Multiple Immune-Inflammatory and Oxidative and Nitrosative Stress Pathways Explain the Frequent Presence of Depression in Multiple Sclerosis

Patients with a diagnosis of multiple sclerosis (MS) or major depressive disorder (MDD) share a wide array of biological abnormalities which are increasingly considered to play a contributory role in the pathogenesis and pathophysiology of both illnesses. Shared abnormalities include peripheral inflammation, neuroinflammation, chronic oxidative and nitrosative stress, mitochondrial dysfunction, gut dysbiosis, increased intestinal barrier permeability with bacterial translocation into the systemic circulation, neuroendocrine abnormalities and microglial pathology. Patients with MS and MDD also display a wide range of neuroimaging abnormalities and patients with MS who display symptoms of depression present with different neuroimaging profiles compared with MS patients who are depression-free. The precise details of such pathology are markedly different however. The recruitment of activated encephalitogenic Th17 T cells and subsequent bidirectional interaction leading to classically activated microglia is now considered to lie at the core of MS-specific pathology. The presence of activated microglia is common to both illnesses although the pattern of such action throughout the brain appears to be different. Upregulation of miRNAs also appears to be involved in microglial neurotoxicity and indeed T cell pathology in MS but does not appear to play a major role in MDD. It is suggested that the antidepressant lofepramine, and in particular its active metabolite desipramine, may be beneficial not only for depressive symptomatology but also for the neurological symptoms of MS. One clinical trial has been carried out thus far with, in particular, promising MRI findings.     

Redox Proteomics of the Inflammatory Secretome Identifies a Common Set of Redoxins and Other Glutathionylated Proteins Released in Inflammation,Influenza Virus Infection and Oxidative Stress

Protein cysteines can form transient disulfides with glutathione (GSH), resulting in the production of glutathionylated proteins, and this process is regarded as a mechanism by which the redox state of the cell can regulate protein function. Most studies on redox regulation of immunity have focused on intracellular proteins. In this study we have used redox proteomics to identify those proteins released in glutathionylated form by macrophages stimulated with lipopolysaccharide (LPS) after pre-loading the cells with biotinylated GSH. Of the several proteins identified in the redox secretome, we have selected a number for validation. Proteomic analysis indicated that LPS stimulated the release of peroxiredoxin (PRDX) 1, PRDX2, vimentin (VIM), profilin1 (PFN1) and thioredoxin 1 (TXN1). For PRDX1 and TXN1, we were able to confirm that the released protein is glutathionylated. PRDX1, PRDX2 and TXN1 were also released by the human pulmonary epithelial cell line, A549, infected with influenza virus. The release of the proteins identified was inhibited by the anti-inflammatory glucocorticoid, dexamethasone (DEX), which also inhibited tumor necrosis factor (TNF)-α release, and by thiol antioxidants (N-butanoyl GSH derivative, GSH-C4, and N-acetylcysteine (NAC), which did not affect TNF-α production. The proteins identified could be useful as biomarkers of oxidative stress associated with inflammation, and further studies will be required to investigate if the extracellular forms of these proteins has immunoregulatory functions.     

Analysis by Transfer Electrophoresis of Reactivity of IgG with Brain Proteins in Multiple Sclerosis

Abstract: Central nervous system proteins, immobilized on cellulose nitrate sheets, can be identified by incubation with specific antisera. Using this technique, no disease-related antibodies against CNS proteins were detected in the cerebrospinal fluid, brain extracts, or sera from cases of multiple sclerosis, although a positive response was seen in sera from animals with experimental autoimmune encephalomyelitis.     

