Charakterisierung der Selenoproteine Thioredoxinreduktase 1 und 2 anhand von Knock-out-Mausmodellen

Thioredoxin Reductases (TR) are ubiquitously expressed Selenium-containing redox enzymes. By reducing different intracellular substrates they protect cells from oxidative stress. For mus musculus two members of the Thioredoxin Reductase gene family have been characterized so far: TR1 and TR2. Whereas the subcellular localization of TR1 is mainly cytoplasmatic, TR2 was only found in mitochondria. The protein functions of TR1 and TR2 were investigated by examining genetically engineered mice. Here I will present the establishment of a conditional TR1-knockout mouse strain and the phenotypical characterization of TR1- and of TR2-knockout mice, that have been established before. Both knockouts, TR1 and TR2, showed to be embryonically lethal. TR1 deficient mouse embryos died at day 9.5, whereas TR2 deficient mouse embryos died around day 13.0 of embryonic gestation. Total deficiency of TR1 leads to strong developmental retardation and malformations in organogenesis mostly affecting the turning of the embryo, closure of neural tube, formation of head structures and formation of somites. Thereby TR1 deficient embryos maximally reach the developmental stage of normal E8.5 wildtype embryos. Neuronal specific TR1 knockout mice show growth retardation and cerebellar hypoplasia. Starting at the postnatal age of 10-14 days they loose weight and show cerebellar ataxia and tremors. Recent results indicate that mice die at the age of approximately 4 weeks. TR2 deficient embryos can already be recognized at embryonic day E11.5 due to a smaller embryo size and paler colour. Mice are retarded in their status of organ differentiation and show malformations in heart and liver development, possibly leading to fatal heart failure or liver insufficiency. Besides that, TR2 deficient embryos show decreased proliferation rates of hematopoetic progenitor cells in the embryonic liver. Taken together these results indicate that TR1 plays an important role in embryonic growth and organogenesis. Besides, deficiency of TR2 is proposed to disturb primarily the integrity of mitochondria and as a consequence affect processes like cell proliferation and transdifferentiation – leading to embryonic death. The functions of Thioredoxin Reductases 1 and 2 in separate organ systems need to be further analyzed by using conditional ablation strategies.Thioredoxinreduktasen (TR) sind ubiquitär exprimierte, Selen enthaltende Redoxenzyme, die Zellen durch Reduktion intrazellulärer Substrate vor oxidativem Stress schützen. Durch gezielte Inaktivierung der vorwiegend zytoplasmatisch lokalisierten Thioredoxinreduktase 1 (TR1) und der mitochondrialen Thioredoxinreduktase 2 (TR2) im Mausmodell sollten die Funktionen der TR in vivo studiert werden. In dieser Arbeit wurden TR1- defiziente Mäuse in Form eines konditionalen Maus-Knock-out-Modells etabliert. Weiterhin wurden die Phänotypen sowohl des TR1- als auch des bereits zuvor etablierten TR2-defizienten Mausstammes beschrieben. Es stellte sich heraus, dass der Mangel einer funktionellen TR1 ebenso wie der Mangel einer funktionellen TR2 zu pränataler Letalität von Mausembryonen führt. Dabei ist ein Überleben der TR1–defizienten Embryonen nur bis zum Tag E9.5 möglich, während die TR2–defizienten Embryonen etwa am Tag E13.0 der Embryonalentwicklung sterben. TR1–defiziente Embryonen zeigen hochgradige Entwicklungsstörungen in der Organogenese. Die Embryonen erreichen dabei höchstens ein mit Tag E8.5 normal entwickelter Mausembryonen vergleichbares Stadium, wobei die embryonale Drehung nicht vollzogen wird, das Neuralrohr sich nicht vollständig schließt, Kopfstrukturen nicht erkennbar und besonders das Fehlen der Somiten auffällig sind. Die neuronenspezifische Ausschaltung der TR1 führte bei neugeborenen Mäusen zu allgemeiner Wachstumsretardation und Hypoplasie des Kleinhirns mit klinischen Symptomen wie Tremor und Ataxie. Die neuronenspezifisch TR1–defizienten Mäuse erreichten nach bisherigen Untersuchungen höchstens ein Alter von etwa 28 Lebenstagen. TR2–defiziente Embryonen zeigen ebenfalls Rückstände in ihrer Entwicklung. Sie sind ab dem Tag E11.0 der Embryonalentwicklung kleiner und blasser als ihre Wurfgeschwister. Alle Organe sind in ihrer Differenzierung retardiert. Die beobachtete Dysplasien im Bereich des Herzens und der Leber haben möglicherweise fatale Auswirkungen auf die Überlebensfähigkeit der TR2-defizienten Embryonen. Außerdem sind reduzierte Proliferationsraten in der embryonalen Hämatopoese, in Herz- und Lebergewebe beobachtet worden. Zusammengefasst zeigte sich eine wichtige Bedeutung der TR1 für embryonales Wachstum und Organogenese. Weiterhin zeigte sich, dass der Mangel einer funktionellen TR2 möglicherweise primär die mitochondriale Integrität der Zellen schädigt und dadurch sekundär Zellproliferation und Gewebsdifferenzierung beeinträchtigt werden, die ein Überleben der Embryonen unmöglich machen. Bei beiden Knock-out-Maus-Modellen handelt es sich um konditionale Konstrukte, sodass die Funktionen der Thioredoxinreduktasen 1 und 2 zukünftig auch separat in einzelnen Organen studiert werden können

