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.

The intrinsic amyloidogenic propensity of cofilin-1 is aggravated by Cys-80 oxidation: A possible link with neurodegenerative diseases.

Cofilin-1, an actin dynamizing protein, forms actin-cofilin rods, which is one of the major events that exacerbates the pathophysiology of amyloidogenic diseases. Cysteine oxidation in cofilin-1 under oxidative stress plays a crucial role in the formation of these rods. Others and we have reported that cofilin-1 possesses a self-oligomerization property in vitro and in vivo under physiological conditions. However, it remains elusive if cofilin-1 itself forms amyloid-like structures. We, therefore, hypothesized that cofilin-1 might form amyloid-like assemblies, with a potential to intensify the pathophysiology of amyloid-linked diseases. We used various in silico and in vitro techniques and examined the amyloid-forming propensity of cofilin-1. The study confirms that cofilin-1 possesses an intrinsic tendency of aggregation and forms amyloid-like structures in vitro. Further, we studied the effect of cysteine oxidation on the stability and structural features of cofilin-1. Our data show that oxidation at Cys-80 renders cofilin-1 unstable, leading to a partial loss of protein structure. The results substantiate our hypothesis and establish a strong possibility that cofilin-1 aggregation might play a role in cofilin-mediated pathology and the progression of several amyloid-linked diseases.

Thiol switches in membrane proteins - Extracellular redox regulation in cell biology.

Redox-mediated signal transduction depends on the enzymatic production of second messengers such as hydrogen peroxide, nitric oxide and hydrogen sulfite, as well as specific, reversible redox modifications of cysteine-residues in proteins. So-called thiol switches induce for instance conformational changes in specific proteins that regulate cellular pathways e.g., cell metabolism, proliferation, migration, gene expression and inflammation. Reduction, oxidation and disulfide isomerization are controlled by oxidoreductases of the thioredoxin family, including thioredoxins, glutaredoxins, peroxiredoxins and protein dsisulfide isomerases. These proteins are located in different cellular compartments, interact with substrates and catalyze specific reactions. Interestingly, some of these proteins are released by cells. Their extracellular functions and generally extracellular redox control have been widely underestimated. Here, we give an insight into extracellular redox signaling, extracellular thiol switches and their regulation by secreted oxidoreductases and thiol-isomerases, a topic whose importance has been scarcely studied so far, likely due to methodological limitations. We focus on the secreted redox proteins and characterized thiol switches in the ectodomains of membrane proteins, such as integrins and the metalloprotease ADAM17, which are among the best-characterized proteins and discuss their underlying mechanisms and biological implications.

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.

Signals of the Neuropilin-1-MET Axis and Cues of Mechanical Force Exertion Converge to Elicit Inflammatory Activation in Coherent Endothelial Cells.

The neuropilin-1 (NRP1)-MET signaling axis regulates the motility of individual endothelial cells (ECs). It is unknown how this signaling pathway affects the endothelial barrier in coherent ECs forming a tight monolayer. We hypothesized that it is involved both in modulation of the endothelial barrier and in EC activation. To investigate the role of NRP1-MET signaling in inflammatory processes (e.g., systemic inflammatory response syndrome [SIRS] or snakebite-induced SIRS-like conditions), we employed the C-type lectin-related protein rhodocetin-αß (RCαß) as a specific trigger of this signal axis in ECs in vitro. In coherent HUVECs, RCαß reinforced the actin cytoskeleton and increased cell stiffness, thus favoring vascular endothelial cadherin-mediated transmission of intercellular forces. Increased cell stiffness was associated with enhanced activation of RhoA and nuclear translocation of NF-κB. Simultaneously, RCαß-triggered signaling via the NRP1-MET axis increased EC monolayer permeability, induced transcription of proinflammatory genes such as ICAM-1 and, consequently, leukocyte tethering. The RCαß-induced transcriptome differed from that induced by hepatocyte growth factor, although in both cases the same tyrosine kinase, MET, was involved. This was due to RCαß-mediated recruitment of the MET coreceptor NRP1 and additional Rho-mediated activation of the actomyosin system. RCαß induced similar transcriptional and cellular changes if external shear forces were applied. These data highlight the modulatory role of NRP1 as MET coreceptor, and they explain how some snake venoms induce SIRS-like conditions. Additionally, this study demonstrates that inflammatory activation of coherent ECs is triggered by converging signals that are induced by NRP1-MET signaling and influenced by intercellular forces.

