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Aging is a major risk factor to develop neurodegenerative diseases and is associated with decreased buffering capacity of the proteostasis network. We investigated the significance of the unfolded protein response (UPR), a major signaling pathway activated to cope with endoplasmic reticulum (ER) stress, in the functional deterioration of the mammalian brain during aging. We report that genetic disruption of the ER stress sensor IRE1 accelerated age-related cognitive decline. In mouse models, overexpressing an active form of the UPR transcription factor XBP1 restored synaptic and cognitive function, in addition to reducing cell senescence. Proteomic profiling of hippocampal tissue showed that XBP1 expression significantly restore changes associated with aging, including factors involved in synaptic function and pathways linked to neurodegenerative diseases. The genes modified by XBP1 in the aged hippocampus where also altered. Collectively, our results demonstrate that strategies to manipulate the UPR in mammals may help sustain healthy brain aging.

Alteration in the buffering capacity of the proteostasis network is an emerging feature of Alzheimer’s disease (AD), highlighting the occurrence of endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) is the main adaptive pathway to cope with protein folding stress at the ER. Inositol requiring enzyme-1 (IRE1) is an ER-located kinase and endoribonuclease that operates as a central ER stress sensor, enabling the establishment of adaptive and repair programs through the control of the expression of the transcription factor X-Box binding protein 1 (XBP1). A polymorphism in the XBP1 promoter has been suggested as a risk factor for AD. To artificially enforce the adaptive capacity of the UPR in the AD brain, we developed strategies to express the active form of XBP1 in neurons using preclinical models. Overexpression of an active form of XBP1 in the nervous system using transgenic mice significantly reduced the load of amyloid deposits in the cerebral cortex and hippocampus and preserved synaptic and cognitive function. Moreover, local delivery of XBP1 into the hippocampus of an AD mice using adeno-associated vectors improved long-term potentiation, memory performance, and dendritic spine density. Quantitative proteomics of the hippocampus revealed that XBP1 expression corrects a large proportion of the alterations observed in the 5xFAD model, restoring the levels of several synaptic proteins and factors involved in actin cytoskeleton regulation and axonal growth. Our results illustrate the therapeutic potential of targeting UPR-dependent gene expression programs as a strategy to ameliorate AD features and sustain synaptic function.

Endoplasmic reticulum (ER) stress is a prominent cellular alteration of diseases impacting the nervous system that are associated to the accumulation of misfolded and aggregated protein species during aging. The unfolded protein response (UPR) is the main pathway mediating adaptation to ER stress, but it can also trigger deleterious cascades of inflammation and cell death leading to cell dysfunction and neurodegeneration. Genetic and pharmacological studies in experimental models shed light into molecular pathways possibly contributing to ER stress and the UPR activation in human neuropathies. Most of experimental models are, however, based on the overexpression of mutant proteins causing familial forms of these diseases or the administration of neurotoxins that induce pathology in young animals. Whether the mechanisms uncovered in these models are relevant for the etiology of the vast majority of age-related sporadic forms of neurodegenerative diseases is an open question. Here, we provide a systematic analysis of the current evidence linking ER stress to human pathology and the main mechanisms elucidated in experimental models. Furthermore, we highlight the recent association of metabolic syndrome to increased risk to undergo neurodegeneration, where ER stress arises as a common denominator in the pathogenic crosstalk between peripheral organs and the nervous system.

Endoplasmic reticulum (ER) stress is a prominent cellular alteration of diseases impacting the nervous system that are associated to the accumulation of misfolded and aggregated protein species during aging. The unfolded protein response (UPR) is the main pathway mediating adaptation to ER stress, but it can also trigger deleterious cascades of inflammation and cell death leading to cell dysfunction and neurodegeneration. Genetic and pharmacological studies in experimental models shed light into molecular pathways possibly contributing to ER stress and the UPR activation in human neuropathies. Most of experimental models are, however, based on the overexpression of mutant proteins causing familial forms of these diseases or the administration of neurotoxins that induce pathology in young animals. Whether the mechanisms uncovered in these models are relevant for the etiology of the vast majority of age-related sporadic forms of neurodegenerative diseases is an open question. Here, we provide a systematic analysis of the current evidence linking ER stress to human pathology and the main mechanisms elucidated in experimental models. Furthermore, we highlight the recent association of metabolic syndrome to increased risk to undergo neurodegeneration, where ER stress arises as a common denominator in the pathogenic crosstalk between peripheral organs and the nervous system.

