Breakdown of Arabidopsis thaliana thioredoxins and glutaredoxins based on electrostatic similarity-Leads to common and unique interaction partners and functions.

The reversible reduction and oxidation of protein thiols was first described as mechanism to control light/dark-dependent metabolic regulation in photosynthetic organisms. Today, it is recognized as an essential mechanism of regulation and signal transduction in all kingdoms of life. Proteins of the thioredoxin (Trx) family, Trxs and glutaredoxins (Grxs) in particular, catalyze thiol-disulfide exchange reactions and are vital players in the operation of thiol switches. Various Trx and Grx isoforms are present in all compartments of the cell. These proteins have a rather broad but at the same time distinct substrate specificity. Understanding the molecular basis of their target specificity is central to the understanding of physiological and pathological redox signaling. Electrostatic complementarity of the redoxins with their target proteins has been proposed as a major reason. Here, we analyzed the electrostatic similarity of all Arabidopsis thaliana Trxs, Grxs, and proteins containing such domains. Clustering of the redoxins based on this comparison suggests overlapping and also distant target specificities and thus functions of the different sub-classes including all Trx isoforms as well as the three classes of Grxs, i.e. CxxC-, CGFS-, and CC-type Grxs. Our analysis also provides a rationale for the tuned substrate specificities of both the ferredoxin- and NADPH-dependent Trx reductases.

Cysteinyl and methionyl redox switches: Structural prerequisites and consequences.

Redox modifications of specific cysteinyl and methionyl residues regulate key enzymes and signal-transducing proteins in various pathways. Here, we analyzed the effect of redox modifications on protein structure screening the RCSB protein data bank for oxidative modifications of proteins, i.e. protein disulfides, mixed disulfides with glutathione, cysteinyl sulfenic acids, cysteinyl S-nitrosylation, and methionyl sulfoxide residues. When available, these structures were compared to the structures of the same proteins in the reduced state with respect to both pre-requirements for the oxidative modifications as well as the structural consequences of the modifications. In general, the conformational changes induced by the redox modification are small, i.e. within the range of normal fluctuations. Some redox modifications, disulfides in particular, induces alterations in the electrostatic properties of the proteins. Solvent accessibility does not seem to be a strict pre-requirement for the redox modification of a particular residue. We identified an enrichment of certain other amino acid residues in the vicinity of the susceptible residues, for disulfide and sulfenic acid modifications, for instance, histidyl and tyrosyl residues. These motifs, as well as the specific features of the susceptible sulfur-containing amino acids, may become helpful for the prediction of redox modifications.

Nucleoredoxin Plays a Key Role in the Maintenance of Retinal Pigmented Epithelium Differentiation.

Nucleoredoxin (Nrx) belongs to the Thioredoxin protein family and functions in redox-mediated signal transduction. It contains the dithiol active site motif Cys-Pro-Pro-Cys and interacts and regulates different proteins in distinct cellular pathways. Nrx was shown to be catalytically active in the insulin assay and recent findings indicate that Nrx functions, in fact, as oxidase. Here, we have analyzed Nrx in the mammalian retina exposed to (perinatal) hypoxia-ischemia/reoxygenation, combining ex vivo and in vitro models. Our data show that Nrx regulates cell differentiation, which is important to (i) increase the number of glial cells and (ii) replenish neurons that are lost following the hypoxic insult. Nrx is essential to maintain cell morphology. These regulatory changes are related to VEGF but do not seem to be linked to the Wnt/ß-catenin pathway, which is not affected by Nrx knock-down. In conclusion, our results strongly suggest that hypoxia-ischemia could lead to alterations in the organization of the retina, related to changes in RPE cell differentiation. Nrx may play an essential role in the maintenance of the RPE cell differentiation state via the regulation of VEGF release.

Cytosolic glutaredoxin 1 is upregulated in AMD and controls retinal pigment epithelial cells proliferation via ß-catenin.

