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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 in a redoxdependent 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. Changes in the actin cytoskeleton also disrupt mitochondria-ER contact sites. This results in lower mitochondrial Ca2+ levels and inhibition of the pyruvate dehydrogenase complex, explaining the metabolic changes upon GDAP1 loss of function. Together, these findings reconcile GDAP1-associated phenotypes and implicate disrupted actin signaling in CMT4A pathophysiology.

Endogenous redox systems not only counteract oxidative damage induced by high levels of hydroxyl radicals (OH·) under pathological conditions, but also shape redox signaling as a key player in the regulation of physiological processes. Second messengers like hydrogen peroxide and nitric oxide, as well as redox enzymes of the Thioredoxin (Trx) family, including Trxs, glutaredoxins (Grxs), and peroxiredoxins (Prxs) modulate reversible, oxidative modifications of proteins. Thereby redox regulation is part of various cellular processes such as the immune response and Trx proteins have been linked in different disorders including inflammatory diseases. Here, we have analyzed the protein distribution of representative oxidoreductases of the Trx fold protein family—Trx1, Grx1, Grx2, and Prx2—in a murine model of allergic asthma bronchiale, as well as their potential therapeutic impact on type-2 driven airway inflammation. Ovalbumin (OVA) sensitization and challenge using the type-2 prone Balb/c mouse strain resulted in increased levels of all investigated proteins in distinct cellular patterns. While concomitant treatment with Grx1 and Prx2 did not show any therapeutic impact on the outcome of the disease, Grx2 or Trx1 treatment before and during the OVA challenge phase displayed pronounced protective effects on the manifestation of allergic airway inflammation. Eosinophil numbers and the type-2 cytokine IL-5 were significantly reduced while lung function parameters profoundly improved. The number of macrophages in the bronchoalveolar lavage (BAL) did not change significantly, however, the release of nitric oxide that was linked to airway inflammation was successfully prevented by enzymatically active Grx2 ex vivo. The Grx2 Cys-X-X-Ser mutant that facilitates de-/glutathionylation, but does not catalyze dithiol/disulfide exchange lost the ability to protect from airway hyper reactivity and to decrease NO release by macrophages, however, it reduced the number of infiltrating immune cells and IL-5 release. Altogether, this study demonstrates that specific redox proteins and particular enzyme activities protect against inflammatory damage. During OVA-induced allergic airway inflammation, administration of Grx2 exerts beneficial and thus potentially therapeutic effects.

Mitochondrial fusion and fission tailors the mitochondrial shape to changes in cellular homeostasis. Players of this process are the mitofusins, which regulate fusion of the outer mitochondrial membrane, and the fission protein DRP1. Upon specific stimuli, DRP1 translocates to the mitochondria, where it interacts with its receptors FIS1, MFF, and MID49/51. Another fission factor of clinical relevance is GDAP1. Here, we identify and discuss cysteine residues of these proteins that are conserved in phylogenetically distant organisms and which represent potential sites of posttranslational redox modifications. We reveal that worms and flies possess only a single mitofusin, which in vertebrates diverged into MFN1 and MFN2. All mitofusins contain four conserved cysteines in addition to cysteine 684 in MFN2, a site involved in mitochondrial hyperfusion. DRP1 and FIS1 are also evolutionarily conserved but only DRP1 contains four conserved cysteine residues besides cysteine 644, a specific site of nitrosylation. MFF and MID49/51 are only present in the vertebrate lineage. GDAP1 is missing in the nematode genome and contains no conserved cysteine residues. Our analysis suggests that the function of the evolutionarily oldest proteins of the mitochondrial fusion and fission machinery, the mitofusins and DRP1 but not FIS1, might be altered by redox modifications.

Redox regulation depends on the enzymatically controlled production and decay of redox active molecules. NADPH oxidases, superoxide dismutases, nitric oxide synthases, and others produce the redox active molecules superoxide, hydrogen peroxide, nitric oxide, and hydrogen sulfide. These react with target proteins inducing spatiotemporal modifications of cysteine residues within different signaling cascades. Thioredoxin family proteins are key regulators of the redox state of proteins. They regulate the formation and removal of oxidative modifications by specific thiol reduction and oxidation. All of these redox enzymes affect inflammatory processes and the innate and adaptive immune response. Interestingly, this regulation involves different mechanisms in different biological compartments and specialized cell types. The localization and activity of distinct proteins including, for instance, the transcription factor NFκB and the immune mediator HMGB1 are redox-regulated. The transmembrane protein ADAM17 releases proinflammatory mediators, such as TNFα, and is itself regulated by a thiol switch. Moreover, extracellular redox enzymes were shown to modulate the activity and migration behavior of various types of immune cells by acting as cytokines and/or chemokines. Within this review article, we will address the concept of redox signaling and the functions of both redox enzymes and redox active molecules in innate and adaptive immune responses.

