3-nitrotyrosine and Parkinson-Disease

Reactive oxygen species are generated as a by-product of routine biochemical reactions. However, dysfunction of the antioxidant system or mutations in gene function may result in the elevated production of the pro-oxidant species. Modified endogenous molecules due to chemical interactions with increased levels of reactive oxygen and nitrogen species in the cellular microenvironment can be termed as biomarkers of oxidative stress. 3-Nitrotyrosine is one such promising biomarker of oxidative stress formed due to nitration of protein-bound and free tyrosine residues by reactive peroxynitrite molecules. Nitration of proteins at the subcellular level results in conformational alterations that damage the cytoskeleton and result in neurodegeneration. In this review, we summarized the role of oxidative/nitrosative processes as a contributing factor for progressive neurodegeneration in Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease and Prion disease. The selective tyrosine protein nitration of the major marker proteins in related pathologies has been discussed. The alteration in 3-Nitrotyrosine profile occurs well before any symptoms appear and can be considered as a potential target for early diagnosis of neurodegenerative diseases. Furthermore, the reduction in 3-Nitrotyrosine levels in response to treatment with neuroprotective has been highlighted which is indicative of the importance of this particular marker in oxidative stress-related brain and central nervous system pathologies.

Topics: Alzheimer Disease; Amyotrophic Lateral Sclerosis; Biomarkers; Humans; Huntington Disease; Parkinson Disease; Prion Diseases; Tyrosine

Parkinson's disease (PD) and Parkinsonian syndromes; Progressive supranuclear palsy (PSP), and Multiple system atrophy (MSA) are debilitating neurodegenerative disorders. Fractalkine is a chemokine involved in neuroinflammation, whereas, 3-nitrotyrosine (3-NT) is a marker of early neurodegenerative cellular-damage. We measured Fractalkine and 3-NT levels in the serum of these patients to examine the neuroinflammation hypothesis and also to decipher the propensity of these biologics to be used as early (5 years from onset) biochemical markers in neurodegenerative Parkinsonism. The diagnoses of PD, PSP and MSA were performed as per the respective clinical criteria. 21 PD, 9 PSP and 8 MSA patients along with controls participated in this study. Serum concentrations of Fractalkine and 3-NT were measured by ELISA. Fractalkine levels were increased in PD, PSP and MSA cohorts in comparison with controls with p < 0.001, p < 0.05 and p < 0.05 respectively. Levels of 3-NT also showed elevation in PD (p < 0.01) vs. controls. However, Pearson plot showed that Fractalkine levels were high in the patients with unified Parkinson's disease rating scale (UPDRS) part III motor score of 1, meaning slight disability, but gradually dropped in patients with motor score of 4, which is a measure of severe motor disability. This negative correlation (- .565, p < .0.01) also accentuates the neuroprotectant/anti-inflammatory nature of Fractalkine in PD. Continuous rise of 3-NT in PD, positively correlating (.512, p < 0.05) with worsening motor symptoms points to deleterious consequences of nitrosative stress. To our knowledge, this is the first report providing evidence that serum Fractalkine and 3-NT have early diagnostic/prognostic significance as PD biomarkers.

Topics: Chemokine CX3CL1; Disabled Persons; Humans; Motor Disorders; Parkinson Disease; Pilot Projects; Severity of Illness Index; 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

