p38/p53-Mediated Bax induction contributes to neurons degeneration in rotenone-induced cellular and rat models of Parkinson’s disease

Rotenone is an environmental neurotoxin that induces degeneration of dopaminergic (DA) neurons in substantia nigra pars compacta (SNpc), which ultimately results in parkinsonism, but the molecular mechanisms of selective degeneration of nigral DA neurons are not fully understood. In the present study, we investigated the induction of p38^MAPK/p53 and Bax in SNpc of Lewis rats after chronic treatment with rotenone and the contribution of Bax to rotenone-induced apoptotic commitment of differentiated PC12 cells. Lewis rats were subcutaneously treated with rotenone (1.5 mg/kg) twice a day for 50 days and the loss of tyrosine hydroxylase (THase), motor function impairment, and expression of p38^MAPK, P-p38^MAPK, p53, and Bax were assessed. After differentiated PC cells were treated with rotenone (500 nM) for 6–36 h, protein levels of p38^MAPK and P-p38^MAPK, p53 nuclear translocation, Bax induction and cell death were measured. The results showed that rotenone administration significantly reduced motor activity and caused a loss of THase immunoreactivity in SNpc of Lewis rats. The degeneration of nigral DA neurons was accompanied by the increases in p38^MAPK, P-p38^MAPK, p53, and Bax protein levels. In cultured PC12 cells, rotenone also induced an upregulation of p38^MAPK, P-p38^MAPK, p53 and Bax. Pharmacological inhibition of p38^MAPK with SB203580 (25 μM) blunted rotenone-induced cell apoptosis. Treatment with SB203580 prevented the p53 nuclear translocation and upregulation of Bax. Inhibition of p53 with pifthrin-alpha or Bax with siRNAs significantly reduced rotenone-induced Bax induction and apoptotic cell death. These results suggest that the p38^MAPK/p53-dependent induction of Bax contributes to rotenone’s neurotoxicity in PD models.     

In vivo real-time measurement of superoxide anion radical with a novel electrochemical sensor

The dynamics of superoxide anion (O₂⁻) in vivo remain to be clarified because no appropriate method exists to directly and continuously monitor and evaluate O₂⁻ in vivo. Here, we establish an in vivo method using a novel electrochemical O₂⁻ sensor. O₂⁻ generated is measured as a current and evaluated as a quantified partial value of electricity (Q_part), which is calculated by integration of the difference between the baseline and the actual reacted current. The accuracy and efficacy of this method were confirmed by dose-dependent O₂⁻ generation in xanthine–xanthine oxidase in vitro in phosphate-buffered saline and human blood. It was then applied to endotoxemic rats in vivo. O₂⁻ current began to increase 1 h after lipopolysaccharide, and Q_part increased significantly for 6 h in endotoxemic rats, in comparison to sham-treated rats. These values were attenuated by superoxide dismutase. The generation and attenuation of O₂⁻ were indirectly confirmed by plasma lipid peroxidation with malondialdehyde, endothelial injury with soluble intercellular adhesion molecule-1, and microcirculatory dysfunction. This is a novel method for measuring O₂⁻ in vivo and could be used to monitor and treat the pathophysiology caused by excessive O₂⁻ generation in animals and humans.     

Nitrated α-Synuclein Induces the Loss of Dopaminergic Neurons in the Substantia Nigra of Rats

Background

The pathology of Parkinson''s disease (PD) is characterized by the degeneration of the nigrostriatal dopaminergic pathway, as well as the formation of intraneuronal inclusions known as Lewy bodies and Lewy neurites in the substantia nigra. Accumulations of nitrated α-synuclein are demonstrated in the signature inclusions of Parkinson''s disease. However, whether the nitration of α-synuclein is relevant to the pathogenesis of PD is unknown.

Methodology/Principal Findings

In this study, effect of nitrated α-synuclein to dopaminergic (DA) neurons was determined by delivering nitrated recombinant TAT-α-synuclein intracellular. We provide evidence to show that the nitrated α-synuclein was toxic to cultured dopaminergic SHSY-5Y neurons and primary mesencephalic DA neurons to a much greater degree than unnitrated α-synuclein. Moreover, we show that administration of nitrated α-synuclein to the substantia nigra pars compacta of rats caused severe reductions in the number of DA neurons therein, and led to the down-regulation of D₂R in the striatum in vivo. Furthermore, when administered to the substantia nigra of rats, nitrated α-synuclein caused PD-like motor dysfunctions, such as reduced locomotion and motor asymmetry, however unmodified α-synuclein had significantly less severe behavioral effects.