Linkage of inflammation and oxidative stress via release of glutathionylated peroxiredoxin-2, which acts as a danger signal.

The mechanism by which oxidative stress induces inflammation and vice versa is unclear but is of great importance, being apparently linked to many chronic inflammatory diseases. We show here that inflammatory stimuli induce release of oxidized peroxiredoxin-2 (PRDX2), a ubiquitous redox-active intracellular enzyme. Once released, the extracellular PRDX2 acts as a redox-dependent inflammatory mediator, triggering macrophages to produce and release TNF-α. The oxidative coupling of glutathione (GSH) to PRDX2 cysteine residues (i.e., protein glutathionylation) occurs before or during PRDX2 release, a process central to the regulation of immunity. We identified PRDX2 among the glutathionylated proteins released in vitro by LPS-stimulated macrophages using mass spectrometry proteomic methods. Consistent with being part of an inflammatory cascade, we find that PRDX2 then induces TNF-α release. Unlike classical inflammatory cytokines, PRDX2 release does not reflect LPS-mediated induction of mRNA or protein synthesis; instead, PRDX2 is constitutively present in macrophages, mainly in the reduced form, and is released in the oxidized form on LPS stimulation. Release of PRDX2 is also observed in human embryonic kidney cells treated with TNF-α. Importantly, the PRDX2 substrate thioredoxin (TRX) is also released along with PRDX2, enabling an oxidative cascade that can alter the -SH status of surface proteins and thereby facilitate activation via cytokine and Toll-like receptors. Thus, our findings suggest a model in which the release of PRDX2 and TRX from macrophages can modify the redox status of cell surface receptors and enable induction of inflammatory responses. This pathway warrants further exploration as a potential novel therapeutic target for chronic inflammatory diseases.

Redox regulation of cytoskeletal dynamics during differentiation and de-differentiation.

BACKGROUND: The cytoskeleton, unlike the bony vertebrate skeleton or the exoskeleton of invertebrates, is a highly dynamic meshwork of protein filaments that spans through the cytosol of eukaryotic cells. Especially actin filaments and microtubuli do not only provide structure and points of attachments, but they also shape cells, they are the basis for intracellular transport and distribution, all types of cell movement, and--through specific junctions and points of adhesion--join cells together to form tissues, organs, and organisms. SCOPE OF REVIEW: The fine tuned regulation of cytoskeletal dynamics is thus indispensible for cell differentiation and all developmental processes. Here, we discussed redox signalling mechanisms that control this dynamic remodeling. Foremost, we emphasised recent discoveries that demonstrated reversible thiol and methionyl switches in the regulation of actin dynamics. MAJOR CONCLUSIONS: Thiol and methionyl switches play an essential role in the regulation of cytoskeletal dynamics. GENERAL SIGNIFICANCE: The dynamic remodeling of the cytoskeleton is controlled by various redox switches. These mechanisms are indispensible during development and organogenesis and might contribute to numerous pathological conditions. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation.

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.

Cysteine Oxidation Targets Peroxiredoxins 1 and 2 for Exosomal Release through a Novel Mechanism of Redox-Dependent Secretion.