Proteins of the thioredoxin (Trx) family are ubiquitously expressed oxidoreductases. They use cysteinyl residues within their active site to modify substrate proteins posttranslationally by reduction/oxidation reactions or de-/glutathionylation. They play a crucial role in regulating cellular functions such as proliferation, differentiation and apoptosis. This thesis entitled “Thioredoxin family proteins in physiology and disease” focusses on these proteins, i.e. the Trx systems, the glutaredoxin (Grx) systems and the peroxiredoxins (Prxs). Because descriptions of cellular functions of redoxins are rare, we aimed at identifying new interaction partners and functions under physiological and hypoxic conditions in various cell culture and animal models. This thesis emphasizes the concept of compartmentalised redox signaling and demonstrates not only the complexity of the Trx family proteins, but the species-, tissue- and cell type-specific responses to oxygen deprivation and the distinct contribution of the redoxins to controling the fate of a cell. In detail: - Human, mitochondrial Grx2 is an electron donor for Prx3. - Vertebrate Grx3 (human and zebrafish) is involved in iron regulation. - Trx family proteins showed a tissue- and cell type-specific expression and distribution in the rat CNS and responded tissue- and cell type-specific to oxygen deprivation in numerous cell lines and models for perinatal asphyxia, renal ischemia/reperfusion (I/R) injury and transplantation of pancreatic β-cells. (compare to summary of the thesis), Proteine der Thioredoxin (Trx) Familie sind ubiquitär exprimierte Oxidoreduktasen, die Cysteine innerhalb ihres aktiven Zentrums dazu nutzen, Proteine posttranslational durch Reduktions-Oxidations Reaktionen oder De-/Glutathionylierung zu modifizieren. Sie regulieren Prozesse wie Proliferation, Differenzierung und Apoptose. Diese Doktorarbeit mit dem Titel “Thioredoxin family proteins in physiology and disease” konzentriert sich auf die Trx Systeme, die Glutaredoxin (Grx) Systeme und auf die Peroxiredoxine (Prx). Da nur wenige zelluläre Funktionen dieser Proteine bekannt sind, galt es neue Interaktionspartner sowie Funktionen unter physiologischen und hypoxischen Bedingungen in verschiedenen Zell- und Tiermodellen aufzudecken. Diese Arbeit unterstreicht das Konzept des kompartimentalisierten Redox-Signalings und zeigt nicht nur die Komplexität der Trx Familie, sondern spezies-, gewebs- und zell-spezifische Antworten auf Hypoxie und den unterschiedlichen Beitrag der Redoxine an der Kontrolle über das Zellschicksal. Im Detail: - Mitochondrielles Grx2 ist ein Elektronendonor für Prx3 identifiziert. - Grx3 in Vertebraten (Mensch, Zebrafisch) fungiert im Eisenstoffwechsel. - Proteine der Trx Familie zeigten eine gewebs- und zell-spezifische Expression und Verteilung im Zentralen Nervensystem der Ratte sowie eine gewebs- und zell-spezifische Antwort auf Sauerstoffmangel in verschiedenen Zelllinien und Tiermodellen für perinatale Asphyxie, renale Ischämie/Reperfusion (I/R) und Betazell-Transplantation. (vergleichlich der Zusammenfassung der Dissertation)

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 Fil: Romero, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Holubiec, Mariana Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Tornatore, Tamara Logica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; Argentina Fil: Rivière, Stéphanie. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; Argentina Fil: Hanschmann, Eva Maria. Heinrich Heine University; Argentina Fil: Kölliker Frers, Rodolfo Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; Argentina Fil: Tau, Julia. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Patología; Argentina Fil: Blanco, Eduardo. Universidad de Lleida; España Fil: Galeano, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Rodríguez de Fonseca, Fernando. Universidad de Málaga; España Fil: Lillig, Christopher Horst. Heinrich Heine University; Alemania Fil: Capani, Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; Argentina. Universidad de Chile; Chile. Universidad Argentina "John F. Kennedy"; Argentina