Conclusions/Significance

Our results provide evidence that α-synuclein, principally in its nitrated form, induce DA neuron death and may be a major factor in the etiology of PD.     

Mechanisms of DJ-1 neuroprotection in a cellular model of Parkinson's disease

Mitochondrial dysfunction, proteasome inhibition, and α-synuclein aggregation are thought to play important roles in the pathogenesis of Parkinson's disease (PD). Rare cases of early-onset PD have been linked to mutations in the gene encoding DJ-1, a protein with antioxidant and chaperone functions. In this study, we examined whether DJ-1 protects against various stresses involved in PD, and we investigated the underlying mechanisms. Expression of wild-type DJ-1 rescued primary dopaminergic neurons from toxicity elicited by rotenone, proteasome inhibitors, and mutant α-synuclein. Neurons with reduced levels of endogenous DJ-1 were sensitized to each of these insults, and DJ-1 mutants involved in familial PD exhibited decreased neuroprotective activity. DJ-1 alleviated rotenone toxicity by up-regulating total intracellular glutathione. In contrast, inhibition of α-synuclein toxicity by DJ-1 correlated with up-regulation of the stress-inducible form of Hsp70. RNA interference studies revealed that this increase in Hsp70 levels was necessary for DJ-1-mediated suppression of α-synuclein aggregation, but not toxicity. Our findings suggest that DJ-1 acts as a versatile pro-survival factor in dopaminergic neurons, activating different protective mechanisms in response to a diverse range of PD-related insults.     

DJ-1 Protects Dopaminergic Neurons against Rotenone-Induced Apoptosis by Enhancing ERK-Dependent Mitophagy

Loss-of-function mutations in the gene encoding the multifunctional protein, DJ-1, have been implicated in the pathogenesis of early-onset familial Parkinson's disease (PD), suggesting that DJ-1 may act as a neuroprotectant for dopaminergic (DA) neurons. Enhanced autophagy may benefit PD by clearing damaged organelles and protein aggregates; thus, we determined if DJ-1 protects DA neurons against mitochondrial dysfunction and oxidative stress through an autophagic pathway. Cultured DA cells (MN9D) overexpressing DJ-1 were treated with the mitochondrial complex I inhibitor, rotenone. In addition, rotenone was injected into the left substantia nigra of rats 4 weeks after injection with a DJ-1 expression vector. Overexpression of DJ-1 protected MN9D cells against apoptosis, significantly enhanced the survival of nigral DA neurons after rotenone treatment in vivo, and rescued rat behavioral abnormalities. Overexpression of DJ-1 enhanced rotenone-evoked expression of the autophagic markers, beclin-1 and LC3II, while transmission electron microscopy and confocal imaging revealed that the ultrastructural signs of autophagy were increased by DJ-1. The neuroprotective effects of DJ-1 were blocked by phosphoinositol 3‐kinase and the autophagy inhibitor, 3-methyladenine, and by the ERK pathway inhibitor, U0126. Confocal imaging revealed that the size of p62-positive puncta decreased significantly in DJ-1 overexpression of MN9D cells 12 h after rotenone treatment, suggesting that DJ-1 reveals the ability to clear aggregated p62 associated with PD. Factors that control autophagy, including DJ-1, may inhibit rotenone-induced apoptosis and present novel targets for therapeutic intervention in PD.     

Production of superoxides and nitric oxide generation in haemocytes of mussel Mytilus galloprovincialis (Lmk.) after exposure to cadmium: A possible involvement of Na/H exchanger in the induction of cadmium toxic effects

The present study investigates cadmium (Cd) ability to enhance superoxides (O₂⁻) and nitric oxide (NO) production (as nitrites) in haemocytes of mussel Mytilus galloprovincialis as well as the possible involvement of Na⁺/H⁺ exchanger (NHE) in the induction of NADPH oxidase and NO synthase activity. PMA, a well-known PKC-mediated NADPH oxidase as well as NO synthase stimulator was also used, in order to verify Cd effects on both O₂⁻ and NO generation. According to the results of the present study, micromolar concentrations of Cd (0.05, 5, 10 and 50 μM) seemed to enhance O₂⁻ and NO generation in haemocytes of mussels. Moreover, O₂⁻ and NO generation in haemocytes exposed to Cd could be enhanced by its ability to induce reactive oxygen species (ROS) but respiratory burst activation as well. Inhibition of NO synthase with 10 μM l-NAME, significantly attenuated Cd ability to enhance O₂⁻ production and diminished NO generation, thus leading to the suggestion that Cd toxic effects, started at concentration of 50 μM, could enhance NADPH oxidase and NO synthase stimulation in haemocytes of mussels. NHE seems to play a regulatory role in the induction of either O₂⁻ or NO generation in haemocytes exposed to the metal, since its inhibition with the use of 10 μM EIPA significantly decrease both O₂⁻ and NO production. The involvement of NHE in the induction of O₂⁻ and NO generation, probably via PKC-mediated NADPH oxidase and NO synthase activation, is likely to be crucial to haemocytes exposed to heavy metals, such as Cd.     