Nonclassical protein secretion is of major importance as a number of cytokines and inflammatory mediators are secreted via this route. Current evidence indicates that there are several mechanistically distinct methods of nonclassical secretion. We have shown recently that peroxiredoxin (Prdx) 1 and Prdx2 are released by various cells upon exposure to inflammatory stimuli such as lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). The released Prdx then acts to induce production of inflammatory cytokines. However, Prdx1 and 2 do not have signal peptides and therefore must be secreted by alternative mechanisms, as has been postulated for the inflammatory mediators interleukin-1ß (IL-1ß) and high mobility group box-1 (HMGB1). We show here that circulating Prdx1 and 2 are present exclusively as disulfide-linked homodimers. Inflammatory stimuli also induce in vitro release of Prdx1 and 2 as disulfide-linked homodimers. Mutation of cysteines Cys51 or Cys172 (but not Cys70) in Prdx2, and Cys52 or Cys173 (but not Cys71 or Cys83) in Prdx1 prevented dimer formation and this was associated with inhibition of their TNF-α-induced release. Thus, the presence and oxidation of key cysteine residues in these proteins are a prerequisite for their secretion in response to TNF-α, and this release can be induced with an oxidant. By contrast, the secretion of the nuclear-associated danger signal HMGB1 is independent of cysteine oxidation, as shown by experiments with a cysteine-free HMGB1 mutant. Release of Prdx1 and 2 is not prevented by inhibitors of the classical secretory pathway, instead, both Prdx1 and 2 are released in exosomes from both human embryonic kidney (HEK) cells and monocytic cells. Serum Prdx1 and 2 also are associated with the exosomes. These results describe a novel pathway of protein secretion mediated by cysteine oxidation that underlines the importance of redox-dependent signaling mechanisms in inflammation.

Identification of a dithiol-disulfide switch in collapsin response mediator protein 2 (CRMP2) that is toggled in a model of neuronal differentiation.

Vertebrate-specific glutaredoxin 2 (Grx2) is expressed in at least two isoforms, mitochondrial Grx2a and cytosolic Grx2c. We have previously shown that cytosolic Grx2 is essential for embryonic development of the brain. In particular, we identified collapsin response mediator protein 2 (CRMP2/DPYSL2), a mediator of the semaphorin-plexin signaling pathway, as redox-regulated target of Grx2c and demonstrated that this regulation is required for normal axonal outgrowth. In this study, we demonstrate the molecular mechanism of this regulation, a specific and reversible intermolecular Cys-504-Cys-504 dithiol-disulfide switch in homotetrameric CRMP2. This switch determines two conformations of the quaternary CRMP2 complex that controls axonal outgrowth and thus neuronal development.

Identification of potential protein dithiol-disulfide substrates of mammalian Grx2.

BACKGROUND: Glutaredoxins (Grxs) catalyze the reduction of protein disulfides via the dithiol mechanism and the de-/glutathionylation of substrates via the monothiol mechanism. These rapid, specific, and generally also reversible modifications are part of various signaling cascades regulating for instance cell proliferation, differentiation and apoptosis. Even though crucial functions of the conserved, mitochondrial Grx2a and the cytosolic/nuclear Grx2c isoforms have been proposed, only a few substrates have been identified in vitro or in vivo. The significance of redox signaling is emerging, yet a general lack of methods for the time-resolved analysis of these distinct and rapid modifications in vivo constitutes the biggest challenge in the redox signaling field. METHODS AND RESULTS: Here, we have identified potential interaction partners for Grx2 isoforms in human HeLa cells and mouse tissues by an intermediate trapping approach. Some of the 50 potential substrates are part of the cytoskeleton or act in protein folding, cellular signaling and metabolism. Part of these interactions were further verified by immunoprecipitation or a newly established 2-D redox blot. CONCLUSIONS: Our study demonstrates that Grx2 catalyzes both the specific oxidation and the reduction of cysteinyl residues in the same compartment at the same time and without affecting the global cellular thiol-redox state. GENERAL SIGNIFICANCE: The knowledge of specific targets will be helpful in understanding the functions of Grx2. The 2-D redox blot may be useful for the analysis of the overall thiol-redox state of proteins with high molecular weight and numerous cysteinyl residues, that evaded analysis by previously described methods.

The significance of glutaredoxins for diabetes mellitus and its complications.