The Presence of Aldehyde-Reacted Proteins in Normal and Multiple Sclerosis White Matter

The incorporation of tritium from NaB3H4 into the major protein components of myelin and the presence of weak fluorescence emission bands at wavelengths of approximately 440 and 500 nm from sodium dodecyl sulfate-solubilized, delipidated white matter are indicative of the presence of the products of aldehyde reactions with proteins. The incorporation of tritium from NaB3H4 into myelin proteins was confirmed by reaction with purified components of myelin basic protein or with lipophilin, a purified fraction of proteolipid protein. From the extent of tritium incorporation into the purified proteins, it is estimated that approximately 0.2 mol of tritium is incorporated/mol of myelin basic protein and approximately 0.4 mol of tritium/mol of proteolipid protein. There is approximately 50% greater incorporation of tritium into a more degraded, less positively charged form of the basic protein. The incorporation of tritium into normal and multiple sclerosis white matter was compared. There is a small but statistically significant difference in the percentage of the total counts incorporated into the major protein fractions for the two groups, with the multiple sclerosis samples showing a higher percentage of the counts in the Wolfgram protein and a lower percentage in the myelin basic protein compared with the normal samples.     

Occurrence of Anti-Drug Antibodies against Interferon-Beta and Natalizumab in Multiple Sclerosis: A Collaborative Cohort Analysis

Immunogenicity of biopharmaceutical products in multiple sclerosis is a frequent side effect which has a multifactorial etiology. Here we study associations between anti-drug antibody (ADA) occurrence and demographic and clinical factors. Retrospective data from routine ADA test laboratories in Sweden, Denmark, Austria and Germany (Dusseldorf group) and from one research study in Germany (Munich group) were gathered to build a collaborative multi-cohort dataset within the framework of the ABIRISK project. A subset of 5638 interferon-beta (IFNβ)-treated and 3440 natalizumab-treated patients having data on at least the first two years of treatment were eligible for interval-censored time-to-event analysis. In multivariate Cox regression, IFNβ-1a subcutaneous and IFNβ-1b subcutaneous treated patients were at higher risk of ADA occurrence compared to IFNβ-1a intramuscular-treated patients (pooled HR = 6.4, 95% CI 4.9–8.4 and pooled HR = 8.7, 95% CI 6.6–11.4 respectively). Patients older than 50 years at start of IFNβ therapy developed ADA more frequently than adult patients younger than 30 (pooled HR = 1.8, 95% CI 1.4–2.3). Men developed ADA more frequently than women (pooled HR = 1.3, 95% CI 1.1–1.6). Interestingly we observed that in Sweden and Germany, patients who started IFNβ in April were at higher risk of developing ADA (HR = 1.6, 95% CI 1.1–2.4 and HR = 2.4, 95% CI 1.5–3.9 respectively). This result is not confirmed in the other cohorts and warrants further investigations. Concerning natalizumab, patients older than 45 years had a higher ADA rate (pooled HR = 1.4, 95% CI 1.0–1.8) and women developed ADA more frequently than men (pooled HR = 1.4, 95% CI 1.0–2.0). We confirmed previously reported differences in immunogenicity of the different types of IFNβ. Differences in ADA occurrence by sex and age are reported here for the first time. These findings should be further investigated taking into account other exposures and biomarkers.     

The Involvement of Copper Transporter in Lead-induced Oxidative Stress in Astroglia

Lead (Pb), depositing primarily in astroglia in the brain, is a well-known neurotoxicant and a risk factor for neurologic disorders. Pb has been reported to induce oxidative stress by probably the disturbance of copper (Cu) homeostasis in astroglia. Thus, we hypothesized that Pb-induced oxidative stress is initiated by interfering with Cu transporter in astroglia. In this study, we observed Pb-induced oxidative stress as indicated by reactive oxygen species (ROS) augmentation and GRP78 and GRP94 protein induction, and it was parallel to Cu accumulation intracellularly by Pb. To further address Cu transporter as a potential Pb target, a heavy metal-binding (HMB) domain of Cu-transporting ATPase (Atp7a) was overexpressed and purified. Evidence showed that one molecule of HMB chelated 11 Pb ions or seven Cu ions and that Pb competed with Cu for binding to HMB. These findings suggest that Pb-induced oxidative stress results from the impairment of Cu metabolism by Pb targeting of Atp7a.     

