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Alzheimer’s disease (AD), the most common age-related neurodegenerative disorder, is clinically characterized by progressive neuronal loss resulting in loss of memory and dementia. AD is histopathologically characterized by the extensive distribution of senile plaques and neurofibrillary tangles, and synapse loss. Amnestic mild cognitive impairment (MCI) is generally accepted to be an early stage of AD. MCI subjects have pathology and symptoms that fall on the scale intermediately between ‘normal’ cognition with little or no pathology and AD. A rare number of individuals, who exhibit normal cognition on psychometric tests but whose brains show widespread postmortem AD pathology, are classified as ‘asymptomatic’ or ‘preclinical’ AD (PCAD). In this study, we evaluated changes in protein phosphorylation states in the inferior parietal lobule of subjects with AD, MCI, PCAD, and control brain using a 2-D PAGE proteomics approach in conjunction with Pro-Q Diamond phosphoprotein staining. Statistically significant changes in phosphorylation levels were found in 19 proteins involved in energy metabolism, neuronal plasticity, signal transduction, and oxidative stress response. Changes in the disease state phosphoproteome may provide insights into underlying mechanisms for the preservation of memory with expansive AD pathology in PCAD and the progressive memory loss in amnestic MCI that escalates to the dementia and the characteristic pathology of AD brain.

Chemotherapy-induced cognitive impairment (CICI) is a quality of life-altering consequence of chemotherapy experienced by a large percentage of cancer survivors. Approximately half of FDA-approved anti-cancer drugs are known to produce ROS. Doxorubicin (Dox), a prototypical ROS-generating chemotherapeutic agent, generates superoxide (O 2 − •) via redox cycling. Our group previously demonstrated that Dox, which does not cross the BBB, induced oxidative damage to plasma proteins leading to TNF-α elevation in the periphery and, subsequently, in brain following cancer chemotherapy. We hypothesize that such processes play a central role in CICI. The current study tested the notion that O 2 − • is involved and likely responsible for Dox-induced plasma protein oxidation and TNF-α release. Addition of O 2 − • as the potassium salt (KO 2 ) to plasma resulted in significantly increased oxidative damage to proteins, indexed by protein carbonyl (PC) and protein-bound HNE levels. We then adapted this protocol for use in cell culture. Incubation of J774A.1 macrophage culture using this KO 2 -18crown6 protocol with 1 and 10 µM KO 2 resulted in dramatically increased levels of TNF-α produced. These findings, together with our prior results, provide strong evidence that O 2 − • and its resulting reactive species are critically involved in Dox-induced plasma protein oxidation and TNF-α release.

The naked mole-rat (NMR) is the longest-lived rodent and possesses several exceptional traits: marked cancer resistance, negligible senescence, prolonged genomic integrity, pronounced proteostasis, and a sustained health span. The underlying molecular mechanisms that contribute to these extraordinary attributes are currently under investigation to gain insights that may conceivably promote and extend human health span and lifespan. The ubiquitin–proteasome and autophagy–lysosomal systems play a vital role in eliminating cellular detritus to maintain proteostasis and have been previously shown to be more robust in NMRs when compared with shorter-lived rodents. Using a 2-D PAGE proteomics approach, differential expression and phosphorylation levels of proteins involved in proteostasis networks were evaluated in the brains of NMRs in an age-dependent manner. We identified 9 proteins with significantly altered levels and/or phosphorylation states that have key roles involved in proteostasis networks. To further investigate the possible role that autophagy may play in maintaining cellular proteostasis, we examined aspects of the PI3K/Akt/mammalian target of rapamycin (mTOR) axis as well as levels of Beclin-1, LC3-I, and LC3-II in the brain of the NMR as a function of age. Together, these results show that NMRs maintain high levels of autophagy throughout the majority of their lifespan and may contribute to the extraordinary health span of these rodents. The potential of augmenting human health span via activating the proteostasis network will require further studies.

Superoxide dismutases (SODs) are the primary reactive oxygen species (ROS)-scavenging enzymes of the cell and catalyze the dismutation of superoxide radicals O2- to H2O2 and molecular oxygen (O2). Among the three forms of SOD identified, manganese-containing SOD (MnSOD, SOD2) is a homotetramer located wholly in the mitochondrial matrix. Because of the SOD2 strategic location, it represents the first mechanism of defense against the augmentation of ROS/reactive nitrogen species levels in the mitochondria for preventing further damage. This study seeks to understand the effects that the partial lack (SOD2(-/+) ) or the overexpression (TgSOD2) of MnSOD produces on oxidative/nitrative stress basal levels in different brain isolated cellular fractions (i.e., mitochondrial, nuclear, cytosolic) as well as in the whole-brain homogenate. Furthermore, because of the known interaction between SOD2 and p53 protein, this study seeks to clarify the impact that the double mutation has on oxidative/nitrative stress levels in the brain of mice carrying the double mutation (p53(-/-) × SOD2(-/+) and p53(-/-) × TgSOD2). We show that each mutation affects mitochondrial, nuclear, and cytosolic oxidative/nitrative stress basal levels differently, but, overall, no change or reduction of oxidative/nitrative stress levels was found in the whole-brain homogenate. The analysis of well-known antioxidant systems such as thioredoxin-1 and Nrf2/HO-1/BVR-A suggests their potential role in the maintenance of the cellular redox homeostasis in the presence of changes of SOD2 and/or p53 protein levels.

Naked mole-rats (NMRs) are the oldest-living rodent species. Living underground in a thermally stable ecological niche, NMRs have evolved certain exceptional traits, resulting in sustained health spans, negligible cognitive decline, and a pronounced resistance to age-related disease. Uncovering insights into mechanisms underlying these extraordinary traits involved in successful aging may conceivably provide crucial clues to extend the human life span and health span. One of the most fundamental processes inside the cell is the production of ATP, which is an essential fuel in driving all other energy-requiring cellular activities. Not surprisingly, a prominent hallmark in age-related diseases, such as neurodegeneration and cancer, is the impairment and dysregulation of metabolic pathways. Using a two-dimensional polyacrylamide gel electrophoresis proteomics approach, alterations in expression and phosphorylation levels of metabolic proteins in the brains of NMRs, aged 2-24 years, were evaluated in an age-dependent manner. We identified 13 proteins with altered levels and/or phosphorylation states that play key roles in various metabolic pathways including glycolysis, β-oxidation, the malate-aspartate shuttle, the Tricarboxylic Acid Cycle (TCA) cycle, the electron transport chain, NADPH production, as well as the production of glutamate. New insights into potential pathways involved in metabolic aspects of successful aging have been obtained by the identification of key proteins through which the NMR brain responds and adapts to the aging process and how the NMR brain adapted to resist age-related degeneration. This study examines the changes in the proteome and phosphoproteome in the brain of the naked mole-rat aged 2-24 years. We identified 13 proteins (labeled in red) with altered expression and/or phosphorylation levels that are conceivably associated with sustained metabolic functions in the oldest NMRs that may promote a sustained health span and life span.

Fatty acid synthase (FASN), a lipogenic enzyme, is upregulated in colorectal cancer (CRC). Increased de novo lipid synthesis is thought to be a metabolic adaptation of cancer cells that promotes survival and metastasis; however, the mechanisms for this phenomenon are not fully understood. We show that FASN plays a role in regulation of energy homeostasis by enhancing cellular respiration in CRC. We demonstrate that endogenously synthesized lipids fuel fatty acid oxidation, particularly during metabolic stress, and maintain energy homeostasis. Increased FASN expression is associated with a decrease in activation of energy-sensing pathways and accumulation of lipid droplets in CRC cells and orthotopic CRCs. Immunohistochemical evaluation demonstrated increased expression of FASN and p62, a marker of autophagy inhibition, in primary CRCs and liver metastases compared to matched normal colonic mucosa. Our findings indicate that overexpression of FASN plays a crucial role in maintaining energy homeostasis in CRC via increased oxidation of endogenously synthesized lipids. Importantly, activation of fatty acid oxidation and consequent downregulation of stress-response signaling pathways may be key adaptation mechanisms that mediate the effects of FASN on cancer cell survival and metastasis, providing a strong rationale for targeting this pathway in advanced CRC.