Diabetes mellitus is a non-communicable metabolic disease hallmarked by chronic hyperglycemia caused by beta-cell failure. Diabetic complications affect the vasculature and result in macro- and microangiopathies, which account for a significantly increased morbidity and mortality. The rising incidence and prevalence of diabetes is a major global health burden. There are no feasible strategies for beta-cell preservation available in daily clinical practice. Therefore, patients rely on antidiabetic drugs or the application of exogenous insulin. Glutaredoxins (Grxs) are ubiquitously expressed and highly conserved members of the thioredoxin family of proteins. They have specific functions in redox-mediated signal transduction, iron homeostasis and biosynthesis of iron-sulfur (FeS) proteins, and the regulation of cell proliferation, survival, and function. The involvement of Grxs in chronic diseases has been a topic of research for several decades, suggesting them as therapeutic targets. Little is known about their role in diabetes and its complications. Therefore, this review summarizes the available literature on the significance of Grxs in diabetes and its complications. In conclusion, Grxs are differentially expressed in the endocrine pancreas and in tissues affected by diabetic complications, such as the heart, the kidneys, the eye, and the vasculature. They are involved in several pathways essential for insulin signaling, metabolic inflammation, glucose and fatty acid uptake and processing, cell survival, and iron and mitochondrial metabolism. Most studies describe significant changes in glutaredoxin expression and/or activity in response to the diabetic metabolism. In general, mitigated levels of Grxs are associated with oxidative distress, cell damage, and even cell death. The induced overexpression is considered a potential part of the cellular stress-response, counteracting oxidative distress and exerting beneficial impact on cell function such as insulin secretion, cytokine expression, and enzyme activity.

Iron-sulfur cluster loss in mitochondrial CISD1 mediates PINK1 loss-of-function phenotypes.

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra of the midbrain. Familial cases of PD are often caused by mutations of PTEN-induced kinase 1 (PINK1) and the ubiquitin ligase Parkin, both pivotal in maintaining mitochondrial quality control. CISD1, a homodimeric mitochondrial iron-sulfur-binding protein, is a major target of Parkin-mediated ubiquitination. We here discovered a heightened propensity of CISD1 to form dimers in Pink1 mutant flies and in dopaminergic neurons from PINK1 mutation patients. The dimer consists of two monomers that are covalently linked by a disulfide bridge. In this conformation CISD1 cannot coordinate the iron-sulfur cofactor. Overexpressing Cisd, the Drosophila ortholog of CISD1, and a mutant Cisd incapable of binding the iron-sulfur cluster in Drosophila reduced climbing ability and lifespan. This was more pronounced with mutant Cisd and aggravated in Pink1 mutant flies. Complete loss of Cisd, in contrast, rescued all detrimental effects of Pink1 mutation on climbing ability, wing posture, dopamine levels, lifespan, and mitochondrial ultrastructure. Our results suggest that Cisd, probably iron-depleted Cisd, operates downstream of Pink1 shedding light on PD pathophysiology and implicating CISD1 as a potential therapeutic target.

Parkinson's disease affects millions of people worldwide, causing progressively worse symptoms like stiffness, tremors and difficulty moving. These issues result from the death of neurons in the brain that produce the neurotransmitter dopamine. While most cases have no known cause, 10 to 15 per cent are due to inherited gene mutations. This includes mutations in the genes that code for the proteins PINK1 and Parkin which are essential for maintaining healthy mitochondria, the powerhouse of the cell. Mutations in this quality control system affect a protein called CISD1, which sits within the outer surface of the mitochondria. CISD1 contains a cluster of iron and sulfur ions, and is involved in regulating iron levels and mitochondrial energy production. However, its role in inherited cases of Parkinson's disease, particularly those related to mutations in PINK1 and Parkin, is poorly understood. To understand the impact of CISD1, Bitar et al. studied genetically modified fruit flies and dopamine-producing neurons from Parkinson's patients with PINK1 mutations. This revealed that losing PINK1 activity led to higher levels of CISD1 proteins which lacked the iron-sulfur cluster due to a bond forming between two CISD1 molecules. Reducing levels of the CISD1-equivalent protein in the flies helped to alleviate most of the symptoms caused by PINK1 and Parkin gene mutations, such as difficulties climbing and impaired wing posture. These findings suggest that iron-depleted CISD1 contributes to the symptoms associated with Parkinson's disease, underscoring its potential as a drug target. Drugs that target CISD1 already exist, which could ease the way for further research. Recent studies have shown that cases of Parkinson's related to mutations in PINK-1 share features with some non-inherited instances of the disease, suggesting that this approach could potentially benefit many patients.