Genetic,Immune-Inflammatory,and Oxidative Stress Biomarkers as Predictors for Disability and Disease Progression in Multiple Sclerosis

The aim of this study was to evaluate the TNFβ NcoI polymorphism (rs909253) and immune-inflammatory, oxidative, and nitrosative stress (IO&NS) biomarkers as predictors of disease progression in multiple sclerosis (MS). We included 212 MS patients (150 female, 62 male, mean (±standard deviation (SD)) age?=?42.7?±?13.8 years) and 249 healthy controls (177 female, 72 male, 36.8?±?11 years). The disability was measured the Expanded Disability Status Scale (EDSS) in 2006 and 2011. We determined the TNFβ NcoI polymorphism and serum levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-4, IL-10, and IL-17, albumin, ferritin, and plasma levels of lipid hydroperoxides (CL-LOOH), carbonyl protein, advanced oxidation protein products (AOPPs), nitric oxide metabolites (NOx), and total radical-trapping antioxidant parameter (TRAP). The mean EDSS (±SD) in 2006 was 1.62?±?2.01 and in 2011 3.16?±?2.29, and disease duration was 7.34?±?7.0 years. IL-10, TNF-α, IFN-γ, AOPP, and NOx levels were significantly higher and IL-4 lower in MS patients with a higher 2011 EDSS scores (≥3) as compared with those with EDSS?<?3. The actual increases in EDSS from 2006 to 2011 were positively associated with TNF-α and IFN-γ. Increased IFN-γ values were associated with higher pyramidal symptoms and increased IL-6 with sensitive symptoms. Increased carbonyl protein and IL-10 but lowered albumin levels predicted cerebellar symptoms. The TNFB1/B2 genotype decreased risk towards progression of pyramidal symptoms. Treatments with IFN-β and glatiramer acetate significantly reduced TNF-α but did not affect the other IO&NS biomarkers or disease progression. Taken together, IO&NS biomarkers and NcoI TNFβ genotypes predict high disability in MS and are associated with different aspects of disease progression. New drugs to treat MS should also target oxidative stress pathways.     

Thioredoxins in Redox Maintenance and Survival during Oxidative Stress of Bacteroides fragilis