Alzheimer's disease (AD) is a progressive form of dementia characterized by increased production of amyloid-β plaques and hyperphosphorylated tau protein, mitochondrial dysfunction, elevated oxidative stress, reduced protein clearance, among other. Several studies showed systemic modifications of immune and inflammatory systems due, in part, to decreased levels of CD3+ lymphocytes in peripheral blood in AD. Considering that oxidative stress, both in the brain and in the periphery, can influence the activation and differentiation of T-cells, we investigated the 3-nitrotyrosine (3-NT) proteome of blood T-cells derived from AD patients compared to non-demented (ND) subjects by using a proteomic approach. 3-NT is a formal protein oxidation and index of nitrosative stress. We identified ten proteins showing increasing levels of 3-NT in CD3+ T-cells from AD patients compared with ND subjects. These proteins are involved in energy metabolism, cytoskeletal structure, intracellular signaling, protein folding and turnover, and antioxidant response and provide new insights into the molecular mechanism that impact reduced T-cell differentiation in AD. Our results highlight the role of peripheral oxidative stress in T-cells related to immune-senescence during AD pathology focusing on the specific targets of protein nitration that conceivably can be suitable to further therapies. Further, our data demonstrate common targets of protein nitration between the brain and the periphery, supporting their significance as disease biomarkers.

Brain insulin resistance is associated with an increased Aβ production in AD although the molecular mechanisms underlying this link are still largely unknown. Biliverdin reductase-A (BVR-A) is a unique Ser/Thr/Tyr kinase regulating insulin signalling. Studies from our group, demonstrated that BVR-A impairment is among the earliest events favoring brain insulin resistance development. Furthermore, reported a negative association between BVR-A protein levels/activation and BACE1 protein levels in the parietal cortex of aged beagles (an animal model of AD), thus suggesting a possible interaction. Therefore, we aimed to demonstrate that BVR-A impairment is a molecular bridge linking brain insulin resistance with increased Aβ production. Age-associated changes of BVR-A, BACE1, insulin signalling cascade and APP processing were evaluated in the parietal cortex of beagles and experiments to confirm the hypothesized mechanism(s) have been performed in vitro in HEK293APPswe cells. Our results show that BVR-A impairment occurs early with age and is associated with brain insulin resistance. Furthermore, we demonstrate that BVR-A impairment favors CK1-mediated Ser phosphorylation of BACE1 (known to mediate BACE1 recycling to plasma membrane) along with increased Aβ production in the parietal cortex, with age. Overall, our results suggest that the impairment of BVR-A is an early molecular event contributing to both (I) the onset of brain insulin resistance and (II) the increased Aβ production observed in AD. We, therefore, suggest that by targeting BVR-A activity it could be possible to delay the onset of brain insulin resistance along with an improved regulation of the APP processing.

Down Syndrome (DS) is the most common genetic form of intellectual disability that leads in the majority of cases to development of early-onset Alzheimer-like dementia (AD). The neuropathology of DS has several common features with AD including alteration of redox homeostasis, mitochondrial deficits, and inflammation among others. Interestingly, some of the genes encoded by chromosome 21 are responsible of increased oxidative stress (OS) conditions that are further exacerbated by decreased antioxidant defense. Previous studies from our groups showed that accumulation of oxidative damage is an early event in DS neurodegeneration and that oxidative modifications of selected proteins affects the integrity of the protein degradative systems, antioxidant response, neuronal integrity and energy metabolism. In particular, the current review elaborates recent findings demonstrating the accumulation of oxidative damage in DS and we focus attention on specific deregulation of iron metabolism, which affects both the central nervous system and the periphery. Iron dysmetabolism is a well-recognized factor that contributes to neurodegeneration; thus we opine that better understanding how and to what extent the concerted loss of iron dyshomeostasis and increased OS occur in DS could provide novel insights for the development of therapeutic strategies for the treatment of Alzheimer-like dementia.

International audience; This study examined the impact of surface functionalization and charge on ceria nanomaterial toxicity to Caenorhabditis elegans. The examined endpoints included mortality, reproduction, protein expression, and protein oxidation profiles. Caenorhabditis elegans were exposed to identical 2-5 nm ceria nanomaterial cores which were coated with cationic (diethylaminoethyl dextran; DEAE), anionic (carboxymethyl dextran; CM), and nonionic (dextran; DEX) polymers. Mortality and reproductive toxicity of DEAF-CeO2 was approximately two orders of magnitude higher than for CM-CeO2 or DEX-CeO2. Two-dimensional gel electrophoresis with orbitrap mass spectrometry identification revealed changes in the expression profiles of several mitochondrial-related proteins and proteins that are expressed in the C. elegans intestine. However, each type of CeO2 material exhibited a distinct protein expression profile. Increases in protein carbonyls and protein-bound 3-nitrotyrosine were also observed for some proteins, indicating oxidative and nitrosative damage. Taken together the results indicate that the magnitude of toxicity and toxicity pathways vary greatly due to surface functionalization of CeO2 nano materials.

Significant advances in the efficacy of cancer therapy have been accompanied by an escalation in side effects that result from therapy-induced injury to normal tissues. Cardiac injury is a major cause of death in cancer survivors, but biomarkers for it are detectable only after tissue injury has occurred. Extracellular vesicles (EVs) sort out oxidized proteins from tissues into the circulation as a compensatory mechanism for preventing cellular proteotoxicity. Thus, the protein contents of EVs released during the pre-degeneration stage reflects oxidative stress phenomenon that is occurring in the early damaged tissue. Here, we evaluate the potential of using circulating EVs as early diagnostic markers for long-term cardiac injury. Using a mouse model of doxorubicin (DOX)-induced cardiac injury, we found a significant increase in circulating EVs (DOX_EVs) compared to saline-treated controls. DOX_EVs exhibited a higher level of 4-hydroxynonenal adducted proteins, a lipid peroxidation product linked to DOX-induced cardiotoxicity. Proteomic profiling of DOX_EVs revealed the distinctive presence of brain/heart, muscle, and liver isoforms of glycogen phosphorylase (GP), and their origins were verified to be heart, skeletal muscle, and liver, respectively. The presence of brain/heart GP (PYGB) in DOX_EVs correlated with a reduction of PYGB in heart, but not brain tissues. Manganese superoxide dismutase (MnSOD) overexpression as well as pretreatment with FDA- approved cardioprotective agents and MnSOD mimetics resulted in a reduction of EV-associated PYGB in mice treated with DOX. Kinetic studies indicated that EVs containing PYGB are released prior to the rise of cardiac troponin (the current standard-of-care marker of cardiac injury) in the blood after DOX treatment, suggesting that PYGB is an earlier indicator of cardiac injury than conventional serum biomarkers. Thus, EVs containing PYGB serve as a liquid biopsy that is clinically attractive diagnostic biomarker for therapeutic interventions.

Thermotherapy, as a method of treating cancer, has recently attracted considerable attention from basic and clinical investigators. A number of studies and clinical trials have shown that thermotherapy can be successfully used as a therapeutic approach for various cancers. However, the effects of temperature on cancer bioenergetics have not been studied in detail with a real time, microplate based, label-free detection approach. This study investigates how changes in temperature affect the bioenergetics characteristics (mitochondrial function and glycolysis) of three colorectal cancer (CRC) cell lines utilizing the Seahorse XF96 technology. Experiments were performed at 32°C, 37°C and 42°C using assay medium conditions and equipment settings adjusted to produce equal oxygen and pH levels ubiquitously at the beginning of all experiments. The results suggest that temperature significantly changes multiple components of glycolytic and mitochondrial function of all cell lines tested. Under hypothermia conditions (32°C), the extracellular acidification rates (ECAR) of CRC cells were significantly lower compared to the same basal ECAR levels measured at 37°C. Mitochondrial stress test for SW480 cells at 37°C vs 42°C demonstrated increased proton leak while all other OCR components remained unchanged (similar results were detected also for the patient-derived xenograft cells Pt.93). Interestingly, the FCCP dose response at 37°C vs 42°C show significant shifts in profiles, suggesting that single dose FCCP experiments might not be sufficient to characterize the mitochondrial metabolic potential when comparing groups, conditions or treatments. These findings provide valuable insights for the metabolic and bioenergetic changes of CRC cells under hypo- and hyperthermia conditions that could potentially lead to development of better targeted and personalized strategies for patients undergoing combined thermotherapy with chemotherapy.