DJ-1 inhibits microglial activation and protects dopaminergic neurons in vitro and in vivo through interacting with microglial p65

Parkinson’s disease (PD), one of the most common neurodegenerative disorders, is characterized by progressive neurodegeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). DJ-1 acts essential roles in neuronal protection and anti-neuroinflammatory response, and its loss of function is tightly associated with a familial recessive form of PD. However, the molecular mechanism of DJ-1 involved in neuroinflammation is largely unclear. Here, we found that wild-type DJ-1, rather than the pathogenic L166P mutant DJ-1, directly binds to the subunit p65 of nuclear factor-κB (NF-κB) in the cytoplasm, and loss of DJ-1 promotes p65 nuclear translocation by facilitating the dissociation between p65 and NF-κB inhibitor α (IκBα). DJ-1 knockout (DJ-1^−/−) mice exhibit more microglial activation compared with wild-type littermate controls, especially in response to lipopolysaccharide (LPS) treatment. In cellular models, knockdown of DJ-1 significantly upregulates the gene expression and increases the release of LPS-treated inflammatory cytokines in primary microglia and BV2 cells. Furthermore, DJ-1 deficiency in microglia significantly enhances the neuronal toxicity in response to LPS stimulus. In addition, pharmacological blockage of NF-κB nuclear translocation by SN-50 prevents microglial activation and alleviates the damage of DA neurons induced by microglial DJ-1 deficiency in vivo and in vitro. Thus, our data illustrate a novel mechanism by which DJ-1 facilitates the interaction between IκBα and p65 by binding to p65 in microglia, and thus repressing microglial activation and exhibiting the protection of DA neurons from neuroinflammation-mediated injury in PD.Subject terms: Cell death in the nervous system, Parkinson''s disease     

Nitrogen dioxide solubility and permeation in lipid membranes

Nitrogen dioxide (NO₂) is an important oxidant molecule in biology that is produced by several biological processes, and it is also an important air pollutant. It can oxidize proteins and lipids with important consequences on their biological functions. Despite its relevance, the interaction of NO₂ with the cell barrier, the lipid membrane, is poorly understood. For instance, can lipid membranes limit NO₂ diffusion? To estimate the permeability of lipid membranes to NO₂ it is necessary to learn more about its solubility in the lipid phase. However, experimental data on NO₂ solubility is very limited. To improve our knowledge on this matter, we used a mixed approach consisting in calculating the solubility of NO₂ and related diatomic and triatomic gases (NO, O₂, CO₂, etc.) in different solvents using quantum calculations and Tomasi’s Polarizable Continuum Model and validating and correcting these results using experimental data available for the related gases. This approach led to an estimated partition coefficient for NO₂ of 2.7 between n-octanol and water, and 1.5 between lipid membranes and water, meaning that NO₂ is a moderately hydrophobic molecule (less than NO, more than CO₂). Based on the solubility-diffusion permeability theory, the permeability coefficient was estimated to be 5 cm s⁻¹, up to 4000 times higher than that of peroxynitrous acid. It is concluded that lipid membranes are not significant barriers to NO₂ transport.     

Vitamin A supplementation at pharmacological doses induces nitrosative stress on the hypothalamus of adult Wistar rats

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 α-synuclein and D2 receptor (dopamine receptor) contents. Additionally, we quantified caspase-3 activity and tumor necrosis factor-α (TNF-α) 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 (O₂⁻) production found here. Other parameters investigated here, i.e. α-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.     