Nitrative stress is a key component of the pathogenic process in Parkinson's disease (PD), but the relative roles of constitutive neuronal nitric oxide synthase (n-NOS) and inducible nitric oxide synthase (i-NOS) in glial cells remain unresolved. We have investigated the effects of a range of concentrations of the selective n-NOS inhibitor ARR17477, and the selective i-NOS inhibitor 1400W, on MPP(+)-induced cell death in foetal ventral mesencephalic (VM) dopaminergic cultures. MPP(+) induced a loss of TH-positive neurones accompanied by an increase in immunoreactivity for GFAP and OX-6 as markers of astrocytes and activated microglia, respectively, and induced i-NOS immunoreactivity. Unexpectedly, MPP(+) treatment did not induce 3-NT immunoreactivity in the cultures. ARR17477 and 1400W alone had no effect on the number of TH-positive cells or on the number of GFAP or OX-6 positive cells. ARR17477 did not prevent the MPP(+)-induced decrease in TH-positive neurones and had no effect on the increased number of GFAP- and OX-6-positive cells. By contrast, 1400W caused a concentration-dependent preservation of TH-positive neurones in the presence of MPP(+). It also significantly reduced the number of OX-6-immunoreactive cells and there was a small reduction in GFAP immunoreactivity. The results suggest a major role for i-NOS-mediated nitrative stress in microglia in MPP(+)-induced dopaminergic cell death and this may have important implications for developing neuroprotective strategies for PD.

Topics: Amidines; Animals; Antibodies, Monoclonal; Astrocytes; Benzylamines; Cell Count; Cells, Cultured; Dopaminergic Neurons; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glial Fibrillary Acidic Protein; Mesencephalon; Microglia; Neuroprotective Agents; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Parkinson Disease; Picolines; Primary Cell Culture; Rats, Wistar; Thiophenes; Tyrosine; Tyrosine 3-Monooxygenase

Numerous factors contribute to the death of substantia nigra (SN) dopamine (DA) neurons in Parkinson's disease (PD). Compelling evidence implicates mitochondrial deficiency, oxidative stress, and inflammation as important pathogenic factors in PD. Chronic exposure of rats to rotenone causes a PD-like syndrome, in part by causing oxidative damage and inflammation in substantia nigra. Pomegranate juice (PJ) has the greatest composite antioxidant potency index among beverages, and it has been demonstrated to have protective effects in a transgenic model of Alzheimer's disease. The present study was designed to examine the potential neuroprotective effects of PJ in the rotenone model of PD. Oral administration of PJ did not mitigate or prevent experimental PD but instead increased nigrostriatal terminal depletion, DA neuron loss, the inflammatory response, and caspase activation, thereby heightening neurodegeneration. The mechanisms underlying this effect are uncertain, but the finding that PJ per se enhanced nitrotyrosine, inducible nitric oxide synthase, and activated caspase-3 expression in nigral DA neurons is consistent with its potential pro-oxidant activity.

Topics: Animals; Caspase 3; Disease Models, Animal; Dopaminergic Neurons; Inflammation; Lythraceae; Male; Mitochondrial Diseases; Nitric Oxide Synthase Type II; Oxidative Stress; Parkinson Disease; Rats; Rats, Inbred Lew; Rotenone; Substantia Nigra; Tyrosine

The research group has detected nitrosative stress and a singular version of nitrosylated serum α-synuclein in serum of Parkinson's disease (PD) patients. Dysfunction of the thyroid gland has been proposed to be linked to this disease. The aim of the study was to know if the thyroid gland is involved in idiopathic PD and nitrosative stress. We studied 50 patients (early and advanced disease patients), 35 controls, and 6 subjects with thyroidectomy. Clinical characteristics, serum thyroperoxidase levels, and 3-nitrotyrosine proteins were analyzed. Enzyme-linked immunosorbent assay and immunoblotting methods were employed. The findings indicated that the prevalence of two thyroid dysfunctions (hyper- or hypothyroidism) was not found to be different in patients relative to controls. However, the levels of the enzyme thyroperoxidase were found to be elevated in early disease patients (p<0.006), not in advanced disease subjects, and these levels were negatively correlated with serum 3-nitrotyrosine proteins (p<0.05), the indicators of nitrosative stress. The thyroidectomized subjects showed very low levels of serum 3-nitrotyrosine proteins (78% reduction vs. controls) and, among these proteins, the nitrosylated serum α-synuclein was nearly absent. These observations lead to the hypothesis that the thyroid gland and thyroperoxidase participate in nitrosylation of serum proteins and they could influence Parkinsonian nitrosative stress as well as nitrosylation of serum α-synuclein, a potentially pathogenic factor.