Mitochondria play a key role in eukaryotic cells, being mediators of energy, biosynthetic and regulatory requirements of these cells. Emerging proteomics techniques have allowed scientists to obtain the differentially expressed proteome or the proteomic redox status in mitochondria. This has unmasked the diversity of proteins with respect to subcellular location, expression and interactions. Mitochondria have become a research 'hot spot' in subcellular proteomics, leading to identification of candidate clinical targets in neurodegenerative diseases in which mitochondria are known to play pathological roles. The extensive efforts to rapidly obtain differentially expressed proteomes and unravel the redox proteomic status in mitochondria have yielded clinical insights into the neuropathological mechanisms of disease, identification of disease early stage and evaluation of disease progression. Although current technical limitations hamper full exploitation of the mitochondrial proteome in neurosciences, future advances are predicted to provide identification of specific therapeutic targets for neurodegenerative disorders.

Microglial cells are brain-resident macrophages engaged in surveillance and maintained in a constant state of relative inactivity. However, their involvement in autoimmune diseases indicates that in pathological conditions microglia gain an inflammatory phenotype. The mechanisms underlying this change in the microglial phenotype are still unclear. Since metabolism is an important modulator of immune cell function, we focused our attention on glutamine synthetase (GS), a modulator of the response to lipopolysaccharide (LPS) activation in other cell types, which is expressed by microglia.GS inhibition enhances release of inflammatory mediators of LPS-activated microglia in vitro, leading to perturbation of the redox balance and decreased viability of cocultured neurons. GS inhibition also decreases insulin-mediated glucose uptake in microglia. In vivo, microglia-specific GS ablation enhances expression of inflammatory markers upon LPS treatment. In the spinal cords from experimental autoimmune encephalomyelitis (EAE), GS expression levels and glutamine/glutamate ratios are reduced.Recently, metabolism has been highlighted as mediator of immune cell function through the discovery of mechanisms that (behind these metabolic changes) modulate the inflammatory response. The present study shows for the first time a metabolic mechanism mediating microglial response to a proinflammatory stimulus, pointing to GS activity as a master modulator of immune cell function and thus unraveling a potential therapeutic target.Our study highlights a new role of GS in modulating immune response in microglia, providing insights into the pathogenic mechanisms associated with inflammation and new strategies of therapeutic intervention. Antioxid. Redox Signal. 26, 351-363.

Down syndrome (DS) is one of the most common causes of intellectual disability, owing to trisomy of all or part of chromosome 21. DS is also associated with the development of Alzheimer disease (AD) neuropathology after the age of 40 years. To better clarify the cellular and metabolic pathways that could contribute to the differences in DS brain, in particular those involved in the onset of neurodegeneration, we analyzed the frontal cortex of DS subjects with or without significant AD pathology in comparison with age-matched controls, using a proteomics approach. Proteomics represents an advantageous tool to investigate the molecular mechanisms underlying the disease. From these analyses, we investigated the effects that age, DS, and AD neuropathology could have on protein expression levels. Our results show overlapping and independent molecular pathways (including energy metabolism, oxidative damage, protein synthesis, and autophagy) contributing to DS, to aging, and to the presence of AD pathology in DS. Investigation of pathomechanisms involved in DS with AD may provide putative targets for therapeutic approaches to slow the development of AD.

Accumulation of oxidative damage is a common feature of neurodegeneration that, together with mitochondrial dysfunction, point to the fact that reactive oxygen species are major contributors to loss of neuronal homoeostasis and cell death. Among several targets of oxidative stress, free-radical-mediated damage to proteins is particularly important in aging and age-related neurodegenerative diseases. In the majority of cases, oxidative-stress-mediated post-translational modifications cause non-reversible modifications of protein structure that consistently lead to impaired function. Redox proteomics methods are powerful tools to unravel the complexity of neurodegeneration, by identifying brain proteins with oxidative post-translational modifications that are detrimental for protein function. The present review discusses the current literature showing evidence of impaired pathways linked to oxidative stress possibly involved in the neurodegenerative process leading to the development of Alzheimer-like dementia. In particular, we focus attention on dysregulated pathways that underlie neurodegeneration in both aging adults with DS (Down's syndrome) and AD (Alzheimer's disease). Since AD pathology is age-dependent in DS and shows similarities with AD, identification of common oxidized proteins by redox proteomics in both DS and AD can improve our understanding of the overlapping mechanisms that lead from normal aging to development of AD. The most relevant proteomics findings highlight that disturbance of protein homoeostasis and energy production are central mechanisms of neurodegeneration and overlap in aging DS and AD. Protein oxidation affects crucial intracellular functions and may be considered a ‘leitmotif’ of degenerating neurons. Therapeutic strategies aimed at preventing/reducing multiple components of processes leading to accumulation of oxidative damage will be critical in future studies. Abbreviations: Aβ, amyloid β peptide; AD, Alzheimer’s disease; AGE, advanced glycation end-product; APP, amyloid precursor protein; CAT, catalase; Chr21, chromosome 21; CMA, chaperone-mediated autophagy; 2,4-DNPH, 2,4-dinitrophenylhydrazine; DS, Down's syndrome; ER, endoplasmic reticulum; ETC, electron transport chain; FBA, fructose bisphosphate aldolase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; GPX, glutathione peroxidase; GRP78, glucose-regulated protein of 78 kDa; HNE, 4-hydroxynonenal; HSC71, heat-shock cognate 71 stress protein; HSP, heat-shock protein; MAPK, mitogen-activated protein kinase; MCI, mild cognitive impairment; MDH, malate dehydrogenase; MeSOX, methionine sulfoxide; NFT, neurofibrillary tangle; 3-NT, 3-nitrotyrosine; OS, oxidative stress; PK, pyruvate kinase; PQC, protein quality control; PTM, post-translational modification; PUFA, polyunsaturated fatty acid; RNS, reactive nitrogen species; ROS, reactive oxygen species; SOD, superoxide dismutase; TCA, tricarboxylic acid; UCH-L1, ubiquitin C-terminal hydrolase L1; UPR, unfolded protein response; UPS, ubiquitin–proteasome system

This retrospective review on discoveries of the roles of oxidative stress in brain of subjects with Alzheimer disease (AD) and animal models thereof as well as brain from animal models of chemotherapy-induced cognitive impairment (CICI) results from the author receiving the 2013 Discovery Award from the Society for Free Radical Biology and Medicine. The paper reviews our laboratory's discovery of protein oxidation and lipid peroxidation in AD brain regions rich in amyloid β-peptide (Aβ) but not in Aβ-poor cerebellum; redox proteomics as a means to identify oxidatively modified brain proteins in AD and its earlier forms that are consistent with the pathology, biochemistry, and clinical presentation of these disorders; how Aβ in in vivo, ex vivo, and in vitro studies can lead to oxidative modification of key proteins that also are oxidatively modified in AD brain; the role of the single methionine residue of Aβ(1-42) in these processes; and some of the potential mechanisms in the pathogenesis and progression of AD. CICI affects a significant fraction of the 14 million American cancer survivors, and due to diminished cognitive function, reduced quality of life of the persons with CICI (called "chemobrain" by patients) often results. A proposed mechanism for CICI employed the prototypical ROS-generating and non-blood brain barrier (BBB)-penetrating chemotherapeutic agent doxorubicin (Dox, also called adriamycin, ADR). Because of the quinone moiety within the structure of Dox, this agent undergoes redox cycling to produce superoxide free radical peripherally. This, in turn, leads to oxidative modification of the key plasma protein, apolipoprotein A1 (ApoA1). Oxidized ApoA1 leads to elevated peripheral TNFα, a proinflammatory cytokine that crosses the BBB to induce oxidative stress in brain parenchyma that affects negatively brain mitochondria. This subsequently leads to apoptotic cell death resulting in CICI. This review outlines aspects of CICI consistent with the clinical presentation, biochemistry, and pathology of this disorder. To the author's knowledge this is the only plausible and self-consistent mechanism to explain CICI. These two different disorders of the CNS affect millions of persons worldwide. Both AD and CICI share free radical-mediated oxidative stress in brain, but the source of oxidative stress is not the same. Continued research is necessary to better understand both AD and CICI. The discoveries about these disorders from the Butterfield Laboratory that led to the 2013 Discovery Award from the Society of Free Radical and Medicine provide a significant foundation from which this future research can be launched.

