein. We also evaluated serum markers of endotoxin exposure including LPS binding protein (LBP). Our data show that our PD subjects exhibit significantly greater intestinal permeability (gut leakiness) than controls. In addition, this intestinal hyperpermeability significantly correlated with increased intestinal mucosa staining for E. coli bacteria, nitrotyrosine, and alpha-synuclein as well as serum LBP levels in PD subjects. These data represent not only the first demonstration of abnormal intestinal permeability in PD subjects but also the first correlation of increased intestinal permeability in PD with intestinal alpha-synuclein (the hallmark of PD), as well as staining for gram negative bacteria and tissue oxidative stress. Our study may thus shed new light on PD pathogenesis as well as provide a new method for earlier diagnosis of PD and suggests potential therapeutic targets in PD subjects.. Clinicaltrials.gov NCT01155492.

Topics: Acute-Phase Proteins; Aged; alpha-Synuclein; Biomarkers; Carrier Proteins; Endotoxins; Escherichia coli; Escherichia coli Infections; Female; Humans; Immunoenzyme Techniques; Intestinal Mucosa; Intestines; Male; Membrane Glycoproteins; Middle Aged; Neurons; Oxidative Stress; Parkinson Disease; Permeability; Sucrose; Tyrosine

Trichloroethylene, a chlorinated solvent widely used as a degreasing agent, is a common environmental contaminant. Emerging evidence suggests that chronic exposure to trichloroethylene may contribute to the development of Parkinson's disease. The purpose of this study was to determine if selective loss of nigrostriatal dopaminergic neurons could be reproduced by systemic exposure of adult Fisher 344 rats to trichloroethylene. In our experiments, oral administration of trichloroethylene induced a significant loss of dopaminergic neurons in the substantia nigra pars compacta in a dose-dependent manner, whereas the number of both cholinergic and GABAergic neurons were not decreased in the striatum. There was a robust decline in striatal levels of 3, 4-dihydroxyphenylacetic acid without a significant depletion of striatal dopamine. Rats treated with trichloroethylene showed defects in rotarod behavior test. We also found a significantly reduced mitochondrial complex I activity with elevated oxidative stress markers and activated microglia in the nigral area. In addition, we observed intracellular alpha-synuclein accumulation in the dorsal motor nucleus of the vagus nerve, with some in nigral neurons, but little in neurons of cerebral cortex. Overall, our animal model exhibits some important features of Parkinsonism, and further supports that trichloroethylene may be an environmental risk factors for Parkinson's disease.

Topics: alpha-Synuclein; Animals; Caspase 3; CD11b Antigen; Choline O-Acetyltransferase; Chromatography, High Pressure Liquid; Disease Models, Animal; Dopamine; Dopamine and cAMP-Regulated Phosphoprotein 32; Dose-Response Relationship, Drug; Electrochemistry; Encephalitis; Gene Expression Regulation; Male; Mitochondria; Neurodegenerative Diseases; Oxidative Stress; Rats; Rats, Inbred F344; Rotarod Performance Test; Solvents; Substantia Nigra; Trichloroethylene; Tyrosine; Tyrosine 3-Monooxygenase

Although the relationship between hyperglycemia (using diabetic animal model) and plasma nitrotyrosine level has been studied, the effect of hypoglycemia on nitrotyrosine level in the brain has not been addressed. Here, we evaluated nitration of protein, the colocalization of nitration with alpha-synuclein, activity of inducible nitric oxide synthase, and nitric oxide content using fasting and diabetic animal models. The results showed that signals of alpha-synuclein were widely distributed in most parts of the pallium, midbrain, hippocampus and cerebellum, as indicated by immunohistochemistry. Most signals of the 3-nitrotyrosine were colocalized with those of alpha-synuclein in the midbrain of fasting rats. The level of proteins containing 3-nitrotyrosine was significantly increased in the brain of fasting rats in Western blotting, especially in the midbrain, compared with control rats. In addition, the 3-nitrotyrosine signals increased in hippocampus of diabetic rats. Immunoprecipitation showed that alpha-synuclein was nitrated in the fasting rats. The iNOS activity and nitric oxide levels were significantly increased in both fasting and diabetic animals. The enhanced 3-nitrotyrosine level in the brain of fasting rats suggests that nitration of protein including alpha-synuclein in the midbrain is more affected by hypoglycemia in fasting than hyperglycemia in diabetic rats.