Brain ageing is the main risk factor to develop dementia and neurodegenerative diseases, associated with a decay in the buffering capacity of the proteostasis network. We investigated the significance of the unfolded protein response (UPR), a major signaling pathway to cope with ER stress, to the functional deterioration of the brain during aging. Genetic disruption of the ER stress sensor IRE1α accelerated cognitive and motor decline during ageing. Exogenous bolstering of the UPR by overexpressing an active form of the UPR transcription factor XBP1 restored synaptic and cognitive function, in addition to reducing cell senescence. Proteomic profiling of hippocampal tissue indicated that XBP1s expression attenuated age-related alterations to synaptic function and pathways linked to neurodegenerative diseases. Overall, our results demonstrate that strategies to manipulate the UPR in mammals may sustain healthy brain ageing.

Aging is the major risk factor for the development of dementia and neurodegenerative disorders, and the aging brain manifests severe deficits in buffering capacity by the proteostasis network. Accordingly, we investigated the significance of the unfolded protein response (UPR), a major signaling pathway that copes with endoplasmic reticulum (ER) stress, to normal mammalian brain aging. Genetic disruption of ER stress sensor IRE1α accelerated cognitive and motor dysfunction during aging. Exogenous bolstering of the UPR by overexpressing an active form of the transcription factor XBP1 restored synaptic and cognitive function in addition to reducing cell senescence. Remarkably, proteomic profiling of hippocampal tissue indicated that XBP1s expression corrected age-related alterations in synaptic function. Collectively, our data demonstrate that strategies to manipulate the UPR sustain healthy brain aging.One Sentence SummaryThe IRE1/XBP1 pathway dictates when and how brain function declines during aging.

The development of the central nervous system requires a series of morphogenetic events that shape brain and spinal cord structures. Several brain regions and neural circuits are formed by differential gene expression patterns and cell migration events involving neurons. During neurogenesis and neuritogenesis, increased demand for protein synthesis occurs to express key neuronal proteins to generate axons, dendrites, and synapsis. The endoplasmic reticulum (ER) is a central hub controlling protein homeostasis (proteostasis), impacting a wide range of cellular processes required for brain function. Although most of the field has focused on studying the role of ER stress in neurodegenerative diseases marked by abnormal protein aggregation, accumulating evidence indicates that ER proteostasis contributes to brain development and may impact neurodevelopmental processes such as neuronal migration, differentiation, and function. Here, we review emerging evidence linking neurodevelopment with ER proteostasis and its relevance to human disorders.

Altered proteostasis is a salient feature of Alzheimer's disease (AD), highlighting the occurrence of endoplasmic reticulum (ER) stress and abnormal protein aggregation. ER stress triggers the activation of the unfolded protein response (UPR), a signaling pathway that enforces adaptive programs to sustain proteostasis or eliminate terminally damaged cells. IRE1 is an ER-located kinase and endoribonuclease that operates as a major stress transducer, mediating both adaptive and proapoptotic programs under ER stress. IRE1 signaling controls the expression of the transcription factor XBP1, in addition to degrade several RNAs. Importantly, a polymorphism in the XBP1 promoter was suggested as a risk factor to develop AD. Here, we demonstrate a positive correlation between the progression of AD histopathology and the activation of IRE1 in human brain tissue. To define the significance of the UPR to AD, we targeted IRE1 expression in a transgenic mouse model of AD. Despite initial expectations that IRE1 signaling may protect against AD, genetic ablation of the RNase domain of IRE1 in the nervous system significantly reduced amyloid deposition, the content of amyloid β oligomers, and astrocyte activation. IRE1 deficiency fully restored the learning and memory capacity of AD mice, associated with improved synaptic function and improved long-term potentiation (LTP). At the molecular level, IRE1 deletion reduced the expression of amyloid precursor protein (APP) in cortical and hippocampal areas of AD mice. In vitro experiments demonstrated that inhibition of IRE1 downstream signaling reduces APP steady-state levels, associated with its retention at the ER followed by proteasome-mediated degradation. Our findings uncovered an unanticipated role of IRE1 in the pathogenesis of AD, offering a novel target for disease intervention.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron death. A 20% of familial ALS cases are associated with mutations in the gene coding for superoxide dismutase 1 (SOD1). The accumulation of abnormal aggregates of different proteins is a common feature in motor neurons of patients and transgenic ALS mice models, which are thought to contribute to disease pathogenesis. Developmental morphogens, such as the Wnt family, regulate numerous features of neuronal physiology in the adult brain and have been implicated in neurodegeneration. β-catenin is a central mediator of both, Wnt signaling activity and cell-cell interactions. We previously reported that the expression of mutant SOD1 in the NSC34 motor neuron cell line decreases basal Wnt pathway activity, which correlates with cytosolic β-catenin accumulation and impaired neuronal differentiation. In this work, we aimed a deeper characterization of β-catenin distribution in models of ALS motor neurons. We observed extensive accumulation of β-catenin supramolecular structures in motor neuron somas of pre-symptomatic mutant SOD1 mice. In cell-cell appositional zones of NSC34 cells expressing mutant SOD1, β-catenin displays a reduced co-distribution with E-cadherin accompanied by an increased association with the gap junction protein Connexin-43; these findings correlate with impaired intercellular adhesion and exacerbated cell coupling. Remarkably, pharmacological inhibition of the glycogen synthase kinase-3β (GSK3β) in both NSC34 cell lines reverted both, β-catenin aggregation and the adverse effects of mutant SOD1 expression on neuronal differentiation. Our findings suggest that early defects in β-catenin distribution could be an underlying factor affecting the onset of neurodegeneration in familial ALS.