Free radical-mediated damage to proteins is particularly important in aging and age-related neurodegenerative diseases, because in the majority of cases it is a non-reversible phenomenon that requires clearance systems for removal. Major consequences of protein oxidation are loss of protein function and the formation of large protein aggregates, which are often toxic to cells if allowed to accumulate. Deposition of aggregated, misfolded, and oxidized proteins may also result from the impairment of protein quality control (PQC) system, including protein unfolded response, proteasome, and autophagy. Perturbations of such components of the proteostasis network that provides a critical protective role against stress conditions are emerging as relevant factor in triggering neuronal death. In this outlook paper, we discuss the role of protein oxidation as a major contributing factor for the impairment of the PQC regulating protein folding, surveillance, and degradation. Recent studies from our group and from others aim to better understand the link between Down syndrome and Alzheimer’s disease neuropathology. We propose oxidative stress and alteration of proteostasis network as a possible unifying mechanism triggering neurodegeneration.

Down syndrome (DS) is the most frequent genetic cause of intellectual disability characterized by the presence of three copies of chromosome 21 (Chr21). Individuals with DS have sufficient neuropathology for a diagnosis of Alzheimer's disease (AD) after the age of 40years. The aim of our study is to gain new insights in the molecular mechanisms impaired in DS subjects that eventually lead to the development of dementia. We evaluate the PI3K/Akt/mTOR axis in the frontal cortex from DS cases (under the age of 40years) and DS with AD neuropathology compared with age-matched controls (Young and Old). The PI3K/Akt/mTOR axis may control several key pathways involved in AD that, if aberrantly regulated, affect amyloid beta (Aβ) deposition and tau phosphorylation. Our results show a hyperactivation of PI3K/Akt/mTOR axis in individuals with DS, with and without AD pathology, in comparison with respective controls. The PI3K/Akt/mTOR deregulation results in decreased autophagy, inhibition of IRS1 and GSK3β activity. Moreover, our data suggest that aberrant activation of the PI3K/Akt/mTOR axis acts in parallel to RCAN1 in phosphorylating tau, in DS and DS/AD. In conclusion, this study provides insights into the neuropathological mechanisms that may be engaged during the development of AD in DS. We suggest that deregulation of this signaling cascade is already evident in young DS cases and persist in the presence of AD pathology. The impairment of the PI3K/Akt/mTOR axis in DS population might represent a key-contributing factor to the neurodegenerative process that culminates in Alzheimer-like dementia.

Alzheimer disease (AD) is a progressive neurodegenerative disorder characterized by severe cognitive impairment, inability to perform activities of daily living and mood changes. Statins, long known to be beneficial in conditions where dyslipidemia occurs by lowering serum cholesterol levels, also have been proposed for use in neurodegenerative conditions, including AD. However, it is not clear that the purported effectiveness of statins in neurodegenerative disorders is directly related to cholesterol-lowering effects of these agents; rather, the pleiotropic functions of statins likely play critical roles. The aim of this review is to provide an overview on the new discoveries about the effects of statin therapy on the oxidative and nitrosative stress levels as well as on the modulation of the heme oxygenase/biliverdin reductase (HO/BVR) system in the brain. We propose a novel mechanism of action for atorvastatin which, through the activation of HO/BVR-A system, may contribute to the neuroprotective effects thus suggesting a potential therapeutic role in AD and potentially accounting for the observation of decreased AD incidence with persons on statin.

Depending on the published study, from 30 % to 70 % of cancer survivors who had undergone chemotherapy reported cognitive dysfunction, usually manifested as memory impairment, difficulty with reasoning, and problems multi-tasking, consistent with deficits of higher executive functioning. This chemotherapy induced cognitive impairment (CICI), often called “chemobrain” by patients, is a major contributor to decreased quality of life for a large percentage of the more than the current 15 million cancer survivors in the USA. More than 50 % of FDA-approved anti-cancer drugs are associated with reactive oxygen species (ROS) as part of their mechanisms of action. Doxorubicin (Dox) is a prototypical ROS generating chemotherapeutic agent used to treat solid tumors and lymphomas as part of multi-drug chemotherapeutic regimens. Dox produces the reactive superoxide radical anion (O2•–) in vivo through quinone to semi-quinone redox cycling. Neither Dox nor its major metabolite crosses the blood brain barrier. We previously reported that intraperitoneal (i.p.) Dox-administration to mice leads to plasma protein damage and elevation of tumor necrosis factor-alpha (TNF-α) in plasma and brain. We further reported that TNF-α elevation in brain leads to further central nervous system toxicity, including oxidative stress, mitochondrial dysfunction, neuronal death, and cognitive impairment. In the present study, we tested the hypothesis that TNF-α in brain was critical to these deleterious effects that contribute to CICI. We demonstrate that oxidative stress is ameliorated and mitochondrial function assessed by the Seahorse-determined oxygen consumption rate (OCR) is preserved in brain of Dox-treated TNF-α knockout (TNFKO) mice. Further, we show in TNFKO mouse brain protection against Dox-related decreased Cho/Cr ratio determined by magnetic resonance spectroscopy (MRS) and protection against loss of phospholipase D (PLD) activity. Coupled with findings from our previous studies, these current results provide important insights into the role of TNF-α in CICI.

It is generally accepted that alterations in metabolism are critical for the metastatic process; however, the mechanisms by which the metabolic changes are controlled by the major drivers of the metastatic process remain elusive. Here, we find that S100A4, a major metastasis-promoting protein, alters metabolic plasticity to drive tumor invasion and metastasis. Investigating how S100A4 regulates metabolism, we find that depletion of S100A4 decreases oxygen consumption rate, mitochondrial activity, and ATP production, and shifts cell metabolism to be more glycolytically active. We further identify that the 49 KD mitochondrial complex I subunit NADH dehydrogenase (ubiquinone) Fe-S protein 2 (NDUFS2) is regulated in an S100A4-dependent manner and that S100A4 and NDUFS2 exhibit co-occurrence at significant levels in various cancer types (cBioPortal). Importantly, we find that knockdown of NDUFS2 inhibits mitochondrial complex I activity, reduces cellular ATP level, and decreases the invasive capacity in three-dimensional (3D) growth, thus mimicking the biological effect resulting from S100A4 knockdown in A549 cells. Likewise, silencing S100A4 and NDUFS2 dramatically decreases the rate of metastasis as well as tumor growth in vivo. Finally, we provide evidence that cells with S100A4 and NDUFS2 depletion shift metabolism toward glycolysis through upregulating hexokinases expression and that suppressing S100A4 signaling sensitizes lung cancer cells to glycolysis inhibition. Together, our findings uncover a novel function of S100A4 in mitochondrial metabolism and highlight the importance of NDUFS2 in regulating the plasticity of mitochondrial metabolism by S100A4 to promote the invasion and metastasis in lung cancer. Citation Format: Lili Liu, Lei Qi, Teresa Knifley, Dava W. Piecoro, Piotr Rychahou, Jinpeng Liu, Mihail I. Mitov, Jeremiah Martin, Chi Wang, Jianrong Wu, Heidi L. Weiss, D. Allan Butterfield, B. Mark Evers, Kathleen L. O'Connor, Min Chen. S100A4 alters mitochondrial metabolism to promote invasion and metastasis of non-small cell lung cancer cells through upregulation of NDUFS2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1119.