Thioredoxin (Trx) family proteins are key players in redox signaling. Here, we have analyzed glutaredoxin (Grx) 1 and Grx2 in age-related macular degeneration (AMD) and in retinal pigment epithelial (ARPE-19) cells. We hypothesized that these redoxins regulate cellular functions and signaling circuits such as cell proliferation, Wnt signaling and VEGF release that have been correlated to the pathophysiology of AMD. ARPE-19 cells were transfected with specific siRNAs to silence the expression of Grx1 and Grx2 and were analyzed for proliferation/viability, migration capacity, ß-catenin activation, and VEGF release. An active site-mutated C-X-X-S Grx1 was utilized to trap interacting proteins present in ARPE-19 cell extracts. In both, AMD retinas and in ARPE-19 cells incubated under hypoxia/reoxygenation conditions, Grx1 showed an increased nuclear localization. Grx1-silenced ARPE-19 cells showed a significantly reduced proliferation and migration rate. Our trapping approach showed that Grx1 interacts with ß-catenin in a dithiol-disulfide exchange reaction. Knock-down of Grx1 led to a reduction in both total and active ß-catenin levels. These findings add redox control to the regulatory mechanisms of ß-catenin signaling in the retinal pigment epithelium and open the door to novel therapeutic approaches in AMD that is currently treated with VEGF-inhibitors.

Molecular Basis for the Interactions of Human Thioredoxins with Their Respective Reductases.

The mammalian cytosolic thioredoxin (Trx) system consists of Trx1 and its reductase, the NADPH-dependent seleno-enzyme TrxR1. These proteins function as electron donor for metabolic enzymes, for instance in DNA synthesis, and the redox regulation of numerous processes. In this work, we analysed the interactions between these two proteins. We proposed electrostatic complementarity as major force controlling the formation of encounter complexes between the proteins and thus the efficiency of the subsequent electron transfer reaction. If our hypothesis is valid, formation of the encounter complex should be independent of the redox reaction. In fact, we were able to confirm that also a redox inactive mutant of Trx1 lacking both active site cysteinyl residues (C32,35S) binds to TrxR1 in a similar manner and with similar kinetics as the wild-type protein. We have generated a number of mutants with alterations in electrostatic properties and characterised their interaction with TrxR1 in kinetic assays. For human Trx1 and TrxR1, complementary electrostatic surfaces within the area covered in the encounter complex appear to control the affinity of the reductase for its substrate Trx. Electrostatic compatibility was even observed in areas that do not form direct molecular interactions in the encounter complex, and our results suggest that the electrostatic complementarity in these areas influences the catalytic efficiency of the reduction. The human genome encodes ten cytosolic Trx-like or Trx domain-containing proteins. In agreement with our hypothesis, the proteins that have been characterised as TrxR1 substrates also show the highest similarity in their electrostatic properties.

Thioredoxin 1 is upregulated in the bone and bone marrow following experimental myocardial infarction: evidence for a remote organ response.

Ischemia and reperfusion events, such as myocardial infarction (MI), are reported to induce remote organ damage severely compromising patient outcomes. Tissue survival and functional restoration relies on the activation of endogenous redox regulatory systems such as the oxidoreductases of the thioredoxin (Trx) family. Trxs and peroxiredoxins (Prxs) are essential for the redox regulation of protein thiol groups and for the reduction of hydrogen peroxide, respectively. Here, we determined whether experimental MI induces changes in Trxs and Prxs in the heart as well as in secondary organs. Levels and localization of Trx1, TrxR1, Trx2, Prx1, and Prx2 were analyzed in the femur, vertebrae, and kidneys of rats following MI or sham surgery. Trx1 levels were significantly increased in the heart (P = 0.0017) and femur (P < 0.0001) of MI animals. In the femur and lumbar vertebrae, Trx1 upregulation was detected in bone-lining cells, osteoblasts, megakaryocytes, and other hematopoietic cells. Serum levels of Trx1 increased significantly 2 days after MI compared to sham animals (P = 0.0085). Differential regulation of Trx1 in the bone was also detected by immunohistochemistry 1 month after MI. N-Acetyl-cysteine treatment over a period of 1 month induced a significant reduction of Trx1 levels in the bone of MI rats compared to sham and to MI vehicle. This study provides first evidence that MI induces remote organ upregulation of the redox protein Trx1 in the bone, as a response to ischemia-reperfusion injury in the heart.