Topics: Aged; Case-Control Studies; Female; Humans; Iodide Peroxidase; Male; Middle Aged; Parkinson Disease; Protein Processing, Post-Translational; Thyroid Gland; 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

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

Oxidative/nitrosative stress plays a crucial role in Parkinson's disease (PD) by triggering mitochondrial dysfunction. Nitrosative stress is mediated by reactive species such as peroxynitrite (PN) which could damage biomolecules thereby impinging on the cellular machinery. We observed that PN (0-1000 μM) inhibited brain mitochondrial complex I (CI) activity in a dose-dependent manner with concomitant tyrosine nitration of proteins. We also observed that exposure to PN at low concentrations (62.5-125 μM) significantly decreased the mitochondrial membrane potential and affected the mitochondrial integrity at higher doses (500-750 μM) as indicated by the mitochondrial swelling experiment. Therefore, it could be surmised that compounds that prevent such mitochondrial damage might have therapeutic value in neurological conditions such as PD. We previously showed that curcumin could detoxify PN and protect against CI inhibition and protein nitration. However, the therapeutic potential of curcumin is constrained by limited bioavailability. To address this issue and obtain improved antioxidants, three bioconjugates of curcumin (Di-demethylenated piperoyl, di-valinoyl and di-glutamoyl esters) were generated and tested against PN-mediated nitrosative stress and mitochondrial damage. We found that among the bioconjugates, the glutamoyl diester of curcumin showed improved protection against PN-dependent CI inhibition and protein nitration compared to other conjugates. Di-glutamoyl curcumin protected dopaminergic neurons against 1-methyl-4-phenylpyridinium (MPP(+))-mediated neuronal death. These effects were improved compared to curcumin alone suggesting that di-glutamoyl curcumin could be a better neuroprotective agent in neurodegenerative diseases such as PD.

Topics: 1-Methyl-4-phenylpyridinium; Animals; Brain; Curcumin; Diet; Electron Transport Complex I; Esters; Flavonoids; Glutamates; Mice; Mitochondria; Mitochondrial Proteins; Mitochondrial Swelling; Parkinson Disease; Peroxynitrous Acid; Phenols; Polyphenols; Protective Agents; 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

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

Current evidence suggests a role of neuroinflammation in the pathogenesis of Parkinson's disease (PD) and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of basal ganglia injury. Reportedly, nonsteroidal anti-inflammatory drugs (NSAIDs) mitigate DAergic neurotoxicity in rodent models of PD. Consistent with these findings, epidemiological analysis indicated that certain NSAIDs may prevent or delay the progression of PD. However, a serious impediment of chronic NSAID therapy, particularly in the elderly, is gastric, renal and cardiac toxicity. Nitric oxide (NO)-donating NSAIDs, have a safer profile while maintaining anti-inflammatory activity of parent compounds. We have investigated the oral activity of the NO-donating derivative of flurbiprofen, [2-fluoro-α-methyl (1,1'-biphenyl)-4-acetic-4-(nitrooxy)butyl ester], HCT1026 (30 mg kg(-1) daily in rodent chow) in mice exposed to the parkinsonian neurotoxin MPTP.. Ageing mice were fed with a control, flurbiprofen, or HCT1026 diet starting ten days before MPTP administration and continuing for all the experimental period. Striatal high affinity synaptosomal dopamine up-take, motor coordination assessed with the rotarod, tyrosine hydroxylase (TH)- and dopamine transporter (DAT) fiber staining, stereological cell counts, immunoblotting and gene expression analyses were used to assess MPTP-induced nigrostriatal DAergic toxicity and glial activation 1-40 days post-MPTP.. HCT1026 was well tolerated and did not cause any measurable toxic effect, whereas flurbiprofen fed mice showed severe gastrointestinal side-effects. HCT1026 efficiently counteracted motor impairment and reversed MPTP-induced decreased synaptosomal [3H]dopamine uptake, TH- and DAT-stained fibers in striatum and TH+ neuron loss in substantia nigra pars compacta (SNpc), as opposed to age-matched mice fed with a control diet. These effects were associated to a significant decrease in reactive macrophage antigen-1 (Mac-1)-positive microglial cells within the striatum and ventral midbrain, decreased expression of iNOS, Mac-1 and NADPH oxidase (PHOX), and downregulation of 3-Nitrotyrosine, a peroxynitrite finger print, in SNpc DAergic neurons.. Oral treatment with HCT1026 has a safe profile and a significant efficacy in counteracting MPTP-induced dopaminergic (DAergic) neurotoxicity, motor impairment and microglia activation in ageing mice. HCT1026 provides a novel promising approach towards the development of effective pharmacological neuroprotective strategies against PD.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Anti-Inflammatory Agents, Non-Steroidal; Corpus Striatum; Disease Models, Animal; Dopamine; Dopamine Agents; Flurbiprofen; Humans; Inflammation; Macrophage-1 Antigen; Male; Mice; Mice, Inbred C57BL; Motor Activity; MPTP Poisoning; Nitric Oxide; Nitric Oxide Synthase Type II; Oxidoreductases; Parkinson Disease; Rotarod Performance Test; Substantia Nigra; Tyrosine