Glycogen synthase kinase (GSK) -3β is a multifunctional protein that has been implicated in the pathological characteristics of Alzheimer’s disease (AD), including the heightened levels of neurofibrillary tangles, amyloid-beta (Aβ) and neurodegeneration. In this study we used 12 month old SAMP8 mice, an AD model, to examine the effects GSK-3β may cause regarding the cognitive impairment and oxidative stress associated with AD. To suppress the level of GSK-3β, SAMP8 mice were treated with an antisense oligonucleotide (GAO) directed at this kinase. We measured a decreased level of GSK-3β in the cortex of the mice, indicating the success of the antisense treatment. Learning and memory assessments of the SAMP8 mice were tested post-antisense treatment using an aversive T-maze and object recognition test, both of which observably improved. In cortex samples of the SAMP8 mice, decreased levels of protein carbonyl and protein-bound HNE were measured indicating decreased oxidative stress. Nuclear factor erythroid -2-related factor 2 (Nrf2) is a transcription factor known to increase the level of many antioxidants, including glutathione-S transferase (GST), and is negatively regulated by the activity of GSK-3β. Our results indicated the increased nuclear localization of Nrf2 and level of GST, suggesting the increased activity of the transcription factor as a result of GSK-3β suppression, consistent with the decreased oxidative stress observed. Consistent with the improved learning and memory, and consistent with GSK-3b being a tau kinase, we observed decreased tau phosphorylation in brain of GAO-treated SAMP8 mice compared to that of RAO-treated SAMP8 mice. Lastly, we examined the ability of GAO to cross the blood-brain barrier and determined it to be possible. The results presented in this study demonstrate that reducing GSK-3 with a phosphorothionated antisense against GSK-3 improves learning and memory, reduces oxidative stress, possibly coincident with increased levels of the antioxidant transcriptional activity of Nrf2, and decreases tau phosphorylation. Our study supports the notion of GAO as a possible treatment for AD.

It is well established that the risk to develop neurodegenerative disorders increases with chronological aging. Accumulating studies contributed to characterize the age-dependent changes either at gene and protein expression level which, taken together, show that aging of the human brain results from the combination of the normal decline of multiple biological functions with environmental factors that contribute to defining disease risk of late-life brain disorders. Finding the "way out" of the labyrinth of such complex molecular interactions may help to fill the gap between "normal" brain aging and development of age-dependent diseases. To this purpose, proteomics studies are a powerful tool to better understand where to set the boundary line of healthy aging and age-related disease by analyzing the variation of protein expression levels and the major post translational modifications that determine "protein" physio/pathological fate. Increasing attention has been focused on oxidative modifications due to the crucial role of oxidative stress in aging, in addition to the fact that this type of modification is irreversible and may alter protein function. Redox proteomics studies contributed to decipher the complexity of brain aging by identifying the proteins that were increasingly oxidized and eventually dysfunctional as a function of age. The purpose of this review is to summarize the most important findings obtained by applying proteomics approaches to murine models of aging with also a brief overview of some human studies, in particular those related to dementia.

In addition to the well-known effects of vitamin D (VitD) in maintaining bone health, there is increasing appreciation that this vitamin may serve important roles in other organs and tissues, including the brain. Given that VitD deficiency is especially widespread among the elderly, it is important to understand how the range of serum VitD levels that mimic those found in humans (from low to high) affects the brain during aging from middle-age to old-age. To address this issue, twenty-seven male F344 rats were split into three groups and fed isocaloric diets containing low (100 IU/kg food), control (1000 IU/kg food), or high (10000 IU/kg food) VitD beginning at middle-age (12 months) and continued for a period of 4–5 months. We compared the effects of these dietary VitD manipulations on oxidative and nitrosative stress measures in posterior brain cortices. The low VitD group showed global elevation of 3-nitrotyrosine (3-NT) compared to control and high VitD treated groups. Further investigation showed that this elevation may involve dysregulation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway and NF-κB mediated transcription of inducible nitric oxide synthase (iNOS) as indicated by translocation of NF-κB to the nucleus and elevation of iNOS levels. Proteomic techniques were used to provide insights into potential mechanisms underlying these effects. Several brain proteins were found at significantly elevated levels in low VitD group compared to the control and high VitD groups. Three of these proteins, 6-phosphofructokinase, triosephosphate isomerase, and pyruvate kinase, are involved directly in glycolysis. Two others, peroxiredoxin-3 and DJ-1/PARK7, have peroxidase activity and are found in mitochondria. Peptidyl-prolyl cis-trans isomerase A (PPIA or cyclophilin A) has been shown to have multiple roles including protein folding, regulation of protein kinases and phosphatases, immunoregulation, cell signaling, and redox status. Together, these results suggest that dietary VitD deficiency contributes to significant nitrosative stress in brain and may promote cognitive decline in middle-aged and elderly adults.

Alzheimer disease is an age-related neurodegenerative condition. AD is histopathologically characterized by the presence of three main hallmarks: senile plaque (SP, rich in amyloid-beta peptide), neuronal fibrillary tangles (NFT, rich in phosphorylated tau protein), and synapse loss. However, definitive biomarkers for this devastating disease in living people are still lacking. In the present study, we showed that levels of oxidative stress markers are significantly increased in the mitochondria isolated from lymphocytes of subjects with mild cognitive impairment (MCI) compared to cognitively normal individuals. Further, increase in mitochondrial oxidative stress in MCI is associated with the MMSE scores, vitamin E components, and beta-carotene. Further, proteomics approach showed that the alterations in the levels of thioredoxin-dependent peroxide reductase, myosin light polypeptide 6, and ATP synthase subunit beta might be important in the progression and pathogenesis of AD. Increased understanding of oxidative stress and protein alterations in easily obtainable peripheral tissues will be helpful in developing biomarkers to combat this devastating disorder.

Amyloid β-peptide (Aβ) plays a central role in the pathophysiology of Alzheimer's disease (AD) through the induction of oxidative stress. This peptide is produced by proteolytic cleavage of amyloid precursor protein (APP) by the action of β- and γ-secretases. Previous studies demonstrated that reduction of Aβ, using an antisense oligonucleotide (AO) directed against the Aβ region of APP, reduced oxidative stress-mediated damage and prevented or reverted cognitive deficits in senescence-accelerated prone mice (SAMP8), a useful animal model for investigating the events related to Aβ pathology and possibly to the early phase of AD. In the current study, aged SAMP8 were treated by AO directed against PS-1, a component of the γ-secretase complex, and tested for learning and memory in T-maze foot shock avoidance and novel object recognition. Brain tissue was collected to identify the decrease of oxidative stress and to evaluate the proteins that are differently expressed and oxidized after the reduction in free radical levels induced by Aβ. We used both expression proteomics and redox proteomics approaches. In brain of AO-treated mice a decrease of oxidative stress markers was found, and the proteins identified by proteomics as expressed differently or nitrated are involved in processes known to be impaired in AD. Our results suggest that the treatment with AO directed against PS-1 in old SAMP8 mice reverses learning and memory deficits and reduces Aβ-mediated oxidative stress with restoration to the normal condition and identifies possible pharmacological targets to combat this devastating dementing disease.