Molecular basis for the distinct functions of redox-active and FeS-transfering glutaredoxins.

Despite their very close structural similarity, CxxC/S-type (class I) glutaredoxins (Grxs) act as oxidoreductases, while CGFS-type (class II) Grxs act as FeS cluster transferases. Here we show that the key determinant of Grx function is a distinct loop structure adjacent to the active site. Engineering of a CxxC/S-type Grx with a CGFS-type loop switched its function from oxidoreductase to FeS transferase. Engineering of a CGFS-type Grx with a CxxC/S-type loop abolished FeS transferase activity and activated the oxidative half reaction of the oxidoreductase. The reductive half-reaction, requiring the interaction with a second GSH molecule, was enabled by switching additional residues in the active site. We explain how subtle structural differences, mostly depending on the structure of one particular loop, act in concert to determine Grx function.

Paracrine regulation and improvement of ß-cell function by thioredoxin.

The failure of insulin-producing ß-cells is the underlying cause of hyperglycemia in diabetes mellitus. ß-cell decay has been linked to hypoxia, chronic inflammation, and oxidative stress. Thioredoxin (Trx) proteins are major actors in redox signaling and essential for signal transduction and the cellular stress response. We have analyzed the cytosolic, mitochondrial, and extracellular Trx system proteins in hypoxic and cytokine-induced stress using ß-cell culture, isolated pancreatic islets, and pancreatic islet transplantation modelling low oxygen supply. Protein levels of cytosolic Trx1 and Trx reductase (TrxR) 1 significantly decreased, while mitochondrial Trx2 and TrxR2 increased upon hypoxia and reoxygenation. Interestingly, Trx1 was secreted by ß-cells during hypoxia. Moreover, murine and human pancreatic islet grafts released Trx1 upon glucose stimulation. Survival of transplanted islets was substantially impaired by the TrxR inhibitor auranofin. Since a release was prominent upon hypoxia, putative paracrine effects of Trx1 on ß-cells were examined. In fact, exogenously added recombinant hTrx1 mitigated apoptosis and preserved glucose sensitivity in pancreatic islets subjected to hypoxia and inflammatory stimuli, dependent on its redox activity. Human subjects were studied, demonstrating a transient increase in extracellular Trx1 in serum after glucose challenge. This increase correlated with better pancreatic islet function. Moreover, hTrx1 inhibited the migration of primary murine macrophages. In conclusion, our study offers evidence for paracrine functions of extracellular Trx1 that improve the survival and function of pancreatic ß-cells.

Signal-regulated oxidation of proteins via MICAL.

Processing of and responding to various signals is an essential cellular function that influences survival, homeostasis, development, and cell death. Extra- or intracellular signals are perceived via specific receptors and transduced in a particular signalling pathway that results in a precise response. Reversible post-translational redox modifications of cysteinyl and methionyl residues have been characterised in countless signal transduction pathways. Due to the low reactivity of most sulfur-containing amino acid side chains with hydrogen peroxide, for instance, and also to ensure specificity, redox signalling requires catalysis, just like phosphorylation signalling requires kinases and phosphatases. While reducing enzymes of both cysteinyl- and methionyl-derivates have been characterised in great detail before, the discovery and characterisation of MICAL proteins evinced the first examples of specific oxidases in signal transduction. This article provides an overview of the functions of MICAL proteins in the redox regulation of cellular functions.

The cytosolic isoform of glutaredoxin 2 promotes cell migration and invasion.