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

Protein nitration due to oxidative and nitrative stress has been linked to the pathogenesis of Parkinson's disease (PD), but its relationship to the loss of dopamine (DA) or tyrosine hydroxylase (TH) activity is not clear. Here we quantified protein-bound 3-nitrotyrosine (3-NT) by a novel gas chromatography/negative chemical ionization tandem mass spectrometry technique and DA and 3,4-dihydroxyphenylalanine (DOPA) by HPLC in tissues or medium of organotypic, mouse mesencephalon cultures after acute or chronic treatments with the peroxynitrite donor 3-morpholino-sydnonimine (SIN-1), the dopaminergic toxin 1-methyl-4-phenylpyridinium (MPP(+)) or the lipophilic complex I inhibitor rotenone. Incubation with SIN-1 (24 h) or MPP(+) treatments (48 h) caused dose-dependent protein nitration reaching a maximum of eightfold increase by 10 mM SIN-1 or twofold by 10 microM MPP(+), but significant DA depletions occurred at much lower concentrations of MPP(+) (1 microM). Chronic MPP(+) or rotenone treatments (3 weeks) caused maximum protein nitration by 1 microM (twofold) or 10nM (fourfold), respectively. Co-treatment with the nitric oxide synthase inhibitor l-NAME (300 microM) prevented protein nitration by MPP(+), but did not protect against MPP(+)-induced DA depletion or inhibition of TH activity. Acute incubation with 100 microM SIN-1 inhibited TH activity, which could be blocked by co-treatment with the tetrahydrobiopterin precursor l-sepiapterin, but tissue DA depletions required higher doses of SIN-1 (>1 mM, 24 h) and longer survival. In conclusion, protein nitration and TH activity or DA depletion are not directly related in these models.

Topics: 1-Methyl-4-phenylpyridinium; Animals; Cell Survival; Dopamine; Dose-Response Relationship, Drug; Enzyme Inhibitors; Mice; Mice, Inbred C57BL; Neurochemistry; Neurotoxins; Nitric Oxide Donors; Nitric Oxide Synthase; Nitro Compounds; Organ Culture Techniques; Oxidative Stress; Parkinson Disease; Proteins; Pterins; Reactive Nitrogen Species; Substantia Nigra; Tyrosine; Tyrosine 3-Monooxygenase; Uncoupling Agents

Long-term or high-dose L-DOPA therapy in patients with Parkinson's disease (PD) may accelerate degeneration of dopaminergic neurons, possibly by increasing oxidative stress. To investigate the effects of cabergoline on peroxynitrite-mediated oxidative damage caused by L-DOPA, the concentration of 3-nitrotyrosine in cerebrospinal fluid (CSF) of 18 PD patients was compared with that in 20 normal controls. The concentration of 3-nitrotyrosine in patients following L-DOPA therapy was significantly higher than in untreated PD patients and controls. On the other hand, the concentration in PD patients after cabergoline therapy was significantly lower than in PD patients after L-DOPA therapy alone. These data suggest that cabergoline scavenges peroxynitrite induced by L-DOPA in patients with PD.