1. Oxidatively Modified Proteins and Proteomic Technologies. 1.1 Chemical Modification of Proteins by Reactive Oxygen Species (E. Stadtman & R. Levine). 1.2 The Chemistry of Protein Modifications Elicited By Nitric Oxide and Related Nitrogen Oxides (D. Thomas, et al.). 1.3 Mass Spectrometry Approaches for the Molecular Characterization of Oxidatively/Nitrosatively Modified Proteins (A. Scaloni). 1.4 Thiol-disulfide Oxidoreduction of Protein Cysteines: Old Methods Revisted for Proteomics (V. Bonetto & P. Ghezzi). 1.5 Carbonylated Proteins and their Implication in Physiology and Pathology (R. Levine & E. Stadtman). 1.6 S-Nitrosation of Cysteine thiols as a Redox Signal (Y. Zhang & N. Hogg). 1.7 Detection of Glycated and GlycoOxidated Proteins (A. Lapolla, et al.). 1.8 MudPIT (Multidimensaional Protein Identification Technology) for Identification of Post-translational Protein Modifications in Complex Biological Mixtures (S. Thomas, et al.). 1.9 Use of a Proteomic Technique to Identify Oxidant-Sensitive Thiol Proteins in Cultured Cells (M. Hampton, et al.). 1.10 ICAT (Isotope-Code Affinity Tag) Approach to Redox Proteomics: Identification and Quantification of Oxidant-Sensitive Protein Thiols (M. Sethuraman, et al.). 1.11 Quantitative Determination of Free and Protein-Associated 3-nitrotyrosine and S-nitrosothiols in the Circulation by Mass Spectrometry and Other Methodologies: A Critical Review and Discussion from the Analytical and Review Point of View (D. Tsikas). 2. Cellular Aspects of Protein Oxidation. 2.1 The Covalent Advantage: A New Paradigm for Cell Signaling Mediated by Thiol Reactive Lipid Oxidation Products (D. Dickinson, et al.). 2.2 Early Molecular Events During Response to Oxidative Stress in Human Cells by Differential Proteomics (G. Tell). 2.3 Oxidative Damage to Proteins: Structural Modifications and Consequences in Cell Function (E. Cabiscol & J. Ros). 2.4 Oxidative Damage and Cellular Senescence: Lessons from Bacteria and Yeast (T. Nystrom). 3. Redox Proteomic Analysis in Human Diseases. 3.1 Proteins as Sensitive Biomarkers of Human Conditions Associated with Oxidative Stress (I. Dalle-Donne, et al.). 3.2 Degradation and Accumulation of Oxidized Proteins in Age-Related Diseases (P. Voss & T. Grune). 3.3 Redox Proteomics: A New Approach to Investgate Oxidative Stress in Alzheimer's Diseases (D. Butterfield, et al.). 3.4 Oxidized Proteins in Cardiac Ischemia-Reperfusion (J. Brennan & P. Eaton). 3.5 Proteome Anaylsis of Oxidative Stress: Glutathionyl Hemoglobin in Diabetic and Uremic Patients (T. Niwa). 3.6 Glyco-Oxidative Biochemistry in Diabetic Renal Injury (T. Miyata). 3.7 Quantitative Screnning of Protein Glycation, Oxidation, and Nitration Adducts by LC-MS/MS: Protein Damage in Diabetes, Uremia, Cirrhosis, and Alzheimer's Disease (P. Thornalley). 3.8 Protein Targets and Functional Consequences of Tyrosine Nitration in Vascular Disease (L. Baker, et al.). 3.9 Oxidation of Artery Wall Proteins by Myeloperoxidase: A proteomics Approach (T. Vaisar & J. Heinecke). 3.10 Oxidative Stress and Protein Oxidation in Pre-Eclampsia (M. Raijmakers, et al.). 3.11 The Involvement of Oxidants in the Etiology of Chronic Airway Diseases: Proteomic Approaches to Identify Redox Processes in Epithelial Cell Signal and Inflammation (A. van der Vliet, et al.). 3.12 Sequestering Agents of Intermediate Reactive Aldehydes as Inhibitors of Advanced Lipoxidation End-Products (ALEs) (M. Carini, et al.).

Proteomics techniques are continuously being developed to further understanding of biology and disease. Many of the pathways that are relevant to disease mechanisms rely on the identification of post-translational modifications (PTMs) such as phosphorylation, acetylation, and glycosylation. Much attention has also been focused on oxidative PTMs which include protein carbonyls, protein nitration, and the incorporation of fatty acids and advanced glycation products to amino acid side chains, amongst others. The introduction of these PTMs in the cell can occur due to the attack of reactive oxygen and nitrogen species (ROS and RNS, respectively) on proteins. ROS and RNS can be present as a result of normal metabolic processes as well as external factors such as UV radiation, disease, and environmental toxins. The imbalance of ROS and RNS with antioxidant cellular defenses leads to a state of oxidative stress, which has been implicated in many diseases. Redox proteomics techniques have been used to characterize oxidative PTMs that result as a part of normal cell signaling processes as well as oxidative stress conditions. This review highlights many of the redox proteomics techniques which are currently available for several oxidative PTMs and brings to the reader's attention the application of redox proteomics for understanding disease pathogenesis in neurodegenerative disorders and others such as cancer, kidney, and heart diseases.

Significance: Alzheimer disease (AD) is an age-related neurodegenerative disease. AD is characterized by progressive cognitive impairment. One of the main histopathological hallmarks of AD brain is the presence of senile plaques (SPs) and another is elevated oxidative stress. The main component of SPs is amyloid beta-peptide (Aβ) that is derived from the proteolytic cleavage of amyloid precursor protein. Recent Advances: Recent studies are consistent with the notion that methionine present at 35 position of Aβ is critical to Aβ-induced oxidative stress and neurotoxicity. Further, we also discuss the signatures of oxidatively modified brain proteins, identified using redox proteomics approaches, during the progression of AD. Critical Issues: The exact relationships of the specifically oxidatively modified proteins in AD pathogenesis require additional investigation. Future Directions: Further studies are needed to address whether the therapies directed toward brain oxidative stress and oxidatively modified key brain proteins might help delay or prevent the progression of AD. Antioxid. Redox Signal. 19, 823–835.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder and represents one of the most disabling conditions. AD shares many features in common with systemic insulin resistance diseases, suggesting that it can be considered as a metabolic disease, characterized by reduced insulin-stimulated growth and survival signaling, increased oxidative stress (OS), proinflammatory cytokine activation, mitochondrial dysfunction, impaired energy metabolism, and altered protein homeostasis. Recent Advances: Reduced glucose utilization and energy metabolism in AD have been associated with the buildup of amyloid-β peptide and hyperphosphorylated tau, increased OS, and the accumulation of unfolded/misfolded proteins. Mammalian target of rapamycin (mTOR), which is aberrantly activated in AD since early stages, plays a key role during AD neurodegeneration by, on one side, inhibiting insulin signaling as a negative feedback mechanism and, on the other side, regulating protein homeostasis (synthesis/clearance).It is likely that the concomitant and mutual alterations of energy metabolism-mTOR signaling-protein homeostasis might represent a self-sustaining triangle of harmful events that trigger the degeneration and death of neurons and the development and progression of AD. Intriguingly, the altered cross-talk between the components of such a triangle of death, beyond altering the redox homeostasis of the neuron, is further exacerbated by increased levels of OS that target and impair key components of the pathways involved. Redox proteomic studies in human samples and animal models of AD-like dementia led to identification of oxidatively modified components of the pathways composing the triangle of death, therefore revealing the crucial role of OS in fueling this aberrant vicious cycle.The identification of compounds able to restore the function of the pathways targeted by oxidative damage might represent a valuable therapeutic approach to slow or delay AD. Antioxid. Redox Signal. 26, 364-387.

Clinical studies suggest a link between peripheral insulin resistance and cognitive dysfunction. Post-mortem analyses of Alzheimer disease (AD) subjects revealed insulin resistance in the brain, suggesting a role of this condition in cognitive deficits observed in AD. In this review, we focus on the glucagon-like peptide-1 (GLP-1) signaling pathway, whose role in the brain is collecting increasing attention because of its association with insulin signaling activation. Areas covered: The role of GLP-1-mediated effects in the brain and how they are affected along the progression of AD pathology is discussed. Furthermore, we provide a comprehensive discussion about the use of GLP-1 mimetics drugs, which have been developed as a treatment for T2DM but seem to possess a number of other physiological properties, including neuroprotective and anti-inflammatory effects, that may be useful to slow AD progression. Expert commentary: The repurposing of antidiabetic drugs for the modulation of brain insulin resistance in AD appears to be of great interest. The beneficial effects on synaptogenesis, neurogenesis, and cell repair as well as the reduction of the chronic inflammatory response, and most importantly the reduction of amyloid plaques in the brain indicate that these drugs have promise as novel treatments for AD.

Oxidative stress is involved in various and numerous pathological states including several age-related neurodegenerative diseases. Peroxidation of the membrane lipid bilayer is one of the major sources of free radical-mediated injury that directly damages neurons causing increased membrane rigidity, decreased activity of membrane-bound enzymes, impairment of membrane receptors and altered membrane permeability and eventual cell death. Moreover, the peroxidation of polyunsaturated fatty acids leads to the formation of aldehydes, which can act as toxic by-products. One of the most abundant and cytotoxic lipid -derived aldehydes is 4-hydroxy 2-nonenal (HNE). HNE toxicity is mainly due to the alterations of cell functions by the formation of covalent adducts of HNE with proteins. A key marker of lipid peroxidation, HNE-protein adducts, were found to be elevated in brain tissues and body fluids of Alzheimer disease, Parkinson disease, Huntington disease and amyotrophic lateral sclerosis subjects and/or models of the respective age-related neurodegenerative diseases. Although only a few proteins were identified as common targets of HNE modification across all these listed disorders, a high overlap of these proteins occurs concerning the alteration of common pathways, such as glucose metabolism or mitochondrial function that are known to contribute to cognitive decline. Within this context, despite the different etiological and pathological mechanisms that lead to the onset of different neurodegenerative diseases, the formation of HNE-protein adducts might represent the shared leit-motif, which aggravates brain damage contributing to disease specific clinical presentation and decline in cognitive performance observed in each case.