Kinetics of reduction of tyrosine phenoxyl radicals by glutathione

Modification of tyrosine (TyrOH) is used as a marker of oxidative and nitrosative stress. 3,3′-Dityrosine formation, in particular, reflects oxidative damage and results from the combination of two tyrosyl phenoxyl radicals (TyrO). This reaction is in competition with reductive processes in the cell which ‘repair’ tyrosyl radicals: possible reductants include thiols and ascorbate. In this study, a rate constant of 2 × 10⁶ M⁻¹ s⁻¹ was estimated for the reaction between tyrosyl radicals and glutathione (GSH) at pH 7.15, generating the radicals by pulse radiolysis and monitoring the tyrosyl radical by kinetic spectrophotometry. Earlier measurements have suggested that this ‘repair’ reaction could be an equilibrium, and to investigate this possibility the reduction (electrode) potential of the (TyrO,H⁺/TyrOH) couple was reinvestigated by observing the fast redox equilibrium with the indicator 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonate). Extrapolation of the reduction potential of TyrO measured at pH 9–11 indicated the mid-point reduction potential of the tyrosyl radical at pH 7, E_m7(TyrO,H⁺/TyrOH) = 0.93 ± 0.02 V. This is close to the reported reduction potential of the glutathione thiyl radical, E_m7 = 0.94 ± 0.03 V, confirming the ‘repair’ equilibrium constant is of the order of unity and suggesting that efficient reduction of TyrO by GSH might require removal of thiyl radicals to move the equilibrium in the direction of repair. Loss of thiyl radicals, facilitating repair of TyrO, can arise either via conjugation of thiyl with thiol/thiolate or oxygen, or unimolecular transformation, the latter important at low concentrations of thiols and oxygen.     

Causes of Parkinson’s disease: genetics of <Emphasis Type="Italic">DJ-1</Emphasis>

The identification of Mendelian mutations in rare forms of familial Parkinsons disease (PD) have provided significant insights into the molecular pathogenesis of this common complex disorder. DJ-1 is the third of four genes known to be definitively causal in familial PD, the three others being -synuclein, parkin and the recently identified PINK1. Mutations in the DJ-1 gene were identified in two European families, a Dutch kindred harbouring a large homozygous genomic deletion encompassing exons 1–5 of the gene and an Italian kindred with a homozygous L166P missense mutation. The clinical phenotype of the two families was similar to that of parkin cases. Age of onset was in the mid-thirties with good responsiveness to l-dopa and slow disease progression. Focal dystonias and blepharospasm were also evident as were behavioural disturbances early in the course of the disease. To date, there are no studies of pathological material from known DJ-1 patients. It therefore remains to be determined whether these patients form Lewy bodies and/or Lewy neurites, the eosinophilic fibrillary inclusions that contain predominantly -synuclein and that are the pathological hallmark of PD.     

Hydrogen peroxide- and nitric oxide-induced systemic antioxidant prime-like activity under NaCl-stress and stress-free conditions in citrus plants

We tested whether pre-treatments of roots with H₂O₂ (10 mM for 8 h) or sodium nitroprusside (SNP; 100 μM for 48 h), a donor of NO, could induce prime antioxidant defense responses in the leaves of citrus plants grown in the absence or presence of 150 mM NaCl for 16 d. Both root pre-treatments increased leaf superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) activities, and induced related-isoform(s) expression under non-NaCl-stress conditions. When followed by salinity, certain enzymatic activities also exhibited an up-regulation in response to H₂O₂ or SNP pre-exposure. An NaCl-stress-provoked decrease in the ascorbate redox state was partially prevented by both pre-treatments, whereas the glutathione redox state under normal and NaCl-stress conditions was increased by SNP. Real-time imaging of NO production was found in vascular tissues and epidermal cells. Furthermore, NaCl-induced inhibition in OH scavenging activity and promotion of OH-mediated DNA strand cleavage was partially prevented by SNP. Moreover, NaCl-dependent protein oxidation (carbonylation) was totally reversed by both pre-treatments as revealed by quantitative assay and protein blotting analysis. These results provide strong evidence that H₂O₂ and NO elicit long-lasting systemic primer-like antioxidant activity in citrus plants under physiological and NaCl-stress conditions.     

The influence of the cation of quaternized chitosans on antioxidant activity

Various quaternized chitosans (QCSs) were synthesized according to previous method. Their reducing power and antioxidant potency against hydroxyl radicals (OH) and hydrogen peroxide (H₂O₂) were explored by the established systems in vitro. The QCSs exhibited markedly antioxidant activity, especially TCEDMCS, whose IC₅₀ on hydroxyl radicals was 0.235 mg/mL. They showed 65–80% scavenging effect on hydrogen peroxide at a dose of 0.5 mg/mL. Generally, the antioxidant activity decreased in the order TCEDMCS > TBEDMCS > EDMCS > PDMCS > IBDMCS > Chitosan. Furthermore, the order of their OH and H₂O₂ scavenging activity was consistent with the electronegativity of different substituted groups in the QCSs. The QCSs showed much stronger antioxidant activity than that of chitosan may be due to the positive charge density of the nitrogen atoms in QCSs strengthened by the substituted groups.     