The anaerobe Bacteroides fragilis is a gram-negative, opportunistic pathogen that is highly aerotolerant and can persist in aerobic environments for extended periods. In this study, the six B. fragilis thioredoxins (Trxs) were investigated to determine their role during oxidative stress. Phylogenetic analyses of Trx protein sequences indicated that four of the six Trxs (TrxA, TrxC, TrxD, and TrxF) belong to the M-type Trx class but were associated with two different M-type lineages. TrxE and TrxG were most closely associated to Y-type Trxs found primarily in cyanobacteria. Single and multiple trx gene deletions were generated to determine functional differences between the Trxs. The trxA gene was essential, but no anaerobic growth defects were observed for any other single trx deletion or for the ΔtrxC ΔtrxD::cfxA ΔtrxE ΔtrxF ΔtrxG quintuple mutant. Regulation of the trx genes was linked to the oxidative stress response, and all were induced by aerobic conditions. The ΔtrxC ΔtrxE ΔtrxF ΔtrxG and the ΔtrxC ΔtrxD::cfxA ΔtrxE ΔtrxF ΔtrxG multiple deletion strains were impaired during growth in oxidized media, but single trx gene mutants did not have a phenotype in this assay. TrxD was protective during exposure to the thiol oxidant diamide, and expression of trxD was induced by diamide. Diamide-induced expression of trxC, trxE, and trxF increased significantly in a trxD mutant strain, suggesting that there is some capacity for compensation in this complex Trx system. These data provide insight into the role of individual Trxs in the B. fragilis oxidative stress response.Protective mechanisms for dealing with oxidative stress are an integral part of any organism that lives in, or is exposed to, an aerobic environment. While aerobic organisms have developed robust systems to contend with the constant threat of destructive oxygen radicals, anaerobic organisms introduced to an aerobic environment are at an elevated risk for damage. Oxygen toxicity in anaerobes is a complex phenomenon involving many aspects of cellular physiology that are impaired as oxidative damage occurs. For example, aerobic exposure of the aerotolerant Bacteroides thetaiotaomicron inhibits growth, in part due to the oxidation of iron-sulfur clusters located within metabolic enzymes (30). To combat this problem, some anaerobic bacteria have evolved multifaceted strategies to manage the production and effects of reactive oxygen species (38). Bacteroides fragilis is a commensal anaerobe found in the human intestine, but it also is the most frequently isolated anaerobe from human infections (10). B. fragilis is unable to multiply in the presence of air (21% O₂); however, it is highly resistant to oxidative stress and can survive for extended periods in a fully aerobic environment. In this regard, B. fragilis is one of the most aerotolerant anaerobes known, and it is able to survive for at least 72 h in the presence of atmospheric oxygen. In contrast, intolerant anaerobes survive for less than 2 h in air (49). This remarkable resistance to oxidative stress is mediated by an oxidative stress response (OSR) which involves a wide array of genes activated during exposure to air or H₂O₂. An ever-growing set of genes and proteins that are induced in response to aerobic exposure have been discovered, and while the function of some have been deduced, many of their contributions to aerotolerance remain to be clarified (15, 36-38, 48). In this regard, a recent expression microarray showed that the B. fragilis thioredoxin (Trx) genes were induced by aerobic conditions, but their role in the OSR has not been adequately explored (48).Trxs are small redox-active proteins (∼12 kDa) found in all phylogenetic branches. Trxs contain a highly conserved Cys-X-X-Cys motif at their active sites, allowing for catalysis of thiol-disulfide reactions (1, 40). The reduction of Trxs is mediated by flavin adenine dinucleotide-dependent Trx reductases (TrxB) which convert oxidized Trxs to their free thiol forms (1). Since the discovery of their role in DNA synthesis and in maintenance of the reduced state of intracellular protein disulfides, Trxs have been shown to be involved in defense against oxidative stress (17). Trxs regenerate oxidatively damaged proteins, modulate the activity of redox stressors, and act as hydrogen donors for detoxification enzymes important during the OSR (7, 9, 25, 27, 28).Analysis of the B. fragilis genome revealed the presence of a single Trx reductase (TrxB) and six Trx homologs. This large repertoire of trx genes appears unusual compared to the typical smaller number of trx genes (two or three) found in other anaerobes (13, 19-21, 33, 42, 29). Previously, Rocha et al. (40) showed that the TrxB/Trx system is the primary thiol/disulfide redox system in B. fragilis; it has an important role in aerotolerance and is essential for survival in an in vivo mouse abscess model. These findings prompted us to propose that while TrxB is required for the function of the system overall, each Trx has important, specific roles in survival and defense against oxidative stress. In this study we present evidence that B. fragilis possesses a complex Trx system in which individual trx genes are differentially regulated but have some capacity to compensate for other trx genes under stress conditions. We also present evidence suggesting that TrxD has a major role in managing thiol oxidation and that trxA is an essential gene.     

Redox Proteomics Changes in the Fungal Pathogen Trichosporon asahii on Arsenic Exposure: Identification of Protein Responses to Metal-Induced Oxidative Stress in an Environmentally-Sampled Isolate