Background Amyotrophic lateral sclerosis (ALS) is a multifactorial fatal motoneuron disease without a cure. Ten percent of ALS cases can be pointed to a clear genetic cause, while the remaining 90% is classified as sporadic. Our study was aimed to uncover new connections within the ALS network through a bioinformatic approach, by which we identified C13orf18, recently named Pacer, as a new component of the autophagic machinery and potentially involved in ALS pathogenesis. Methods Initially, we identified Pacer using a network-based bioinformatic analysis. Expression of Pacer was then investigated in vivo using spinal cord tissue from two ALS mouse models (SOD1G93A and TDP43A315T) and sporadic ALS patients. Mechanistic studies were performed in cell culture using the mouse motoneuron cell line NSC34. Loss of function of Pacer was achieved by knockdown using short-hairpin constructs. The effect of Pacer repression was investigated in the context of autophagy, SOD1 aggregation, and neuronal death. Results Using an unbiased network-based approach, we integrated all available ALS data to identify new functional interactions involved in ALS pathogenesis. We found that Pacer associates to an ALS-specific subnetwork composed of components of the autophagy pathway, one of the main cellular processes affected in the disease. Interestingly, we found that Pacer levels are significantly reduced in spinal cord tissue from sporadic ALS patients and in tissues from two ALS mouse models. In vitro, Pacer deficiency lead to impaired autophagy and accumulation of ALS-associated protein aggregates, which correlated with the induction of cell death. Conclusions This study, therefore, identifies Pacer as a new regulator of proteostasis associated with ALS pathology. Electronic supplementary material The online version of this article (10.1186/s13024-019-0313-9) contains supplementary material, which is available to authorized users.

Abnormal modifications to mutant superoxide dismutase 1 (SOD1) are linked to familial amyotrophic lateral sclerosis (fALS). Misfolding of wild-type SOD1 (SOD1 WT ) is also observed in postmortem tissue of a subset of sporadic ALS (sALS) cases, but cellular and molecular mechanisms generating abnormal SOD1 WT species are unknown. We analyzed aberrant human SOD1 WT species over the lifetime of transgenic mice and found the accumulation of disulfide–cross-linked high–molecular-weight SOD1 WT aggregates during aging. Subcellular fractionation of spinal cord tissue and protein overexpression in NSC-34 motoneuron-like cells revealed that endoplasmic reticulum (ER) localization favors oxidation and disulfide-dependent aggregation of SOD1 WT . We established a pharmacological paradigm of chronic ER stress in vivo, which recapitulated SOD1 WT aggregation in young transgenic mice. These species were soluble in nondenaturing detergents and did not react with a SOD1 conformation-specific antibody. Interestingly, SOD1 WT aggregation under ER stress correlated with astrocyte activation in the spinal cord of transgenic mice. Finally, the disulfide–cross-linked SOD1 WT species were also found augmented in spinal cord tissue of sALS patients, correlating with the presence of ER stress markers. Overall, this study suggests that ER stress increases the susceptibility of SOD1 WT to aggregate during aging, operating as a possible risk factor for developing ALS.