Thioredoxin and glutaredoxin system proteins-immunolocalization in the rat central nervous system.

BACKGROUND: The oxidoreductases of the thioredoxin (Trx) family of proteins play a major role in the cellular response to oxidative stress. Redox imbalance is a major feature of brain damage. For instance, neuronal damage and glial reaction induced by a hypoxic-ischemic episode is highly related to glutamate excitotoxicity, oxidative stress and mitochondrial dysfunction. Most animal models of hypoxia-ischemia in the central nervous system (CNS) use rats to study the mechanisms involved in neuronal cell death, however, no comprehensive study on the localization of the redox proteins in the rat CNS was available. METHODS: The aim of this work was to study the distribution of the following proteins of the thioredoxin and glutathione/glutaredoxin (Grx) systems in the rat CNS by immunohistochemistry: Trx1, Trx2, TrxR1, TrxR2, Txnip, Grx1, Grx2, Grx3, Grx5, and γ-GCS, peroxiredoxin 1 (Prx1), Prx2, Prx3, Prx4, Prx5, and Prx6. We have focused on areas most sensitive to a hypoxia-ischemic insult: Cerebellum, striatum, hippocampus, spinal cord, substantia nigra, cortex and retina. RESULTS AND CONCLUSIONS: Previous studies implied that these redox proteins may be distributed in most cell types and regions of the CNS. Here, we have observed several remarkable differences in both abundance and regional distribution that point to a complex interplay and crosstalk between the proteins of this family. GENERAL SIGNIFICANCE: We think that these data might be helpful to reveal new insights into the role of thiol redox pathways in the pathogenesis of hypoxia-ischemia insults and other disorders of the CNS. This article is part of a Special Issue entitled Human and Murine Redox Protein Atlases.

Thiol Modifications in the Extracellular Space-Key Proteins in Inflammation and Viral Infection.

Posttranslational modifications (PTMs) allow to control molecular and cellular functions in response to specific signals and changes in the microenvironment of cells. They regulate structure, localization, stability, and function of proteins in a spatial and temporal manner. Among them, specific thiol modifications of cysteine (Cys) residues facilitate rapid signal transduction. In fact, Cys is unique because it contains the highly reactive thiol group that can undergo different reversible and irreversible modifications. Upon inflammation and changes in the cellular microenvironment, many extracellular soluble and membrane proteins undergo thiol modifications, particularly dithiol-disulfide exchange, S-glutathionylation, and S-nitrosylation. Among others, these thiol switches are essential for inflammatory signaling, regulation of gene expression, cytokine release, immunoglobulin function and isoform variation, and antigen presentation. Interestingly, also the redox state of bacterial and viral proteins depends on host cell-mediated redox reactions that are critical for invasion and infection. Here, we highlight mechanistic thiol switches in inflammatory pathways and infections including cholera, diphtheria, hepatitis, human immunodeficiency virus (HIV), influenza, and coronavirus disease 2019 (COVID-19).

Changing Perspectives from Oxidative Stress to Redox Signaling-Extracellular Redox Control in Translational Medicine.

Extensive research has changed the understanding of oxidative stress that has been linked to every major disease. Today we distinguish oxidative eu- and distress, acknowledging that redox modifications are crucial for signal transduction in the form of specific thiol switches. Long underestimated, reactive species and redox proteins of the Thioredoxin (Trx) family are indeed essential for physiological processes. Moreover, extracellular redox proteins, low molecular weight thiols and thiol switches affect signal transduction and cell-cell communication. Here, we highlight the impact of extracellular redox regulation for health, intermediate pathophenotypes and disease. Of note, recent advances allow the analysis of redox changes in body fluids without using invasive and expensive techniques. With this new knowledge in redox biochemistry, translational strategies can lead to innovative new preventive and diagnostic tools and treatments in life sciences and medicine.

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.

GDAP1 loss of function inhibits the mitochondrial pyruvate dehydrogenase complex by altering the actin cytoskeleton.