Topics: Aged; Antiparkinson Agents; Cabergoline; Chromatography, High Pressure Liquid; Ergolines; Female; Free Radical Scavengers; Humans; Levodopa; Male; Oxidative Stress; Parkinson Disease; Peroxynitrous Acid; Tyrosine

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

Green tea polyphenols (GTP) are thought to help prevent oxidative stress-related diseases, such as cancer, cardiovascular disease, neurodegenerative disease, and aging. We here investigate the protective mechanisms of GTP on SH-SY5Y cells against apoptosis induced by the pro-parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA). GTP rescued the changes in condensed nuclear and apoptotic bodies, attenuated 6-OHDA-induced early apoptosis, prevented the decrease in mitochondrial membrane potential, and suppressed accumulation of reactive oxygen species (ROS) and of intracellular free Ca(2+). GTP also counteracted the 6-OHDA-induced nitric oxide increase and overexpression of nNOS and iNOS, and decreased the level of protein-bound 3-nitrotyrosine (3-NT). In addition, GTP inhibited the autooxidation of 6-OHDA and scavenged oxygen free radicals in a dose- and time-dependent manner. Our results show that the protective effects of GTP on SH-SY5Y cells are mediated, at least in part, by controlling the ROS-NO pathway.

Topics: Annexin A5; Apoptosis; Blotting, Western; Calcium; Cell Line, Tumor; Cell Survival; Coloring Agents; Dose-Response Relationship, Drug; Flavonoids; Flow Cytometry; Free Radicals; Guanosine Triphosphate; Humans; Membrane Potentials; Mitochondria; Models, Biological; Neurons; Nitric Oxide; Oxidopamine; Oxygen; Parkinson Disease; Phenols; Polyphenols; Quinones; Reactive Oxygen Species; Tea; Tetrazolium Salts; Thiazoles; Time Factors; Tyrosine

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

Peroxynitrite can nitrate tyrosine residues of proteins. We examined nitrotyrosine-containing proteins in cerebrospinal fluid of 66 patients with neurogenic disease by immunoblot analysis. Nitrated tyrosine residue-containing protein was observed in the cerebrospinal fluid and was concluded to be manganese superoxide dismutase (Mn-SOD). The nitrated Mn-SOD level was strikingly elevated in amyotrophic lateral sclerosis patients and was slightly increased in Alzheimer's and Parkinson's disease patients, whereas an elevated Mn-SOD level was observed only in progressive supranuclear palsy group.

Topics: Adult; Aged; Alzheimer Disease; Antibodies; Biomarkers; Female; Humans; Male; Middle Aged; Nitrates; Nitrogen; Oxidative Stress; Parkinson Disease; Precipitin Tests; Superoxide Dismutase; Tyrosine

Much evidence supports a role of nitric oxide (.NO) and peroxynitrite (ONOO(-)) in experimental and idiopathic Parkinson's disease (PD); moreover, an overexpression of neuronal nitric oxide synthase (nNOS) was recently reported in the basal ganglia of PD patients. In accord, we previously found a 50% increased.NO production rate during the respiratory burst of circulating neutrophils (PMN) from PD patients. As PMN express the nNOS isoform, the objective of the present study was to ascertain whether this increased.NO production is representative of nNOS gene upregulation. PMN were isolated from blood samples obtained from seven PD patients and seven age- and sex-matched healthy donors; nNOS mRNA was amplified by reverse transcriptase-polymerase chain reaction and the products were hybridized with a probe for nNOS. Nitrotyrosine-containing proteins and nNOS were detected by Western blot and NO production rate was measured spectrophotometrically by the conversion of oxymyoglobin to metmyoglobin. The results showed that both.NO production and protein tyrosine nitration were significantly increased in PMN isolated from PD patients (PD 0.09 +/- 0.01 vs 0.06 +/- 0.008 nmol min(-1) 10(6) cells(-1); P < 0.05). In addition, five of the seven PD patients showed about 10-fold nNOS mRNA overexpression; while two of the seven PD patients showed an expression level similar to that of the controls; detection of nNOS protein was more evident in the former group. In summary, it is likely that overexpression of nNOS and formation of ONOO(-) in PMN cells from PD patients emphasizes a potential causal role of.NO in the physiopathology of the illness.