Disturbance of endoplasmic reticulum (ER) proteostasis is a common feature of amyotrophic lateral sclerosis (ALS). Protein disulfide isomerases (PDIs) are ER foldases identified as possible ALS biomarkers, as well as neuroprotective factors. However, no functional studies have addressed their impact on the disease process. Here, we functionally characterized four ALS‐linked mutations recently identified in two major PDI genes, PDIA1 and PDIA3/ERp57. Phenotypic screening in zebrafish revealed that the expression of these PDI variants induce motor defects associated with a disruption of motoneuron connectivity. Similarly, the expression of mutant PDIs impaired dendritic outgrowth in motoneuron cell culture models. Cellular and biochemical studies identified distinct molecular defects underlying the pathogenicity of these PDI mutants. Finally, targeting ERp57 in the nervous system led to severe motor dysfunction in mice associated with a loss of neuromuscular synapses. This study identifies ER proteostasis imbalance as a risk factor for ALS, driving initial stages of the disease.

Disturbance of endoplasmic reticulum (ER) proteostasis is observed in Prion-related disorders (PrDs). The protein disulfide isomerase ERp57 is a stress-responsive ER chaperone up-regulated in the brain of Creutzfeldt-Jakob disease patients. However, the actual role of ERp57 in prion protein (PrP) biogenesis and the ER stress response remained poorly defined. We have recently addressed this question using gain- and loss-of-function approaches in vitro and animal models, observing that ERp57 regulates steady-state levels of PrP. Our results revealed that ERp57 modulates the biosynthesis and maturation of PrP but, surprisingly, does not contribute to the global cellular reaction against ER stress in neurons. Here we discuss the relevance of ERp57 as a possible therapeutic target in PrDs and other protein misfolding disorders.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motoneurons in the brain and spinal cord leading to paralysis and death. Although the etiology of ALS remains poorly understood, abnormal protein aggregation and altered proteostasis are common features of sporadic and familial ALS forms. The proteostasis network is decomposed into different modules highly conserved across species and comprehends a collection of mechanisms related to protein synthesis, folding, trafficking, secretion and degradation that is distributed in different compartments inside the cell. Functional studies in various ALS models are revealing a complex scenario where distinct and even opposite effects in disease progression are observed depending on the targeted component of the proteostasis network. Importantly, alteration of the folding capacity of the endoplasmic reticulum (ER) is becoming a common pathological alteration in ALS, representing one of the earliest defects observed in disease models, contributing to denervation and motoneuron dysfunction. Strategies to target-specific components of the proteostasis network using small molecules and gene therapy are under development, and promise interesting avenues for future interventions to delay or stop ALS progression.

Since xenobiotics enter the organism via the liver, hepatocytes must cope with numerous perturbations, including modifications of proteins leading to endoplasmic reticulum stress (ER-stress). This triggers a signaling pathway termed unfolded protein response (UPR) that aims to restore homeostasis or to eliminate disturbed hepatocytes by apoptosis. In the present study, we used the well-established CCl4 hepatotoxicity model in mice to address the questions whether CCl4 induces ER-stress and, if so, whether the well-known ER-stress effector CHOP is responsible for CCl4-induced apoptosis. For this purpose, we treated mice with a high dose of CCl4 injected i.p. and followed gene expression profile over time using Affymetrix gene array analysis. This time resolved gene expression analysis allowed the identification of gene clusters with overrepresented binding sites for the three most important ER-stress induced transcription factors, CHOP, XBP1 and ATF4. Such result was confirmed by the demonstration of CCl4-induced XBP1 splicing, upregulation of CHOP at mRNA and protein levels, and translocation of CHOP to the nucleus. Two observations indicated that CHOP may be responsible for CCl4-induced cell death: (1) Nuclear translocation of CHOP was exclusively observed in the pericentral fraction of hepatocytes that deteriorate in response to CCl4 and (2) CHOP-regulated genes with previously reported pro-apoptotic function such as GADD34, TRB3 and ERO1L were induced in the pericentral zone as well. Therefore, we compared CCl4 induced hepatotoxicity in CHOP knockout versus wild-type mice. Surprisingly, genetic depletion of CHOP did not afford protection against CCl4-induced damage as evidenced by serum GOT and GPT as well as quantification of dead tissue areas. The negative result was obtained at several time points (8, 24 and 72 h) and different CCl4 doses (1.6 and 0.132 g/kg). Overall, our results demonstrate that all branches of the UPR are activated in mouse liver upon CCl4 treatment. However, CHOP does not play a critical role in CCl4-induced cell death and cannot be considered as a biomarker strictly linked to hepatotoxicity. The role of alternative UPR effectors such as XBP1 remains to be investigated.