Charcot-Marie-Tooth (CMT) disease 4A is an autosomal-recessive polyneuropathy caused by mutations of ganglioside-induced differentiation-associated protein 1 (GDAP1), a putative glutathione transferase, which affects mitochondrial shape and alters cellular Ca2+ homeostasis. Here, we identify the underlying mechanism. We found that patient-derived motoneurons and GDAP1 knockdown SH-SY5Y cells display two phenotypes: more tubular mitochondria and a metabolism characterized by glutamine dependence and fewer cytosolic lipid droplets. GDAP1 interacts with the actin-depolymerizing protein Cofilin-1 and beta-tubulin in a redox-dependent manner, suggesting a role for actin signaling. Consistently, GDAP1 loss causes less F-actin close to mitochondria, which restricts mitochondrial localization of the fission factor dynamin-related protein 1, instigating tubularity. GDAP1 silencing also disrupts mitochondria-ER contact sites. These changes result in lower mitochondrial Ca2+ levels and inhibition of the pyruvate dehydrogenase complex, explaining the metabolic changes upon GDAP1 loss of function. Together, our findings reconcile GDAP1-associated phenotypes and implicate disrupted actin signaling in CMT4A pathophysiology.

Both thioredoxin 2 and glutaredoxin 2 contribute to the reduction of the mitochondrial 2-Cys peroxiredoxin Prx3.

The proteins from the thioredoxin family are crucial actors in redox signaling and the cellular response to oxidative stress. The major intracellular source for oxygen radicals are the components of the respiratory chain in mitochondria. Here, we show that the mitochondrial 2-Cys peroxiredoxin (Prx3) is not only substrate for thioredoxin 2 (Trx2), but can also be reduced by glutaredoxin 2 (Grx2) via the dithiol reaction mechanism. Grx2 reduces Prx3 exhibiting catalytic constants (K(m), 23.8 µmol·liter(-1); V(max), 1.2 µmol·(mg·min)(-1)) similar to Trx2 (K(m), 11.2 µmol·liter(-1); V(max), 1.1 µmol·(mg·min)(-1)). The reduction of the catalytic disulfide of the atypical 2-Cys Prx5 is limited to the Trx system. Silencing the expression of either Trx2 or Grx2 in HeLa cells using specific siRNAs did not change the monomer:dimer ratio of Prx3 detected by a specific 2-Cys Prx redox blot. Only combined silencing of the expression of both proteins led to an accumulation of oxidized protein. We further demonstrate that the distribution of Prx3 in different mouse tissues is either linked to the distribution of Trx2 or Grx2. These results introduce Grx2 as a novel electron donor for Prx3, providing further insights into pivotal cellular redox signaling mechanisms.

Cofilin1 oxidation links oxidative distress to mitochondrial demise and neuronal cell death.

Many cell death pathways, including apoptosis, regulated necrosis, and ferroptosis, are relevant for neuronal cell death and share common mechanisms such as the formation of reactive oxygen species (ROS) and mitochondrial damage. Here, we present the role of the actin-regulating protein cofilin1 in regulating mitochondrial pathways in oxidative neuronal death. Cofilin1 deletion in neuronal HT22 cells exerted increased mitochondrial resilience, assessed by quantification of mitochondrial ROS production, mitochondrial membrane potential, and ATP levels. Further, cofilin1-deficient cells met their energy demand through enhanced glycolysis, whereas control cells were metabolically impaired when challenged by ferroptosis. Further, cofilin1 was confirmed as a key player in glutamate-mediated excitotoxicity and associated mitochondrial damage in primary cortical neurons. Using isolated mitochondria and recombinant cofilin1, we provide a further link to toxicity-related mitochondrial impairment mediated by oxidized cofilin1. Our data revealed that the detrimental impact of cofilin1 on mitochondria depends on the oxidation of cysteine residues at positions 139 and 147. Overall, our findings show that cofilin1 acts as a redox sensor in oxidative cell death pathways of ferroptosis, and also promotes glutamate excitotoxicity. Protective effects by cofilin1 inhibition are particularly attributed to preserved mitochondrial integrity and function. Thus, interfering with the oxidation and pathological activation of cofilin1 may offer an effective therapeutic strategy in neurodegenerative diseases.