Proteomics has a wide range of applications, including determination of differences in the proteome in terms of expression and post-translational protein modifications. Redox proteomics allows the identification of specific targets of protein oxidation in a biological sample. Using proteomic techniques, apolipoprotein A-I (ApoA-I) has been found at decreased levels in subjects with a variety of neurodegenerative disorders including in the serum and cerebrospinal fluid (CSF) of Alzheimer disease (AD), Parkinson disease (PD), and Down syndrome (DS) with gout subjects. ApoA-I plays roles in cholesterol transport and regulation of inflammation. Redox proteomics further showed ApoA-I to be highly oxidatively modified and particularly susceptible to modification by 4-hydroxy-2-trans-nonenal (HNE), a lipid peroxidation product. In the current review, we discuss the consequences of oxidation of ApoA-I in terms of neurodegeneration. ROS-associated chemotherapy related ApoA-I oxidation leads to elevation of peripheral levels of tumor necrosis factor-α (TNF-α) that can cross the blood-brain barrier (BBB) causing a signaling cascade that can contribute to neuronal death, likely a contributor to what patients refer to as "chemobrain." Current evidence suggests ApoA-I to be a promising diagnostic marker as well as a potential target for therapeutic strategies in these neurodegenerative disorders.

Alzheimer disease (AD) is an age-related neurodegenerative disease characterized by the presence of three pathological hallmarks: synapse loss, extracellular senile plaques (SP) and intracellular neurofibrillary tangles (NFTs). The major component of SP is amyloid β-peptide (Aβ), which has been shown to induce oxidative stress. The AD brain shows increased levels of lipid peroxidation products, including 4-hydroxy-2-nonenal (HNE). HNE can react covalently with Cys, His, or Lys residues on proteins, altering structure and function of the latter. In the present study we measured the levels of the HNE-modified lipoic acid in brain of subjects with AD and age-matched controls. Lipoic acid is a key co-factor for a number of proteins including pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, key complexes for cellular energetics. We observed a significant decrease in the levels of HNE-lipoic acid in the AD brain compared to that of age-matched controls. To investigate this phenomenon further, the levels and activity of lipoamide dehydrogenase (LADH) were measured in AD and control brains. Additionally, LADH activities were measured after in-vitro HNE-treatment to mice brains. Both LADH levels and activities were found to be significantly reduced in AD brain compared to age-matched control. HNE-treatment also reduced the LADH activity in mice brain. These data are consistent with a two-hit hypothesis of AD: oxidative stress leads to lipid peroxidation that, in turn, causes oxidative dysfunction of key energy-related complexes in mitochondria, triggering neurodegeneration. This study is consonant with the notion that lipoic acid supplementation could be a potential treatment for the observed loss of cellular energetics in AD and potentiate the antioxidant defense system to prevent or delay the oxidative stress in and progression of this devastating dementing disorder., Graphical abstract The NADH-dependent oxido-reductase enzyme lipoamide dehydrogenase (LADH) is an important member of the mitochondrial energy generation complex. Alteration of the structure and activity of LADH by elevated reactive oxygen species (ROS) may hamper energy metabolism and ATP production. Lipoic acid (LA) must be in the reduced form as part of its co-factor function for mitochondrial TCA complexes such as α-ketoglutarate dehydrogenase. However, oxidized LADH is unable to reduce LA to DHLA, and therefore HNE is unable to bind to DHLA efficiently. Consequently, in this study, decreased LA-HNE binding was observed in Alzheimer disease brain. Severe effects on learning, memory, and higher executive functioning, all significantly lost in AD patients, would be expected. Supplementation of LA conceivably may protect LADH from ROS or end products of ROS (e.g., HNE) by self-sacrifice mechanism, potentially providing protection against dementia or slowing the rate of progression of AD. Highlights ► HNE-bound lipoic acid (HNE-LA) levels were decreased in the IPL region of AD brain. ► The TCA enzyme LADH converts inactive lipoic acid to its active-antioxidant form. ► LADH levels and activity were deceased in AD in IPL brain region. ► In-vitro HNE-treatment to mouse brain reduced LADH activity. ► The results fit the two-hit hypothesis of AD.

Down syndrome (DS), trisomy of chromosome 21, is the most common genetic form of intellectual disability. The neuropathology of DS involves multiple molecular mechanisms, similar to AD, including the deposition of beta-amyloid (Aβ) into senile plaques and tau hyperphosphorylationg in neurofibrillary tangles. Interestingly, many genes encoded by chromosome 21, in addition to being primarily linked to amyloid-beta peptide (Aβ) pathology, are responsible for increased oxidative stress (OS) conditions that also result as a consequence of reduced antioxidant system efficiency. However, redox homeostasis is disturbed by overproduction of Aβ, which accumulates into plaques across the lifespan in DS as well as in AD, thus generating a vicious cycle that amplifies OS-induced intracellular changes. The present review describes the current literature that demonstrates the accumulation of oxidative damage in DS with a focus on the lipid peroxidation by-product, 4-hydroxy-2-nonenal (HNE). HNE reacts with proteins and can irreversibly impair their functions. We suggest that among different post-translational modifications, HNE-adducts on proteins accumulate in DS brain and play a crucial role in causing the impairment of glucose metabolism, neuronal trafficking, protein quality control and antioxidant response. We hypothesize that dysfunction of these specific pathways contribute to accelerated neurodegeneration associated with AD neuropathology.

AimsAmong the putative mechanisms proposed to be common factors in Down syndrome (DS) and Alzheimer's disease (AD) neuropathology, deficits in protein quality control (PQC) have emerged as a unifying mechanism of neurodegeneration. Considering that disturbance of protein degradation systems is present in DS and that oxidized/misfolded proteins require polyubiquitinylation for degradation via the ubiquitin proteasome system, this study investigated if dysregulation of protein polyubiquitinylation is associated with AD neurodegeneration in DS.ResultsPostmortem brains from DS cases before and after development of AD neuropathology and age-matched controls were analyzed. By selectively isolating polyubiquitinated proteins, we were able to identify specific proteins with an altered pattern of polyubiquitinylation as a function of age. Interestingly, we found that oxidation is coupled with polyubiquitinylation for most proteins mainly involved in PQC and energy metabolism.InnovationThis is the first study showing alteration of the polyubiquitinylation profile as a function of aging in DS brain compared with healthy controls. Understanding the onset of the altered ubiquitome profile in DS brain may contribute to identification of key molecular regulators of age-associated cognitive decline.ConclusionsDisturbance of the polyubiquitinylation machinery may be a key feature of aging and neurodegeneration. In DS, age-associated deficits of the proteolytic system may further exacerbate the accumulation of oxidized/misfolded/polyubiquitinated proteins, which is not efficiently degraded and may become harmful to neurons and contribute to AD neuropathology. Antioxid. Redox Signal. 26, 280-298.

Mitochondria are considered to be the “power plants” of the cell, but can also initiate and execute cell death, stimulated by oxidative stress (OS). OS induced mitochondrial dysfunction is characterized by a loss in oxygen consumption and reduced ATP production. Curcumin, as a potential therapeutic, has been explored as a candidate for mitochondrial OS suppression, but rapid metabolism and aqueous insolubility has prevented it from being effective. Further, efficient delivery of curcumin via the incorporation into nanocarriers has again been limited due to low drug loading capacities and/or significant burst release, resulting in acute cytotoxicity. Hence, to increase the therapeutic potential and reduce the toxic effects of curcumin, curcumin conjugated poly(β-amino ester) nanogels (CNGs) were synthesized using Michael addition chemistry. This approach provided easy control over the nanogel size, with CNGs showing a uniform release of active curcumin over 48 h with no burst release. This controlled release system significantly increased the safety limit for curcumin, with a ten fold increase in the cytotoxic threshold, as compared to free curcumin. Further, real-time mitochondrial response analysis with the Seahorse XF96 showed effective and prolonged suppression of H 2 O 2 induced mitochondrial oxidative stress upon pre-treating endothelial cells with CNGs and this potential of nanogels was studied at different pre-treatment times prior to H 2 O 2 exposure.