Topics: alpha-Synuclein; Animals; Brain; Diabetes Mellitus, Experimental; Fasting; Male; Mitochondrial Proteins; Nitric Oxide; Nitric Oxide Synthase Type II; Rats; Rats, Sprague-Dawley; Tyrosine

Several lines of evidence implicate a central role for alpha-synuclein (aSN) in the pathogenesis of Parkinson's disease (PD). Besides rare genetic mutations, post-translational mechanisms, such as oxidative stress-related nitration, may alter the protein properties in terms of propensity to aggregate or be degraded. Our group previously described increased reactive oxygen species (ROS) production within easily accessible peripheral blood mononuclear cells (PBMCs) in PD patients compared to healthy elderly subjects. In the present work, we demonstrated a significant induction of nitrotyrosine (NT)-modifications of aSN within PBMCs derived from individuals with idiopathic PD compared to controls, while aSN protein appeared similarly expressed in the two populations. The amount of NT-modified aSN within PBMCs was positively correlated with intracellular ROS concentration and inversely related to daily dosage of levodopa, making its measurement potentially relevant for disease-intervention studies. Neither aSN expression nor its NT-modifications showed any correlation to specific REP1 genotypes, polymorphic variants within aSN gene promoter whose association to PD susceptibility may occur through the modulation of aSN protein expression. Moreover, although NT-modified aSN has been linked to enhanced propensity to aggregate, we failed to detect an increased presence of insoluble aSN aggregates in PBMCs from PD subjects relative to controls, despite a lack of changes in the ubiquitin-proteasome expression or activity. Nonetheless, a significant activation of the autophagy response was identified within PBMCs from PD individuals, which could represent a protective mechanism against abnormal protein accumulation and may explain the lack of aSN aggregation. We discuss the relevance of these findings with respect to PD pathogenesis and biomarker development.

Topics: Aged; alpha-Synuclein; Antiparkinson Agents; Autophagy; Female; Gene Frequency; Genotype; Humans; Levodopa; Male; Middle Aged; Parkinson Disease; Polymorphism, Genetic; Promoter Regions, Genetic; Reactive Oxygen Species; Sex Distribution; Tyrosine

Vitamin A at moderate to high doses is applied in the treatment of some life threatening pathological conditions, for instance cancers. Additionally, vitamin A at low concentrations is a known antioxidant molecule. However, by increasing vitamin A (or its derivatives) concentrations, there is an increase in the levels of oxidative stress markers in several experimental models. Furthermore, it was reported that vitamin A therapy at high doses might induce cognitive decline among the patients, which may become anxious or depressive, for example, depending on vitamin A levels intake. We have previously reported increased levels of oxidative stress markers in rat substantia nigra and striatum. However, the mechanism by which this vitamin altered the redox environment in such rat brain regions remains to be elucidated. In the herein presented work, we have investigated the effects of vitamin A supplementation at clinical doses (1000-9000 IU/kg day(-1)) for 28 days on rat substantia nigra and striatum mitochondrial electron transfer chain (METC) activity, which may produce superoxide anion radical (O(2)(-*)) when impaired. Additionally, the levels of non-enzymatic antioxidant defenses were evaluated, as well as 3-nitrotyrosine, alpha- and beta-synucleins and TNF-alpha levels through ELISA assay. We observed impaired METC in both rat brain regions. Moreover, we found increased O(2)(-*) production and nitrotyrosine content in the nigrostriatal axis of vitamin A-treated rats, suggesting that the use of vitamin A at therapeutic doses may be rethought due to this toxic effects found here.

Topics: alpha-Synuclein; Animals; Caspase 3; Dietary Supplements; Electron Transport; Electron Transport Complex I; Electron Transport Complex II; Electron Transport Complex III; Electron Transport Complex IV; Energy Metabolism; Free Radical Scavengers; Glutathione Transferase; Indicators and Reagents; Mitochondria; Neostriatum; Oxidation-Reduction; Rats; Receptors, Dopamine D2; Substantia Nigra; Tumor Necrosis Factor-alpha; Tyrosine; Vitamin A; Vitamins