Creutzfeldt-Jakob disease has a higher incidence in Chile than in other countries. The post mortem pathological characterization of brain tissue is necessary to reach a definitive diagnosis. We report a 73 years old man with a history compatible with of a rapidly progressive dementia, in which the first electroencephalographic study showed a pattern consistent with non-convulsive status epilepticus. Besides discarding this diagnosis, it was necessary to rule out other causes of rapidly progressive dementia such as Hashimoto encephalopathy. Finally, the sustained clinical deterioration with no response to anticonvulsants and corticosteroids, the imaging studies, a serial electroencephalographic monitoring study and the detection of 14-3-3 protein in cerebrospinal fluid were the keys to achieve the diagnosis of the disease.

Unlike intermolecular disulfide bonds, other protein cross-links arising from oxidative modifications cannot be reversed and are presumably more toxic to cells because they may accumulate and induce protein aggregation. However, most of these irreversible protein cross-links remain poorly characterized. For instance, the antioxidant enzyme human superoxide dismutase 1 (hSod1) has been reported to undergo non-disulfide covalent dimerization and further oligomerization during its bicarbonate-dependent peroxidase activity. The dimerization was shown to be dependent on the oxidation of the single, solvent-exposed Trp(32) residue of hSod1, but the covalent dimer was not isolated nor was its structure determined. In this work, the hSod1 covalent dimer was isolated, digested with trypsin in H(2)O and H(2)(18)O, and analyzed by UV-Vis spectroscopy and mass spectrometry (MS). The results demonstrate that the covalent dimer consists of two hSod1 subunits cross-linked by a ditryptophan, which contains a bond between C3 and N1 of the respective Trp(32) residues. We further demonstrate that the cross-link cleaves under usual MS/MS conditions leading to apparently unmodified Trp(32), partially hinders proteolysis, and provides a mechanism to explain the formation of hSod1 covalent trimers and tetramers. This characterization of the covalent hSod1 dimer identifies a novel oxidative modification of protein Trp residues and provides clues for studying its occurrence in vivo.

Amyotrophic lateral sclerosis (ALS) is a fatal late-onset neurodegenerative disease characterized by the selective loss of motoneurons. The mechanisms underlying neuronal degeneration in ALS are starting to be elucidated, highlighting abnormal protein aggregation and altered mRNA metabolism as common phenomena. ALS involves the selective vulnerablility of a subpopulation of motoneurons, suggesting that intrinsic factors may determine ALS pathogenesis. Accumulating evidence indicates that alterations to endoplasmic reticulum (ER) proteostasis play a critical role on disease progression, representing one of the earliests pathological signatures of the disease. Here we discuss recent studies uncovering a fundamental role of ER stress as the driver of selective neuronal vulnerability in ALS and discuss the potential of targeting the unfolded protein response (UPR) as a therapeutic strategy to treat ALS.

The unequivocal demonstration that the carbonate radical (CO(3) (.-)) is produced from the reaction between the ubiquitous carbon dioxide and peroxynitrite, renewed the interest in the pathogenic roles of oxidants derived from the main physiological buffer, the bicarbonate-carbon dioxide pair. Here, we review the biochemical properties of both the carbonate radical and peroxymonocarbonate (HCO(4) (-)), and discuss the evidence of their formation under physiological conditions. Overall, the review emphasizes the recognition of the biological relevance of oxidants derived from the main physiological buffer as a crucial step into the understanding and control of numerous pathological states.