BACKROUND: Cytosolic glutaredoxin 2 (Grx2c) controls axonal outgrowth and is specifically induced in many cancer cell lines. We thus hypothesized that Grx2c promotes cell motility and invasiveness. METHODS: We characterized the impact of Grx2c expression in cell culture models. We combined stable isotope labeling, phosphopeptide enrichment, and high-accuracy mass spectrometry to characterize the underlying mechanisms. RESULTS: The most prominent associations were found with actin dynamics, cellular adhesion, and receptor-mediated signal transduction, processes that are crucial for cell motility. For instance, collapsin response mediator protein 2, a protein involved in the regulation of cytoskeletal dynamics, is regulated by Grx2c through a redox switch that controls the phosphorylation state of the protein as well. Cell lines expressing Grx2c showed dramatic alterations in morphology. These cells migrated two-fold faster and gained the ability to infiltrate a collagen matrix. CONCLUSIONS: The expression of Grx2c promotes cell migration, and may negatively correlate with cancer-specific survival. GENERAL SIGNIFICANCE: Our results imply critical roles of Grx2c in cytoskeletal dynamics, cell adhesion, and cancer cell invasiveness.

Substrate specificity of thioredoxins and glutaredoxins - towards a functional classification.

The spatio-temporal reduction and oxidation of protein thiols is an essential mechanism in signal transduction in all kingdoms of life. Thioredoxin (Trx) family proteins efficiently catalyze thiol-disulfide exchange reactions and the proteins are widely recognized for their importance in the operation of thiol switches. Trx family proteins have a broad and at the same time very distinct substrate specificity - a prerequisite for redox switching. Despite of multiple efforts, the true nature for this specificity is still under debate. Here, we comprehensively compare the classification/clustering of various redoxins from all domains of life based on their similarity in amino acid sequence, tertiary structure, and their electrostatic properties. We correlate these similarities to the existence of common interaction partners, identified in various previous studies and suggested by proteomic screenings. These analyses confirm that primary and tertiary structure similarity, and thereby all common classification systems, do not correlate to the target specificity of the proteins as thiol-disulfide oxidoreductases. Instead, a number of examples clearly demonstrate the importance of electrostatic similarity for their target specificity, independent of their belonging to the Trx or glutaredoxin subfamilies.

Nucleoredoxin-Dependent Targets and Processes in Neuronal Cells.

Nucleoredoxin (Nrx) is an oxidoreductase of the thioredoxin family of proteins. It was shown to act as a signal transducer in some pathways; however, so far, no comprehensive analysis of its regulated substrates and functions was available. Here, we used a combination of two different strategies to fill this gap. First, we analyzed the thiol-redox state of the proteome of SH-SY5Y neuroblastoma cells depleted of Nrx compared to control cells using a differential thiol-labeling technique and quantitative mass spectrometry. 171 proteins were identified with an altered redox state; 161 of these were more reduced in the absence of Nrx. This suggests functions of Nrx in the oxidation of protein thiols. Second, we utilized the active site mutant Cys208Ser of Nrx, which stabilizes a mixed disulfide intermediate with its substrates and therefore trapped interacting proteins from the mouse brain (identifying 1710 proteins) and neuronal cell culture extracts (identifying 609 proteins). Profiling of the affected biological processes and molecular functions in cells of neuronal origin suggests numerous functions of Nrx in the redox regulation of metabolic pathways, cellular morphology, and signal transduction. These results characterize Nrx as a cellular oxidase that itself may be oxidized by the formation of disulfide relays with peroxiredoxins.

Molecular dynamics simulations and in vitro analysis of the CRMP2 thiol switch.

The collapsin response mediator protein CRMP2 (gene: DPYSL2) is crucial for neuronal development. The homotetrameric CRMP2 complex is regulated via two mechanisms: first by phosphorylation and second by the reduction and oxidation of the Cys504 residues of two adjacent subunits. Here, we have analysed the effects of this redox switch on the protein in vitro combined with force field molecular dynamics (MD). Earlier X-ray data reveal the structure of the rigid body of the molecule but lack the flexible C-terminus with the important sites for phosphorylation and redox regulation. An in silico model for this part was established by replica exchange simulations and homology modelling, which is consistent with the CD spectroscopy results of the recombinant protein. Thermofluor data indicated that the protein aggregates at bivalent ion concentrations below 200 mM. In simulations the protein surface was covered under these conditions by a large number of ions, which most likely prevent aggregation. A tryptophan residue (Trp295) in close proximity to the forming disulphide allowed the measurement of the structural relaxation of the rigid body upon reduction by fluorescence quenching. We were also able to determine the second-order rate constant of CRMP2 oxidation by H2O2. The simulated solvent accessible surface of the hydroxyl group of Ser518 significantly increased upon reduction of the disulphide bond. Our results give the first detailed insight into the profound structural changes of the tetrameric CRMP2 due to oxidation and indicate a tightly connected regulation by phosphorylation and redox modification.