Vitamin A is a micronutrient involved in the regulation of a normal mammalian brain function. In spite of this, it has been demonstrated that vitamin A exerts a wide range of deleterious effects regarding neuronal homeostasis, for instance impairing brain metabolism and suppressing neurogenesis, to cite a few. In addition, vitamin A is a redox active molecule, i.e. it is both anti- and pro-oxidant, depending on its concentration. In the herein presented work, we performed some experiments aiming to investigate the effects of clinically applied doses of vitamin A (1000-9000 IU/kg/day during 28 days) on rat hypothalamic redox state and mitochondrial electron transfer chain (METC) activity, as well as on hypothalamic alpha-synuclein and D2 receptor (dopamine receptor) contents. Additionally, we quantified caspase-3 activity and tumor necrosis factor-alpha (TNF-alpha) levels to assess either neuronal death or an inflammatory state in such brain area. We found that vitamin A supplementation increased free radical production, as well as oxidative and nitrosative stress, in rat hypothalamus. Also, we observed increased complex I-III activity, but decreased complex IV activity in the hypothalamus of vitamin A-treated rats, which may give rise to the increased superoxide anion (O(2)(-)) production found here. Other parameters investigated here, i.e. alpha-synuclein and D2 receptor contents did not change. Even though we did not observe signs of increased cell death or inflammation in the rat hypothalamus, more attention is needed when vitamin A is the choice of treatment in certain pathologies.

Topics: Administration, Oral; alpha-Synuclein; Animals; Brain; Caspase 3; Electron Transport Chain Complex Proteins; Hypothalamus; Oxidation-Reduction; Oxidative Stress; Rats; Rats, Wistar; Receptors, Dopamine D2; Superoxides; Tumor Necrosis Factor-alpha; Tyrosine; Vitamin A

Alpha-synuclein is a major component of Lewy bodies, proteinacious inclusions which are a major hallmark of Parkinson's disease (PD). Lewy bodies contain high levels of nitrated tyrosine residues as determined by antibodies specific for 3-nitrotyrosine (3NT) and via mass spectrometry (MS). We have developed a multiple reaction monitoring (MRM) mass spectrometry method to sensitively quantitate the 3NT levels of specific alpha-synuclein tyrosine residues. We found a 9-fold increase (relative to controls) in levels of 3NT at Tyr-39 of alpha-synuclein in an inducible transgenic cellular model of Parkinson's disease in which monoamine oxidase B (MAO-B) is overexpressed and which emulates several features of PD. Increased nitration of Tyr-39 on endogenous alpha-synuclein via elevations in MAO-B levels could be abrogated by the addition of deprenyl, a specific MAO-B inhibitor. The increased levels of 3NT was selective for Tyr-39 as no significant increases in 3NT levels were detected at other tyrosine residues present in the protein (Tyr-125, Tyr-133, and Tyr-136). This is the first report of increased 3NT levels of a specific tyrosine in a PD model and the first use of MRM mass spectrometry to quantify changes in 3NT modifications at specific sites within a target protein.

Topics: alpha-Synuclein; Animals; Lewy Bodies; Mass Spectrometry; Models, Biological; Monoamine Oxidase; Nitrates; Oxidative Stress; Parkinson Disease; PC12 Cells; Rats; Tyrosine

Tertiary contact formation rates in alpha-synuclein, an intrinsically disordered polypeptide implicated in Parkinson's disease, have been determined from measurements of diffusion-limited electron-transfer kinetics between triplet-excited tryptophan:3-nitrotyrosine pairs separated by 10, 12, 55, and 90 residues. Calculations based on a Markovian lattice model developed to describe intrachain diffusion dynamics for a disordered polypeptide give contact quenching rates for various loop sizes ranging from 6 to 48 that are in reasonable agreement with experimentally determined values for small loops (10-20 residues). Contrary to expectations, measured contact rates in alpha-synuclein do not continue to decrease as the loop size increases (>/=35 residues), and substantial deviations from calculated rates are found for the pairs W4-Y94, Y39-W94, and W4-Y136. The contact rates for these large loops indicate much shorter average donor-acceptor separations than expected for a random polymer.