ERp57 (also known as grp58 and PDIA3) is a protein disulfide isomerase that catalyzes disulfide bonds formation of glycoproteins as part of the calnexin and calreticulin cycle. ERp57 is markedly upregulated in most common neurodegenerative diseases downstream of the endoplasmic reticulum (ER) stress response. Despite accumulating correlative evidence supporting a neuroprotective role of ERp57, the contribution of this foldase to the physiology of the nervous system remains unknown. Here we developed a transgenic mouse model that overexpresses ERp57 in the nervous system under the control of the prion promoter. We analyzed the susceptibility of ERp57 transgenic mice to undergo neurodegeneration. Unexpectedly, ERp57 overexpression did not affect dopaminergic neuron loss and striatal denervation after injection of a Parkinson's disease-inducing neurotoxin. In sharp contrast, ERp57 transgenic animals presented enhanced locomotor recovery after mechanical injury to the sciatic nerve. These protective effects were associated with enhanced myelin removal, macrophage infiltration and axonal regeneration. Our results suggest that ERp57 specifically contributes to peripheral nerve regeneration, whereas its activity is dispensable for the survival of a specific neuronal population of the central nervous system. These results demonstrate for the first time a functional role of a component of the ER proteostasis network in peripheral nerve regeneration.

Docosahexaenoic acid (C22:6, n-3, DHA) is a polyunsaturated fatty acid highly enriched in the brain. This fatty acid can be easily oxidized yielding hydroperoxides as primary products. Cu, Zn-Superoxide dismutase (SOD1) aggregation is a common hallmark of Amyotrophic Lateral Sclerosis (ALS) and the molecular mechanisms behind their formation are not completely understood. Here we investigated the effect of DHA and its hydroperoxides (DHAOOH) on human SOD1 oligomerization in vitro. DHA induced the formation of high-molecular-weight (HMW) SOD1 species (>700 kDa). Aggregation was dependent on free thiols and occurred primarily with the protein in its apo-form. SOD1 incubation with DHA was accompanied by changes in protein structure leading to exposure of protein hydrophobic patches and formation of non-amyloid aggregates. Site-directed mutagenesis studies demonstrated that Cys 6 and Cys 111 in wild-type and Cys 6 in ALS-linked G93A mutant are required for aggregation. In contrast, DHAOOH did not induce HMW species formation but promoted abnormal covalent dimerization of apo-SOD1 that was resistant to SDS and thiol reductants. Overall, our data demonstrate that DHA and DHAOOH induce distinct types of apo-SOD1 oligomerization leading to the formation of HMW and low-molecular-weight species, respectively.

Autophagy is becoming an attractive target to treat neurodegenerative diseases involving protein misfolding and abnormal aggregation. However, the mechanisms underlying the precise role of autophagy in the pathology are still unclear. Accumulating evidence indicates that autophagy impairment may actually contribute to the neurodegeneration process. In this chapter, we discuss several examples indicating the therapeutic effects of manipulating (enhancing) autophagy in preclinical models of neurodegeneration, in addition to identifying key molecular alterations in the autophagy pathway at different levels in these diseases. Challenges in the future development of therapeutic strategies to target the autophagy pathway are discussed.

The development of curative therapies for genetically complex diseases such as ALS has been delayed by the lack of relevant disease models. Recent advances using induced-pluripotent-stem-cell-derived motoneurons from patients harboring distinct ALS mutations have recapitulated essential disease features and have identified some common pathways driving disease pathogenesis.

Amyotrophic lateral sclerosis (ALS) is the most frequent paralytic disease in adults. Most ALS cases are considered sporadic with no clear genetic component. The disruption of protein homeostasis due to chronic stress responses at the endoplasmic reticulum (ER) and the accumulation of abnormal protein inclusions are extensively described in ALS mouse models and patient-derived tissue. Recent studies using pharmacological and genetic manipulation of the unfolded protein response (UPR), an adaptive reaction against ER stress, have demonstrated a complex involvement of the pathway in experimental models of ALS. In addition, quantitative changes in ER stress-responsive chaperones in body fluids have been proposed as possible biomarkers to monitor the disease progression. Here we review most recent advances attributing a causal role of ER stress in ALS.