Trichosporon asahii is a yeast pathogen implicated in opportunistic infections. Cultures of an isolate collected from industrial wastewater were exposed for 2 days to 100 mg/L sodium arsenite (NaAsO₂) and cadmium (CdCl₂). Both metals reduced glutathione transferase (GST) activity but had no effect on superoxide dismutase or catalase. NaAsO₂ exposure increased glutathione reductase activity while CdCl₂ had no effect. Protein thiols were labeled with 5-iodoacetamido fluorescein followed by one dimensional electrophoresis which revealed extensive protein thiol oxidation in response to CdCl₂ treatment but thiol reduction in response to NaAsO₂. Two dimensional electrophoresis analyses showed that the intensity of some protein spots was enhanced on treatment as judged by SameSpots image analysis software. In addition, some spots showed decreased IAF fluorescence suggesting thiol oxidation. Selected spots were excised and tryptic digested for identification by MALDI-TOF/TOF MS. Twenty unique T. asahii proteins were identified of which the following proteins were up-regulated in response to NaAsO₂: 3-isopropylmalate dehydrogenase, phospholipase B, alanine-glyoxylate aminotransferase, ATP synthase alpha chain, 20S proteasome beta-type subunit Pre3p and the hypothetical proteins A1Q1_08001, A1Q2_03020, A1Q1_06950, A1Q1_06913. In addition, the following showed decreased thiol-associated fluorescence consistent with thiol oxidation; aconitase; aldehyde reductase I; phosphoglycerate kinase; translation elongation factor 2; heat shock protein 70 and hypothetical protein A1Q2_04745. Some proteins showed both increase in abundance coupled with decrease in IAF fluorescence; 3-hydroxyisobutyryl- CoA hydrolase; homoserine dehydrogenase Hom6 and hypothetical proteins A1Q2_03020 and A1Q1_00754. Targets implicated in redox response included 10 unique metabolic enzymes, heat shock proteins, a component of the 20S proteasome and translation elongation factor 2. These data suggest extensive proteomic alterations in response to metal-induced oxidative stress in T. asahii. Amino acid metabolism, protein folding and degradation are principally affected.     

阿尔茨海默氏病相关蛋白质及其作用机制研究进展

蛋白质组学技术被广泛的用于阿尔茨海默氏病(AD)的研究中.本文综述了已发现的AD脑组织、外周组织和动物模型中蛋白质差异表达和翻译后修饰变化,结合生物信息学分析结果,初步阐明了参与AD发病机制的关键蛋白质及其信号通路,为深入研究AD的病理生理机制和治疗提供了依据.     

The Role of Citrullinated Proteins Suggests a Novel Mechanism in the Pathogenesis of Multiple Sclerosis

The pathogenesis of MS is unknown. In our studies, we have demonstrated an important role for citrullinated myelin basic protein (MBP). The accompanying loss of positive charge compromises the ability of MBP to interact with the lipid bilayer. The conversion of arginine to citrulline in brain is carried out by an enzyme peptidyl arginine deiminase (PAD) 2. The amount of PAD 2 in brain was increased in MS normal-appearing white matter. The mechanism responsible for this increase involved hypomethylation of the promoter region in the PAD 2 gene in MS, but no change (compared to normal) was found in thymus tissue DNA from the same MS patients. In addition, no change was observed in other neurological diseases, including Alzheimer’s, Parkinson’s, and Huntington’s. We propose that citrullinated MBP, resulting from elevated levels of PAD 2 represents an important biochemical pathway in the pathogenesis of MS. Special issue dedicated to Anthony Campagnoni.     

Delayed P100-Like Latencies in Multiple Sclerosis: A Preliminary Investigation Using Visual Evoked Spread Spectrum Analysis

Conduction along the optic nerve is often slowed in multiple sclerosis (MS). This is typically assessed by measuring the latency of the P100 component of the Visual Evoked Potential (VEP) using electroencephalography. The Visual Evoked Spread Spectrum Analysis (VESPA) method, which involves modulating the contrast of a continuous visual stimulus over time, can produce a visually evoked response analogous to the P100 but with a higher signal-to-noise ratio and potentially higher sensitivity to individual differences in comparison to the VEP. The main objective of the study was to conduct a preliminary investigation into the utility of the VESPA method for probing and monitoring visual dysfunction in multiple sclerosis. The latencies and amplitudes of the P100-like VESPA component were compared between healthy controls and multiple sclerosis patients, and multiple sclerosis subgroups. The P100-like VESPA component activations were examined at baseline and over a 3-year period. The study included 43 multiple sclerosis patients (23 relapsing-remitting MS, 20 secondary-progressive MS) and 42 healthy controls who completed the VESPA at baseline. The follow-up sessions were conducted 12 months after baseline with 24 MS patients (15 relapsing-remitting MS, 9 secondary-progressive MS) and 23 controls, and again at 24 months post-baseline with 19 MS patients (13 relapsing-remitting MS, 6 secondary-progressive MS) and 14 controls. The results showed P100-like VESPA latencies to be delayed in multiple sclerosis compared to healthy controls over the 24-month period. Secondary-progressive MS patients had most pronounced delay in P100-like VESPA latency relative to relapsing-remitting MS and controls. There were no longitudinal P100-like VESPA response differences. These findings suggest that the VESPA method is a reproducible electrophysiological method that may have potential utility in the assessment of visual dysfunction in multiple sclerosis.     