Topics: alpha-Synuclein; Amino Acid Sequence; Electron Transport; Molecular Sequence Data; Protein Conformation; Tryptophan; Tyrosine

Equine pituitary pars intermedia dysfunction (PPID) is a spontaneously occurring progressive disease affecting aged horses and ponies. The pathogenesis of PPID is poorly understood, but the available evidence supports a loss of dopaminergic inhibition of the melanotropes of the pars intermedia. Horses with PPID have increased plasma concentrations of pars intermedia pro-opiomelanocortin-derived peptides that decrease in response to dopamine or dopamine agonist administration. Dopamine and dopamine metabolite concentrations are decreased in the pars intermedia of affected horses compared to age-matched control horses. Horses with disease that are treated with the dopamine agonist pergolide show improvement in clinical signs and normalisation of diagnostic test results. In the present study, immunohistochemical evaluation of pituitary and hypothalamic tissue demonstrated reduced tyrosine hydroxylase immunoreactivity in affected horses compared to age-matched and young controls, supporting the role of dopaminergic neurodegeneration in PPID. In addition, immunohistochemical evaluation revealed an increase in the oxidative stress marker, 3-nitrotyrosine and in nerve terminal protein, alpha-synuclein that colocalised in the pars intermedia of horses with disease. These findings suggest a role for nitration of overexpressed alpha-synuclein in the pathogenesis of neurodegeneration in PPID.

Topics: alpha-Synuclein; Animals; Blotting, Western; Chronic Disease; Dopamine; Horse Diseases; Horses; Immunohistochemistry; Nerve Degeneration; Nerve Tissue Proteins; Nitrogen; Oxidative Stress; Pituitary ACTH Hypersecretion; Pituitary Gland; Synucleins; Tyrosine; Tyrosine 3-Monooxygenase

Alpha-synuclein (alpha-syn) is a major protein component of the neuropathological hallmarks of Parkinson's disease and related neurodegenerative disorders termed synucleinopathies. Neither the mechanism of alpha-syn fibrillization nor the degradative process for alpha-syn has been elucidated. Previously, we showed that wild-type, mutated, and fibrillar alpha-syn proteins are substrates of calpain I in vitro. In this study, we demonstrate that calpain-mediated cleavage near and within the middle region of soluble alpha-syn with/without tyrosine nitration and oxidation generates fragments that are unable to self-fibrillize. More importantly, these fragments prevent full-length alpha-syn from fibrillizing. Calpain-mediated cleavage of alpha-syn fibrils composed of wild-type or nitrated alpha-syn generate C-terminally truncated fragments that retain their fibrillar structure and induce soluble full-length alpha-syn to co-assemble. Therefore, calpain-cleaved soluble alpha-syn inhibits fibrillization, whereas calpain-cleaved fibrillar alpha-syn promotes further co-assembly. These results provide insight into possible disease mechanisms underlying synucleinopathies since the formation of alpha-syn fibrils could be causally linked to the onset/progression of these disorders.

Topics: alpha-Synuclein; Calpain; Chymotrypsin; Humans; Hydrolysis; Microscopy, Immunoelectron; Nerve Degeneration; Nerve Tissue Proteins; Nitrates; Parkinson Disease; Peptide Fragments; Peroxynitrous Acid; Recombinant Proteins; Solubility; Synucleins; Tyrosine

Serofendic acid was recently identified as a neuroprotective factor from fetal calf serum. This study was designed to evaluate the neuroprotective effects of an intranigral microinjection of serofendic acid based on behavioral, neurochemical and histochemical studies in hemi-parkinsonian rats using 6-hydroxydopamine (6-OHDA). Rats were injected with 6-OHDA in the presence or absence of serofendic acid, or were treated with serofendic acid on the same lateral side, at 12, 24 or 72 h after 6-OHDA lesion. Intranigral injection of 6-OHDA alone induced a massive loss of tyrosine hydroxylase (TH)-immunopositive neurons in the substantia nigra pars compacta (SNpc). Either simultaneous or 12 h post-administration of serofendic acid significantly prevented both dopaminergic neurodegeneration and drug-induced rotational asymmetry. Immunoreactivities for oxidative stress markers, such as 3-nitrotyrosine (3-NT) and 4-hydroxy-2-nonenal (4-HNE), were markedly detected in the SNpc of rats injected with 6-OHDA alone. These immunoreactivities were markedly suppressed by the co-administration of serofendic acid, similar to the results in vehicle-treated control rats. In addition, serofendic acid inhibited 6-OHDA-induced alpha-synuclein expression and glial activation in the SNpc. These results suggest that serofendic acid protects against 6-OHDA-induced SNpc dopaminergic neurodegeneration in a rat model of Parkinson's disease.