Diacetyl, methylglyoxal, and glyoxal are α-dicarbonyl catabolites prone to nucleophilic additions of amino groups of proteins and nucleobases, thereby triggering adverse biological responses. Because of their electrophilicity, in aqueous medium, they exist in a phosphate-catalyzed dynamic equilibrium with their hydrate forms. Diacetyl and methylglyoxal can be attacked by peroxynitrite (k(2) ≈ 1.0 × 10(4) M(-1) s(-1) and k(2) ≈ 1.0 × 10(5) M(-1) s(-1), respectively), a potent biological nucleophile and oxidant, yielding the acetyl radical from the homolysis of peroxynitrosocarbonyl adducts, and acetate or formate ions, respectively. We report here that glyoxal also reacts with peroxynitrite, yielding formate ion at rates at least 1 order of magnitude greater than does methylglyoxal. A triplet EPR signal (1:2:1; a(H) = 0.78 mT) attributable to hydrated formyl radical was detected by direct flow experiments. In the presence of the spin trap 2-methyl-2-nitrosopropane, the EPR spectrum displays the di-tert-butyl nitroxide signal, another signal assignable to the spin trapping adduct with hydrogen radical (a(N) = a(H) = 1.44 mT), probably formed from formyl radical decarbonylation, and a third EPR signal assignable to the formyl radical adduct of the spin trap (a(N) = 0.71 mT and a(H) = 0.14 mT). The novelty here is the detection of singlet oxygen ((1)Δ(g)) monomol light emission at 1270 nm during the reaction, probably formed by subsequent dioxygen addition to formyl radical and a Russell reaction of nascent formylperoxyl radicals. Accordingly, the near-infrared emission increases upon raising the peroxynitrite concentration in D(2)O buffer and is suppressed upon addition of O(2) ((1)Δ(g)) quenchers (NaN(3), l-His, H(2)O). Unequivocal evidence of O(2) ((1)Δ(g)) generation was also obtained by chemical trapping of (18)O(2) ((1)Δ(g)) with anthracene-9,10-divinylsulfonate, using HPLC/MS/MS for detection of the corresponding 9,10-endoperoxide derivative. Our studies add insights into the molecular events underlying nitrosative, oxidative, and carbonyl stress in inflammatory processes and aging-associated maladies.

Understanding the mechanisms that determine cell fate under endoplasmic reticulum (ER) stress had been hampered by the lack of models to study unfolded protein response (UPR) adaptive phases. The development of an engineered protein to conditionally induce its misfolding allowed the establishment of a resolvable ER stress condition.

Despite being one of the most important antioxidant defenses, Cu,Zn-superoxide dismutase (Sod1) has been frequently associated with harmful effects, including neurotoxicity. This toxicity has been attributed to immature forms of Sod1 and extraneous catalytic activities. Among these, the ability of Sod1 to function as a peroxidase may be particularly relevant because it is increased in bicarbonate buffer and produces the reactive carbonate radical. Despite many studies, how this radical forms remains unknown. To address this question, we systematically studied hSod1 peroxidase activity in the presence of nitrite, formate, and bicarbonate-carbon dioxide. Kinetic analyses of hydrogen peroxide consumption and of nitrite, formate, and bicarbonate-carbon dioxide oxidation showed that the Sod1-bound hydroxyl-like oxidant functions in the presence of nitrite and formate. In the presence of bicarbonate-carbon dioxide, this oxidant is replaced by peroxymonocarbonate, which is then reduced to the carbonate radical. Peroxymonocarbonate intermediacy was evidenced by (13)C NMR experiments showing line broadening of its peak in the presence of Zn,ZnSod1. In agreement, peroxymonocarbonate was docked into the hSod1 active site, where it interacted with the conserved Arg(143). Also, a reaction between peroxymonocarbonate and Cu(I)Sod1 was demonstrated by stopped-flow experiments. Kinetic simulations indicated that peroxymonocarbonate is produced during Sod1 turnover and not in bulk solution. In the presence of bicarbonate-carbon dioxide, sustained hSod1-mediated oxidations occurred with low steady-state concentrations of hydrogen peroxide (4-10 microM). Thus, carbonate radical formation through peroxymonocarbonate may be a key event in Sod1-induced toxicity.