Alzheimer disease (AD) is a progressive neurodegenerative disease that affects the elderly population with complex etiology. Many hypotheses have been proposed to explain different causes of AD, but the exact mechanisms remain unclear. In this review, we focus attention on the oxidative-stress hypothesis of neurodegeneration and we discuss redox proteomics approaches to analyze post-mortem human brain from AD brain. Collectively, these studies have provided valuable insights into the molecular mechanisms involved both in the pathogenesis and progression of AD, demonstrating the impairment of numerous cellular processes such as energy production, cellular structure, signal transduction, synaptic function, mitochondrial function, cell cycle progression, and degradative systems. Each of these cellular functions normally contributes to maintain healthy neuronal homeostasis, so the deregulation of one or more of these functions could contribute to the pathology and clinical presentation of AD. In particular, we discuss the evidence demonstrating the oxidation/dysfunction of a number of enzymes specifically involved in energy metabolism that support the view that reduced glucose metabolism and loss of ATP are crucial events triggering neurodegeneration and progression of AD.

Traumatic brain injury (TBI) is a spontaneous event in which sudden trauma and secondary injury cause brain damage. Symptoms of TBI can range from mild to severe depending on extent of injury. The outcome can span from complete patient recovery to permanent memory loss and neurological decline. Currently, there is no known cure for TBI; however, immediate medical attention after injury is most beneficial for patient recovery. It is a well-established concept that imbalances in the production of reactive oxygen species (ROS), reactive nitrogen species (RNS), and native antioxidant mechanisms have been shown to increase oxidative stress. Over the years, proteomics has been used to identify specific biomarkers in diseases such as cancers and neurological disorders such as Alzheimer disease and Parkinson disease. As TBI is a risk factor for a multitude of neurological diseases, biomarkers for this phenomenon are a likely field of study in order to confirm diagnosis. This review highlights the current proteomics studies that investigated excessively nitrated proteins and those altered by lipid peroxidation in TBI. This review also highlights possible diagnostic measures and provides insights for future treatment strategies.

Aging is the major risk factor associated with neurodegenerative diseases, including Alzheimer's disease (AD). Until now no clear understanding of the mechanisms of initiation and progression of this dementing disorder exists. Based on the studies that have been conducted so far amyloid β-peptide (Aβ), a protein found in senile plaques, one of the key pathological hallmarks of AD, has been reported to be critical in the pathogenesis of AD. Studies from our laboratory and others showed that Aβ can induce oxidative stress, which leads to oxidative modification of biomolecules, thereby diminishing the normal functions of neuronal cells and eventually leading to loss of neurons and AD. In this review paper, we summarize evidence of oxidative stress in brains of AD and mild cognitive impairment patients, as well as the results from redox proteomics studies. The investigations have provided insights into the downstream effects of oxidative modification of key brain proteins in the pathogenesis of AD. Based on these redox proteomics results, we suggest future areas of research that could be considered to better understand this devastating dementing disorder.

Several studies showed increased oxidative and nitrosative stress in plasma from patients with Alzheimer's disease (AD), however, little and controversial knowledge has emerged about the antioxidant functionality of the heme oxygenase- 1/biliverdin reductase-A (HO-1/BVR-A) system in blood. The current study reports increased levels of both HO-1 and BVR-A in plasma from probable AD patients, as a result of the increased oxidative environment. However, the increase of oxidative stress in plasma result also in the increase of BVR-A 3-nitrotyrosine levels and the decrease of BVR-A phosphotyrosine levels and reductase activity, suggesting that nitrosative stress play the prominent oxidative role in plasma during AD. Our data on HO- 1/BVR-A status in plasma closely correlate with recent reports in hippocampus of subjects with AD and arguably its early form, mild cognitive impairment. Moreover, we show that alterations on HO-1/BVR-A system are tightly connected with cognitive decline indexed by Mini-Mental Status Exam scores. We hypothesize that the HO-1/BVR-A system status in plasma might reflect the ongoing situation in the brain, offering an important biochemical tool for the potential prediction of AD at the earliest stages of the disease.

Oxidative stress has been associated with the onset and progression of mild cognitive impairment (MCI) and Alzheimer disease (AD). AD and MCI brain and plasma display extensive oxidative stress as indexed by protein oxidation, lipid peroxidation, free radical formation, DNA oxidation, and decreased antioxidants. The most abundant endogenous antioxidant, glutathione, plays a significant role in combating oxidative stress. The ratio of oxidized to reduced glutathione is utilized as a measure of intensity of oxidative stress. Antioxidants have long been considered as an approach to slow down AD progression. In this review, we focus on the elevation on glutathione through N-acetyl-cysteine (NAC) and γ-glutamylcysteine ethyl ester (GCEE) as a potential therapeutic approach for Alzheimer disease. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.

Objective Doxorubicin-induced acute cardiotoxicity is associated with the Gly671Val (G671V; rs45511401) variant of multidrug resistance-associated protein 1 (MRP1). Doxorubicin redox cycling causes lipid peroxidation and generation of the reactive electrophile, 4-hydroxy-2-trans-nonenal (HNE). Glutathione forms conjugates with HNE, yielding an MRP1 substrate, GS-HNE, whose intracellular accumulation can cause toxicity. Methods We established stable HEK293 cell lines overexpressing wild-type MRP1 (HEKMRP1), G671V (HEKG671V), and R433S (HEKR433S), a variant not associated with doxorubicin-induced cardiotoxicity and investigated the sensitivity of HEKG671V cells to doxorubicin and transport capacity of G671V toward GS-HNE. Results In ATP-dependent transport studies using plasma membrane-derived vesicles, the Vmax (pmol/min/mg) for GS-HNE transport was the lowest for G671V (69±4) and the highest for R433S (972±213) compared with wild-type MRP1 (416±22), whereas the Km values were 2.8±0.4, 6.0 or more, and 1.7±0.2 µmol/l, respectively. In cells, the doxorubicin IC50 (48 h) was not different in HEKMRP1 (463 nmol/l) versus HEKR433S (645 nmol/l), but this parameter was significantly lower in HEKG671V (181 nmol/l). HEKG671V retained significantly (approximately 20%) more, whereas HEKR433S retained significantly less intracellular doxorubicin than HEKMRP1. Similarly, HEKG671V cells treated with 1.5 µmol/l of doxorubicin for 24 h retained significantly more GS-HNE. In cells treated with 0.5 µmol/l of doxorubicin for 48 , glutathione and glutathione disulfide levels and the glutathione/glutathione disulfide ratio were significantly decreased in HEKG671V versus HEKMRP1; these values were similar in HEKR433S versus HEKMRP1. Conclusion These data suggest that decreased MRP1-dependent GS-HNE efflux contributes to increased doxorubicin toxicity in HEKG671V and potentially in individuals carrying the G671V variant.

Adriamycin (ADR) is a potent anticancer drug used to treat a variety of cancers. Patients treated with ADR have experienced side effects such as heart failure, cardiomyopathy, and "chemobrain", which have been correlated to changes in protein expression in the heart and brain. In order to better understand cellular responses that are disrupted following ADR treatment in immune tissues, this work focuses on spleen. Significantly reduced spleen sizes were found in ADR-treated mice. Global isotopic labeling of tryptic peptides and nanoflow reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) were employed to determine differences in the relative abundances of proteins from ADR-treated mice relative to controls. Fifty-nine proteins of the 388 unique proteins identified showed statistically significant differences in expression levels following acute ADR treatment. Differentially expressed proteins are involved in processes such as cytoskeletal structural integrity, cellular signaling and transport, transcription and translation, immune response, and Ca(2+) binding. These are the first studies to provide insight to the downstream effects of ADR treatment in a peripheral immune organ such as spleen using proteomics.