Topics: Blotting, Western; Enzyme Induction; Female; Humans; Hydrogen Peroxide; Male; Middle Aged; Neutrophils; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Parkinson Disease; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spectrophotometry; 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

Oxidative stress is implicated in the death of dopaminergic neurons in Parkinson's disease and in the 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) model of Parkinson's disease. Oxidative species that might mediate this damage include hydroxyl radical, tyrosyl radical, or reactive nitrogen species such as peroxynitrite. In mice, we showed that MPTP markedly increased levels of o, o'-dityrosine and 3-nitrotyrosine in the striatum and midbrain but not in brain regions resistant to MPTP. These two stable compounds indicate that tyrosyl radical and reactive nitrogen species have attacked tyrosine residues. In contrast, MPTP failed to alter levels of ortho-tyrosine in any brain region we studied. This marker accumulates when hydroxyl radical oxidizes protein-bound phenylalanine residues. We also showed that treating whole-brain proteins with hydroxyl radical markedly increased levels of ortho-tyrosine in vitro. Under identical conditions, tyrosyl radical, produced by the heme protein myeloperoxidase, selectively increased levels of o,o'-dityrosine, whereas peroxynitrite increased levels of 3-nitrotyrosine and, to a lesser extent, of ortho-tyrosine. These in vivo and in vitro findings implicate reactive nitrogen species and tyrosyl radical in MPTP neurotoxicity but argue against a deleterious role for hydroxyl radical in this model. They also show that reactive nitrogen species and tyrosyl radical (and consequently protein oxidation) represent an early and previously unidentified biochemical event in MPTP-induced brain injury. This finding may be significant for understanding the pathogenesis of Parkinson's disease and developing neuroprotective therapies.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Amino Acids; Animals; Brain; Chelating Agents; Dopamine Agents; Free Radicals; Gas Chromatography-Mass Spectrometry; Male; Mice; Mice, Inbred C57BL; Nitrates; Oxidation-Reduction; Oxidative Stress; Parkinson Disease; Pentetic Acid; Tissue Distribution; Tyrosine

Oxidative stress has been proposed as a pathogenetic mechanism in Parkinson's disease (PD). One mechanism of oxidative cellular injury is the nitration of protein tyrosine residues, mediated by peroxynitrite, a reaction product of nitric oxide and superoxide radicals. We demonstrate here the presence of nitrotyrosine immunoreactivity in Lewy bodies within melanized neurons and in amorphous deposits associated with intact and degenerating neurons. The core of the Lewy body was frequently intensely immunolabeled, while the rim was lightly labeled or unlabeled. This likely reflects the fact that tyrosine residues of neurofilament proteins are primarily localized to Lewy body cores, and suggests that nitrotyrosine is present in neurofilament protein itself. Although these observations are as yet unable to provide a definitive link between oxidative stress and neuronal dysfunction, they demonstrate that oxidative stress has occurred within the vulnerable neurons of PD, leaving a permanent marker of oxidative modification of neuronal proteins within the target cells of neurodegeneration. In addition, these observations provide a potential link between excitotoxicity and oxidative stress within the vulnerable neurons of PD and represent a pathogenetic mechanism in common with the 2 other major age-related neurodegenerative diseases, Alzheimer disease and amyotrophic lateral sclerosis.

Topics: Adult; Aged; Aged, 80 and over; Humans; Immunohistochemistry; Lewy Bodies; Middle Aged; Neurons; Oxidative Stress; Parkinson Disease; Substantia Nigra; Tyrosine