Tempol has been shown to protect experimental animals from injuries associated with excessive nitric oxide production. In parallel, tempol decreased the levels of protein-3-nitrotyrosine in the injured tissues, suggesting that it interacted with nitric oxide-derived oxidants such as nitrogen dioxide and peroxynitrite. Relevantly, a few recent studies have shown that tempol catalytically diverts peroxynitrite/carbon dioxide reactivity toward phenol from nitration to nitrosation. To examine whether this shift occurs in biological environments, we studied the effects of tempol (10–100 μM) on peroxynitrite/carbon dioxide (1 mM/2 mM) reactivity toward proteins, native bovine serum albumin (BSA) (0.5–0.7 cys/mol) and reductively denatured BSA (7–19 cys/mol), and cells (J774 macrophages). Although not a true catalyst, tempol strongly inhibited protein–tyrosine nitration (70–90%) and protein–cysteine oxidation (20–50%) caused by peroxynitrite/carbon dioxide in BSA, denatured BSA, and cells while increasing protein–cysteine nitrosation (200–400%). Tempol consumption was attributed mainly to its reaction with protein–cysteinyl radicals. Most of the tempol, however, reacted with the radicals produced from peroxynitrite/carbon dioxide, that is, nitrogen dioxide and carbonate radical anion. Accordingly, tempol decreased the yields of BSA–cysteinyl and BSA–tyrosyl/tryptophanyl radicals, as well their decay products such as protein–3-nitrotyrosine. The parallel increase in protein–nitrosocysteine yields demonstrated that part of the peroxynitrite is oxidized to nitric oxide by the oxammonium cation produced from tempol oxidation by peroxynitrite/carbon dioxide-derived radicals. Protein–nitrosocysteine formation was shown to occur by radical and nonradical mechanisms in studies with a protein–cysteinyl radical trapper. These studies may contribute to the understanding of the protective effects of tempol in animal models of inflammation.

Although the accumulation of a misfolded and protease-resistant form of the prion protein (PrP) is a key event in Prion pathogenesis, the cellular factors involved in its folding and quality control are poorly understood. PrP is a glycosylated and disulfide-bonded protein synthesized at the endoplasmic reticulum (ER). The ER foldase ERp57 (also known as Grp58) is highly expressed in the brain of sporadic and infectious forms of Prion-related disorders. ERp57 is a disulfide isomerase involved in the folding of a subset of glycoproteins in the ER as part of the calnexin/calreticulin cycle. Here we show that levels of ERp57 increase mainly in neurons of Creutzfeldt-Jacob patients. Using gain- and loss-of-function approaches in cell culture we demonstrate that ERp57 expression directly controls the maturation and total levels of wild- type PrP and mutant forms associated with human disease. In addition, we found that PrP physically interacts with ERp57, and also with the closest family member PDIA1, but not ERp72. Furthermore, we generated a conditional knockout mouse for ERp57 in the nervous system and detected a reduction in the steady-state levels of the mono- and non-glycosylated forms of PrP in the brain. In contrast, ERp57 transgenic mice showed increased levels of endogenous PrP. Unexpectedly, ERp57 expression did not affect the susceptibility of cells to ER stress in vitro and in vivo. This study identifies ERp57 as a new modulator of PrP levels and may help understanding the consequences of ERp57 upregulation observed in human disease.

Tar DNA binding protein 43 (TDP-43) is the principal component of ubiquitinated protein inclusions present in nervous tissue of most cases of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Previous studies described a TDP-43A315T transgenic mouse model that develops progressive motor dysfunction in the absence of protein aggregation or significant motoneuron loss, questioning its validity to study ALS. Here we have further characterized the course of the disease in TDP-43A315T mice using a battery of tests and biochemical approaches. We confirmed that TDP-43 mutant mice develop impaired motor performance, accompanied by progressive body weight loss. Significant differences were observed in life span between genders, where females survived longer than males. Histopathological analysis of the spinal cord demonstrated a significant motoneurons loss, accompanied by axonal degeneration, astrogliosis and microglial activation. Importantly, histopathological alterations observed in TDP-43 mutant mice were similar to some characteristic changes observed in mutant SOD1 mice. Unexpectedly, we identified the presence of different species of disulfide-dependent TDP-43 aggregates in cortex and spinal cord tissue. Overall, this study indicates that TDP-43A315T transgenic mice develop key features resembling key aspects of ALS, highlighting its relevance to study disease pathogenesis.