Topics: Adrenergic Agents; Aldehydes; alpha-Synuclein; Animals; Behavior, Animal; Blotting, Western; CD11b Antigen; Cell Count; Cell Line; Disease Models, Animal; Diterpenes; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Interactions; Functional Laterality; Glial Fibrillary Acidic Protein; Humans; Immunohistochemistry; Male; Neurodegenerative Diseases; Neuroprotective Agents; Oxidopamine; Parkinson Disease, Secondary; Parkinsonian Disorders; Rats; Rats, Wistar; Reactive Oxygen Species; Rotarod Performance Test; Rotation; Substantia Nigra; Synaptophysin; Time Factors; Tyrosine; Tyrosine 3-Monooxygenase

Reduced activity of the mitochondrial respiratory chain--particularly complex I--may be implicated in the etiology of both Parkinson's disease and progressive supranuclear palsy, although these neurodegenerative diseases differ substantially as to their distinctive pattern of neuronal cell loss and the predominance of cerebral alpha-synuclein or tau protein pathology. To determine experimentally whether chronic generalized complex I inhibition has an effect on the distribution of alpha-synuclein or tau, we infused rats systemically with the plant-derived isoflavonoid rotenone. Rotenone-treated rats with a pronounced metabolic impairment had reduced locomotor activity, dystonic limb posture and postural instability. They lost neurons in the substantia nigra and in the striatum. Spherical deposits of alpha-synuclein were observed in a few cells, but cells with abnormal cytoplasmic accumulations of tau immunoreactivity were significantly more numerous in the striatum of severely lesioned rats. Abnormally high levels of tau immunoreactivity were found in the cytoplasm of neurons, oligodendrocytes and astrocytes. Ultrastructurally, tau-immunoreactive material consisted of straight 15-nm filaments decorated by antibodies against phosphorylated tau. Many tau+ cell bodies also stained positive for thioflavin S, nitrotyrosine and ubiquitin. Some cells with abnormal tau immunoreactivity contained activated caspase 3. Our data suggest that chronic respiratory chain dysfunction might trigger a form of neurodegeneration in which accumulation of hyperphosphorylated tau protein predominates over deposits of alpha-synuclein.

Topics: alpha-Synuclein; Amyloid beta-Peptides; Analysis of Variance; Animals; Antineoplastic Combined Chemotherapy Protocols; Behavior, Animal; Benzothiazoles; Body Weight; Caspase 3; Caspases; Cell Death; Cerebral Cortex; Cytarabine; Diagnostic Imaging; Dopamine and cAMP-Regulated Phosphoprotein 32; Doxorubicin; Dystonia; Electron Transport Complex III; Enzyme Activation; Glial Fibrillary Acidic Protein; Immunohistochemistry; Locomotion; Male; Microscopy, Electron, Transmission; Mitochondria; Neurons; Phosphopyruvate Hydratase; Phosphorylation; Posture; Psychomotor Performance; Rats; Rats, Inbred Lew; Rotenone; tau Proteins; Tauopathies; Thiazoles; Time Factors; Tyrosine; Tyrosine 3-Monooxygenase; Ubiquitin; Uncoupling Agents

Many models of Parkinson's disease (PD) have succeeded in replicating dopaminergic neuron loss or alpha-synuclein aggregation but not the formation of classical Lewy bodies, the pathological hallmark of PD. Our cybrid model of sporadic PD was created by introducing the mitochondrial genes from PD patients into neuroblastoma cells that lack mitochondrial DNA. Previous studies using cybrids have shown that information encoded by mitochondrial DNA in patients contributes to many pathogenic features of sporadic PD. In this paper, we report the generation of fibrillar and vesicular inclusions in a long-term cybrid cell culture model that replicates the essential antigenic and structural features of Lewy bodies in PD brain without the need for exogenous protein expression or inhibition of mitochondrial or proteasomal function. The inclusions generated by PD cybrid cells stained with eosin, thioflavin S, and antibodies to alpha-synuclein, ubiquitin, parkin, synphilin-1, neurofilament, beta-tubulin, the proteasome, nitrotyrosine, and cytochrome c. Future studies of these cybrids will enable us to better understand how Lewy bodies form and what role they play in the pathogenesis of PD.