Induction of Oxidatively Modified Proteins in Skeletal Muscle by Electrical Stimulation and Its Suppression by Dietary Supplementation of (-)-Epigallocatechin Gallate

The oxidative stress produced by electrical stimulation-induced muscle contraction was examined in the skeletal muscle proteins of rats that had been fed on the dietary flavonoid, (-)-epigallocatechin gallate (EGCg). Electrical stimulation of the rat leg muscle every second day for a two-week period resulted in an increased (p<0.05) muscle weight and accumulation of oxidatively induced modified proteins. Similar stimulation conducted every day for only one week had no effect on the muscle weight or protein oxidation, although the rate of protein degradation increased. Rats fed on a 20% casein diet supplemented with 0.1% EGCg for 2 weeks responded to the electrical stimulation of muscle contraction by reducing the increased muscle protein carbonyl content when compared to their counterparts fed on a control diet. There was no change in activity of antioxidative enzymes in muscle tissue of the EGCg-fed rats receiving electrical stimulation. The results of this study show that the antioxidative property of EGCg was effective for suppressing oxidative modification of the skeletal muscle protein induced by electrical stimulation. This finding demonstrates that EGCg has a beneficial effect in vivo on the free radical-mediated oxidative damage to muscle proteins.     

Shotgun Redox Proteomics: Identification and Quantitation of Carbonylated Proteins in the UVB-Resistant Marine Bacterium,Photobacterium angustum S14

UVB oxidizes proteins through the generation of reactive oxygen species. One consequence of UVB irradiation is carbonylation, the irreversible formation of a carbonyl group on proline, lysine, arginine or threonine residues. In this study, redox proteomics was performed to identify carbonylated proteins in the UVB resistant marine bacterium Photobacterium angustum. Mass-spectrometry was performed with either biotin-labeled or dinitrophenylhydrazide (DNPH) derivatized proteins. The DNPH redox proteomics method enabled the identification of 62 carbonylated proteins (5% of 1221 identified proteins) in cells exposed to UVB or darkness. Eleven carbonylated proteins were quantified and the UVB/dark abundance ratio was determined at both the protein and peptide levels. As a result we determined which functional classes of proteins were carbonylated, which residues were preferentially modified, and what the implications of the carbonylation were for protein function. As the first large scale, shotgun redox proteomics analysis examining carbonylation to be performed on bacteria, our study provides a new level of understanding about the effects of UVB on cellular proteins, and provides a methodology for advancing studies in other biological systems.     

Linoleate and Fatty-acid Patterns of Serum Lipids in Multiple Sclerosis and Other Diseases

The linoleic acid content of serum lipids was measured in 47 patients with multiple sclerosis, 29 patients with other neurological diseases, 35 patients with acute non-neurological illnesses, and 49 healthy control subjects. Reduced linoleic acid content of serum lipids was not specific to multiple sclerosis and occurred in all ill patients with acute non-neurological illness. The fatty-acid pattern of serum lipids in illness resembles that of essential fatty-acid deficiency. It seems that this pattern of reduced linoleic acid content with increased oleic, palmitic, and palmitoleic acid content may be a general phenomenon in ill patients.