Neuronal Damage Induced by Perinatal Asphyxia Is Attenuated by Postinjury Glutaredoxin-2 Administration.

The general disruption of redox signaling following an ischemia-reperfusion episode has been proposed as a crucial component in neuronal death and consequently brain damage. Thioredoxin (Trx) family proteins control redox reactions and ensure protein regulation via specific, oxidative posttranslational modifications as part of cellular signaling processes. Trx proteins function in the manifestation, progression, and recovery following hypoxic/ischemic damage. Here, we analyzed the neuroprotective effects of postinjury, exogenous administration of Grx2 and Trx1 in a neonatal hypoxia/ischemia model. P7 Sprague-Dawley rats were subjected to right common carotid ligation or sham surgery, followed by an exposure to nitrogen. 1 h later, animals were injected i.p. with saline solution, 10 mg/kg recombinant Grx2 or Trx1, and euthanized 72 h postinjury. Results showed that Grx2 administration, and to some extent Trx1, attenuated part of the neuronal damage associated with a perinatal hypoxic/ischemic damage, such as glutamate excitotoxicity, axonal integrity, and astrogliosis. Moreover, these treatments also prevented some of the consequences of the induced neural injury, such as the delay of neurobehavioral development. To our knowledge, this is the first study demonstrating neuroprotective effects of recombinant Trx proteins on the outcome of neonatal hypoxia/ischemia, implying clinical potential as neuroprotective agents that might counteract neonatal hypoxia/ischemia injury.

Iron-sulfur glutaredoxin 2 protects oligodendrocytes against damage induced by nitric oxide release from activated microglia.

Demyelinated brain lesions, a hallmark of autoimmune neuroinflammatory diseases like multiple sclerosis, result from oligodendroglial cell damage. Activated microglia are considered a major source of nitric oxide and subsequent peroxynitrite-mediated damage of myelin. Here, we provide biochemical and biophysical evidence that the oxidoreductase glutaredoxin 2 inhibits peroxynitrite formation by transforming nitric oxide into dinitrosyl-diglutathionyl-iron-complexes. Glutaredoxin 2 levels influence both survival rates of primary oligodendrocyte progenitor cells and preservation of myelin structure in cerebellar organotypic slice cultures challenged with activated microglia or nitric oxide donors. Of note, glutaredoxin 2-mediated protection is not linked to its enzymatic activity as oxidoreductase, but to the disassembly of its uniquely coordinated iron-sulfur cluster using glutathione as non-protein ligand. The protective effect of glutaredoxin 2 is connected to decreased protein carbonylation and nitration. In line, brain lesions of mice suffering from experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, show decreased glutaredoxin 2 expression and increased nitrotyrosine formation indicating that this type of protection is missing in the inflamed central nervous system. Our findings link inorganic biochemistry to neuroinflammation and identify glutaredoxin 2 as a protective factor against neuroinflammation-mediated myelin damage. Thus, improved availability of glutathione-coordinated iron-sulfur clusters emerges as a potential therapeutic approach in inflammatory demyelination.

Glutathione, Glutaredoxins, and Iron.