Topics: Aged; alpha-Synuclein; Blotting, Western; Carrier Proteins; Case-Control Studies; Cell Line; Cysteine Endopeptidases; Cytochromes c; DNA, Mitochondrial; Electron Transport Complex I; Female; Humans; Immunohistochemistry; Lewy Bodies; Male; Microscopy, Confocal; Microscopy, Electron; Middle Aged; Multienzyme Complexes; Nerve Tissue Proteins; Neuroblastoma; Neurofilament Proteins; Neurons; Parkinson Disease; Precipitin Tests; Proteasome Endopeptidase Complex; Staining and Labeling; Synucleins; Transgenes; Tubulin; Tyrosine; Ubiquitin; Ubiquitin-Protein Ligases

Oxidation of catecholamines is suggested to contribute to oxidative stress in Parkinson's disease. Nitric oxide (*NO) is able to oxidize cyclic compounds like ubiquinol; moreover, recent lines of evidence proposed a direct role of *NO and its by-product peroxynitrite in the pathophysiology of Parkinson's disease. The aim of this study was to analyze the potential reaction between 6-hydroxydopamine, a classic inducer of Parkinson's disease, and *NO. The results showed that *NO reacts with the deprotonated form of 6-hydroxydopamine at pH 7 and 37 degrees C with a second-order rate constant of 1.5 x 10(3) M(-1) x s(-1) as calculated by the rate of *NO decay measured with an amperometric sensor. Accordingly, the rates of formation of 6-hydroxy-dopamine quinone were dependent on *NO concentration. The coincubation of *NO and 6-hydroxydopamine with either bovine serum albumin or alpha-synuclein led to tyrosine nitration of the protein, in a concentration dependent-manner and sensitive to superoxide dismutase. These findings suggest the formation of peroxynitrite during the redox reactions following the interaction of 6-hydroxydopamine with *NO. The implications of this reaction for in vivo models are discussed in terms of the generation of reactive nitrogen and oxygen species within a propagation process that may play a significant role in neurodegenerative diseases.

Topics: alpha-Synuclein; Animals; Cattle; Nerve Tissue Proteins; Nitric Oxide; Oxidation-Reduction; Oxidopamine; Parkinson Disease; Parkinson Disease, Secondary; Peroxynitrous Acid; Serum Albumin, Bovine; Superoxide Dismutase; Synucleins; Tyrosine

The synucleinopathies are a group of neurodegenerative disorders characterized by the presence of alpha-synuclein inclusions in neurons (Lewy body diseases, LBD) or glial cells (multiple system atrophies, MSA). Recently, nitration of alpha-synuclein has been reported as the possible modification that induces its aggregation and deposition in these disorders. In this study we investigated the distribution and relationships of alpha-synuclein inclusions and 3-nitrotyrosine (3-NT), a marker of protein nitration through oxidative mechanisms, in brains diagnosed with LBD or MSA and control brains using double immunohistochemical techniques. In LBD cases, 3-NT colocalized with alpha-synuclein immunoreactivity in classic and cortical Lewy bodies and in dystrophic neurites in substantia nigra. However, most pale bodies and diffuse deposits in substantia nigra and Lewy neurites in hippocampus lack 3-NT immunoreactivity. A majority of cases showed diffuse cytoplasmic 3-NT staining in pyramidal cells of the CA2-3 regions of the hippocampus that was independent of alpha-synuclein deposits. All MSA cases showed 3-NT immunoreactivity in glial inclusions. 3-NT neuronal staining was restricted to pontine nuclei with three cases showing nuclear and one case cytoplasmic staining. There was no colocalization of 3-NT nuclear immunoreactivity with alpha-synuclein-immunopositive nuclear inclusions in pontine neurons. These data show that protein nitration in LBD and MSA cases has a widespread distribution and is not only associated with the alpha-synuclein deposits. The presence of alpha-synuclein-positive deposits lacking 3-NT immunoreactivity suggests that nitration is not a prerequisite for alpha-synuclein deposition.