Highly hydroxylated or γ-cyclodextrin-bicapped water-soluble derivative of fullerene: The antioxidant ability assessed by electron spin resonance method and β-carotene bleaching assay

Antioxidant ability of the water-soluble derivative of fullerene (C60), prepared by high-degree hydroxylation [C60-(OH)₃₂·8H₂O] or C60/γ-cyclodextrin (1:2 mol/mol) clathrate formation [C60/(γ-CD)₂], was assessed by electron spin resonance method and β-carotene bleaching assay. These C60 derivatives have an ability to diminish a 1:2:2:1 quartet ESR spectrum attributed to hydroxyl radicals (OH) as shown by DMPO-spin trap/ESR method. Meanwhile, a singlet radical-signal different from OH-attributed signals increased in a manner dependent on concentrations of C60-(OH)₃₂·8H₂O. This might suggest that C60-(OH)₃₂·8H₂O scavenges OH owing to dehydrogenation of C60-(OH)₃₂·8H₂O, and is simultaneously oxidized to a stable radical species, which may be a dehydrogenated fullerenol radical (C60-O). Furthermore, these water-soluble derivatives of C60 suppressed fading of yellowish color characteristic of intact β-carotene in β-carotene bleaching assay. Antioxidant abilities of these derivatives were assessed as retention of yellowish color (viz absorbance at 470 nm) for 180 min. Namely, β-carotene-attributed chromaticity (% relative absorbance at 470 nm compared with the control) after 180 min was 69% for C60-(OH)₃₂·8H₂O (400 μM: C60-eq.), and 32% for C60/(γ-CD)₂ (400 μM: C60-eq.), whereas it was 6% for l(+)-ascorbic acid (400 μM) which is hydrophilic, and 85% for (±)-α-tocopherol (400 μM) which is lipophilic, respectively. Thus C60-(OH)₃₂·8H₂O and C60/(γ-CD)₂ can scavenge OH, and have a distinct antioxidative activity in the aqueous system containing linoleic acid which is abundantly contained in the cell membrane together with other unsaturated lipids. These C60 derivatives have a potential to protect the cell membrane from oxidative stress due to OH.     

Comparison of antioxidant abilities of magnolol and honokiol to scavenge radicals and to protect DNA

The antioxidant properties of magnolol and honokiol were evaluated in the experimental systems of reducing ONOO⁻ and ¹O₂, bleaching β-carotene in linoleic acid (LH) emulsion, and trapping 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS⁺) and 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH), and then were applied to inhibit the oxidation of DNA induced by Cu²⁺/glutathione (GSH) and 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH). Magnolol and honokiol were active to reduce ONOO⁻ and ¹O₂. Honokiol showed a little higher activity to protect LH and to inhibit Cu²⁺/GSH-induced oxidation of DNA than magnolol. In addition, honokiol exhibited higher activities to trap ABTS⁺ and DPPH than magnolol. In particular, honokiol trapped 2.5 radicals while magnolol only trapped 1.8 radicals in protecting DNA against AAPH-induced oxidation. The obtained results suggested that low antioxidant ability of magnolol may be related to the intramolecular hydrogen bond formed between di-ortho-hydroxyl groups, which hindered the hydrogen atom in hydroxyl group to be abstracted by radicals. Therefore, the antioxidant capacity of magnolol was lower than that of honokiol.     

Decreased S-nitrosation of peptide thiols in the membrane interior

It has been proposed that autoxidation of nitric oxide (NO) stimulates S-nitrosation of thiols located in the hydrophobic milieu. We tested whether thiols located in hydrophobic membranes undergo enhanced S-nitrosation in the presence of NO/O₂. The transmembrane cysteinyl peptides C₄ (AcNH-KKACALA(LA)₆KK-CONH₂) and C₈ (AcNH-KKALALACALA(LA)₃KK-CONH₂) were incorporated into dilauroylphosphatidylcholine bilayers; their location in the membrane was determined by EPR spin labeling. The peptides, C₈ and C₄, and glutathione (GSH; 300 μM) were treated with a NO donor, DEA-NONOate, and nitrosothiol formation was determined under various O₂ levels. Surprisingly, the more hydrophobic cysteinyl peptide, C₈, did not yield any S-nitrosated product compared to GSH in the aqueous phase or C₄ peptide in the liposomes in the presence of NO/O₂. These data suggest that thiols located deeply in the hydrophobic core of the membrane may be less likely to undergo S-nitrosation in the presence of NO/O₂.