SIGNIFICANCE: Glutathione (GSH) is the most abundant cellular low-molecular-weight thiol in the majority of organisms in all kingdoms of life. Therefore, functions of GSH and disturbed regulation of its concentration are associated with numerous physiological and pathological situations. Recent Advances: The function of GSH as redox buffer or antioxidant is increasingly being questioned. New functions, especially functions connected to the cellular iron homeostasis, were elucidated. Via the formation of iron complexes, GSH is an important player in all aspects of iron metabolism: sensing and regulation of iron levels, iron trafficking, and biosynthesis of iron cofactors. The variety of GSH coordinated iron complexes and their functions with a special focus on FeS-glutaredoxins are summarized in this review. Interestingly, GSH analogues that function as major low-molecular-weight thiols in organisms lacking GSH resemble the functions in iron homeostasis. CRITICAL ISSUES: Since these iron-related functions are most likely also connected to thiol redox chemistry, it is difficult to distinguish between mechanisms related to either redox or iron metabolisms. FUTURE DIRECTIONS: The ability of GSH to coordinate iron in different complexes with or without proteins needs further investigation. The discovery of new Fe-GSH complexes and their physiological functions will significantly advance our understanding of cellular iron homeostasis. Antioxid. Redox Signal. 27, 1235-1251.

Redoxins in peripheral neurons after sciatic nerve injury.

Peripheral nerve injury causes redox stress in injured neurons by upregulations of pro-oxidative enzymes, but most neurons survive suggesting an activation of endogenous defense against the imbalance. As potential candidates we assessed thioredoxin-fold proteins, called redoxins, which maintain redox homeostasis by reduction of hydrogen peroxide or protein dithiol-disulfide exchange. Using a histologic approach, we show that the peroxiredoxins (Prdx1-6), the glutaredoxins (Glrx1, 2, 3 and 5), thioredoxin (Txn1 and 2) and their reductases (Txnrd1 and 2) are expressed in neurons, glial and/or vascular cells of the dorsal root ganglia (DRGs) and in the spinal cord. They show distinct cellular and subcellular locations in agreement with the GO terms for "cellular component". The expression and localization of Glrx, Txn and Txnrd proteins was not affected by sciatic nerve injury but peroxiredoxins were upregulated in the DRGs, Prdx1 and Prdx6 mainly in non-neuronal cells and Prdx4 and Prdx5 in DRG neurons, the latter associated with an increase of respective mRNAs and protein accumulation in peripheral and/or central fibers. The upregulation of Prdx4 and Prdx5 in DRG neurons was reduced in mice with a cre-loxP mediated deficiency of hypoxia inducible factor 1 alpha (HIF1α) in these neurons. The results identify Prdx4 and Prdx5 as endogenous HIF1α-dependent, transcriptionally regulated defenders of nerve injury evoked redox stress that may be important for neuronal survival and regeneration.

Redox proteomics of the inflammatory secretome identifies a common set of redoxins and other glutathionylated proteins released in inflammation, influenza virus infection and oxidative stress.

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

Thioredoxin 1 and glutaredoxin 2 contribute to maintain the phenotype and integrity of neurons following perinatal asphyxia.

BACKGROUND: Thioredoxin (Trx) family proteins are crucial mediators of cell functions via regulation of the thiol redox state of various key proteins and the levels of the intracellular second messenger hydrogen peroxide. Their expression, localization and functions are altered in various pathologies. Here, we have analyzed the impact of Trx family proteins in neuronal development and recovery, following hypoxia/ischemia and reperfusion. METHODS: We have analyzed the regulation and potential functions of Trx family proteins during hypoxia/ischemia and reoxygenation of the developing brain in both an animal and a cellular model of perinatal asphyxia. We have analyzed the distribution of 14 Trx family and related proteins in the cerebellum, striatum, and hippocampus, three areas of the rat brain that are especially susceptible to hypoxia. Using SH-SY5Y cells subjected to hypoxia and reoxygenation, we have analyzed the functions of some redoxins suggested by the animal experiment. RESULTS AND CONCLUSIONS: We have described/discovered a complex, cell-type and tissue-specific expression pattern following the hypoxia/ischemia and reoxygenation. Particularly, Grx2 and Trx1 showed distinct changes during tissue recovery following hypoxia/ischemia and reoxygenation. Silencing of these proteins in SH-SY5Y cells subjected to hypoxia-reoxygenation confirmed that these proteins are required to maintain the normal neuronal phenotype. GENERAL SIGNIFICANCE: These findings demonstrate the significance of redox signaling in cellular pathways. Grx2 and Trx1 contribute significantly to neuronal integrity and could be clinically relevant in neuronal damage following perinatal asphyxia and other neuronal disorders.