Topics: Aged; alpha-Synuclein; Brain; Cell Nucleus; Female; Humans; Inclusion Bodies; Lewy Bodies; Lewy Body Disease; Male; Middle Aged; Nerve Tissue Proteins; Neurites; Neuroglia; Neurons; Nitrates; Oxidative Stress; Striatonigral Degeneration; Synucleins; Tyrosine

Mutations in alpha-synuclein (A30P and A53T) are involved in some cases of familial Parkinson's disease (FPD), but it is not known how they result in nigral cell death. We examined the effect of alpha-synuclein overexpression on the response of cells to various insults. Wild-type alpha-synuclein and alpha-synuclein mutations associated with FPD were overexpressed in NT-2/D1 and SK-N-MC cells. Overexpression of wild-type alpha-synuclein delayed cell death induced by serum withdrawal or H(2)O(2), but did not delay cell death induced by 1-methyl-4-phenylpyridinium ion (MPP(+)). By contrast, wild-type alpha-synuclein transfectants were sensitive to viability loss induced by staurosporine, lactacystin or 4-hydroxy-2-trans-nonenal (HNE). Decreases in glutathione (GSH) levels were attenuated by wild-type alpha-synuclein after serum deprivation, but were aggravated following lactacystin or staurosporine treatment. Mutant alpha-synucleins increased levels of 8-hydroxyguanine, protein carbonyls, lipid peroxidation and 3-nitrotyrosine, and markedly accelerated cell death in response to all the insults examined. The decrease in GSH levels was enhanced in mutant alpha-synuclein transfectants. The loss of viability induced by toxic insults was by apoptosic mechanism. The presence of abnormal alpha-synucleins in substantia nigra in PD may increase neuronal vulnerability to a range of toxic agents.

Topics: 1-Methyl-4-phenylpyridinium; Aldehydes; alpha-Synuclein; Cell Division; Cell Line; Cell Survival; Clone Cells; Culture Media, Serum-Free; Enzyme Inhibitors; Gene Expression; Glutathione; Guanine; Humans; Hydrogen Peroxide; Ketones; Lipid Peroxidation; Mitochondria; Mutation; Nerve Tissue Proteins; Neuroblastoma; Oxidants; Oxidative Stress; Parkinsonian Disorders; Synucleins; Teratocarcinoma; Transfection; Tyrosine

Aggregated alpha-synuclein proteins form brain lesions that are hallmarks of neurodegenerative synucleinopathies, and oxidative stress has been implicated in the pathogenesis of some of these disorders. Using antibodies to specific nitrated tyrosine residues in alpha-synuclein, we demonstrate extensive and widespread accumulations of nitrated alpha-synuclein in the signature inclusions of Parkinson's disease, dementia with Lewy bodies, the Lewy body variant of Alzheimer's disease, and multiple system atrophy brains. We also show that nitrated alpha-synuclein is present in the major filamentous building blocks of these inclusions, as well as in the insoluble fractions of affected brain regions of synucleinopathies. The selective and specific nitration of alpha-synuclein in these disorders provides evidence to directly link oxidative and nitrative damage to the onset and progression of neurodegenerative synucleinopathies.

Topics: alpha-Synuclein; Alzheimer Disease; Antibodies, Monoclonal; Blotting, Western; Brain; Brain Chemistry; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Humans; Immunohistochemistry; Lewy Bodies; Lewy Body Disease; Microscopy, Immunoelectron; Multiple System Atrophy; Nerve Tissue Proteins; Neurodegenerative Diseases; Neurons; Oxidative Stress; Parkinson Disease; Synucleins; Tyrosine

Reactive nitrogen species may play a mechanistic role in neurodegenerative diseases by posttranslationally altering normal brain proteins. In support of this hypothesis, we demonstrate that an anti-3-nitrotyrosine polyclonal antibody stains all of the major hallmark lesions of synucleinopathies including Lewy bodies, Lewy neurites and neuraxonal spheroids in dementia with Lewy bodies, the Lewy body variant of Alzheimer's disease, and neurodegeneration with brain iron accumulation type 1, as well as glial and neuronal cytoplasmic inclusions in multiple system atrophy. This antibody predominantly recognized nitrated alpha-synuclein when compared to other in vitro nitrated constituents of these pathological lesions, such as neurofilament subunits and microtubules. Collectively, these findings imply that alpha-synuclein is nitrated in pathological lesions. The widespread presence of nitrated alpha-synuclein in diverse intracellular inclusions suggests that oxidation/nitration is involved in the onset and/or progression of neurodegenerative diseases.

Topics: Adult; Aged; Aged, 80 and over; alpha-Synuclein; Antibodies; Blotting, Western; Brain Diseases; Female; Humans; Immunohistochemistry; Inclusion Bodies; Lewy Bodies; Male; Middle Aged; Nerve Tissue Proteins; Neurodegenerative Diseases; Neuroglia; Nitrates; Synucleins; Tissue Distribution; Tyrosine