The Role of Thioredoxin Reductases in Brain Development

The thioredoxin-dependent system is an essential regulator of cellular redox balance. Since oxidative stress has been linked with neurodegenerative disease, we studied the roles of thioredoxin reductases in brain using mice with nervous system (NS)-specific deletion of cytosolic (Txnrd1) and mitochondrial (Txnrd2) thioredoxin reductase. While NS-specific Txnrd2 null mice develop normally, mice lacking Txnrd1 in the NS were significantly smaller and displayed ataxia and tremor. A striking patterned cerebellar hypoplasia was observed. Proliferation of the external granular layer (EGL) was strongly reduced and fissure formation and laminar organisation of the cerebellar cortex was impaired in the rostral portion of the cerebellum. Purkinje cells were ectopically located and their dendrites stunted. The Bergmann glial network was disorganized and showed a pronounced reduction in fiber strength. Cerebellar hypoplasia did not result from increased apoptosis, but from decreased proliferation of granule cell precursors within the EGL. Of note, neuron-specific inactivation of Txnrd1 did not result in cerebellar hypoplasia, suggesting a vital role for Txnrd1 in Bergmann glia or neuronal precursor cells

Identification of a signature motif for the eIF4a3–SECIS interaction

eIF4a3, a DEAD-box protein family member, is a component of the exon junction complex which assembles on spliced mRNAs. The protein also acts as a transcript-selective translational repressor of selenoprotein synthesis during selenium deficiency. Selenocysteine (Sec) incorporation into selenoproteins requires a Sec Insertion Sequence (SECIS) element in the 3′ untranslated region. During selenium deficiency, eIF4a3 binds SECIS elements from non-essential selenoproteins, preventing Sec insertion. We identified a molecular signature for the eIF4a3-SECIS interaction using RNA gel shifts, surface plasmon resonance and enzymatic foot printing. Our results support a two-site interaction model, where eIF4a3 binds the internal and apical loops of the SECIS. Additionally, the stability of the complex requires uridine in the SECIS core. In terms of protein requirements, the two globular domains of eIF4a3, which are connected by a linker, are both critical for SECIS binding. Compared to full-length eIF4a3, the two domains in trans bind with a lower association rate but notably, the uridine is no longer important for complex stability. These results provide insight into how eIF4a3 discriminates among SECIS elements and represses translation

Osteo-Chondroprogenitor–Specific Deletion of the Selenocysteine tRNA Gene, Trsp, Leads to Chondronecrosis and Abnormal Skeletal Development: A Putative Model for Kashin-Beck Disease

Kashin-Beck disease, a syndrome characterized by short stature, skeletal deformities, and arthropathy of multiple joints, is highly prevalent in specific regions of Asia. The disease has been postulated to result from a combination of different environmental factors, including contamination of barley by mold mycotoxins, iodine deficiency, presence of humic substances in drinking water, and, importantly, deficiency of selenium. This multifunctional trace element, in the form of selenocysteine, is essential for normal selenoprotein function, including attenuation of excessive oxidative stress, and for the control of redox-sensitive molecules involved in cell growth and differentiation. To investigate the effects of skeletal selenoprotein deficiency, a Cre recombinase transgenic mouse line was used to trigger Trsp gene deletions in osteo-chondroprogenitors. Trsp encodes selenocysteine tRNA[Ser]Sec, required for the incorporation of selenocysteine residues into selenoproteins. The mutant mice exhibited growth retardation, epiphyseal growth plate abnormalities, and delayed skeletal ossification, as well as marked chondronecrosis of articular, auricular, and tracheal cartilages. Phenotypically, the mice thus replicated a number of the pathological features of Kashin-Beck disease, supporting the notion that selenium deficiency is important to the development of this syndrome

Redox homeostasis and age-related deficits in neuromuscular integrity and function

Skeletal muscle is a major site of metabolic activity and is the most abundant tissue in the human body. Age-related muscleatrophy (sarcopenia) and weakness, characterized by progressive loss of lean muscle mass and function, is a major contributorto morbidity and has a profound effect on the quality of life of older people. With a continuously growing older population(estimated 2 billion of people aged >60 by 2050), demand for medical and social care due to functional deficits, associatedwith neuromuscular ageing, will inevitably increase. Desp ite the importance of this ‘epidemic’ problem, the primarybiochemical and molecular mechanisms underlying age-related deficits in neuromuscular integrity and function have not beenfully determined. Skeleta l muscle generates reactive oxygen and nitrogen species (RONS) from a variety of subcellular sources,and age-associated oxidative damage has been suggested to be a major fac tor contributing to the initiation and progression ofmuscle atrophy inherent with ageing. RONS can modulate a variety of intracellular signal transduction processes, anddisruption of these events over time due to altered redox control has been proposed as an underlying mechanis m of ageing.The role of oxidants in ageing has been extensively examined in different model organisms that have undergone geneticmanipulations with inconsistent findings. Transgenic and knockout rodent studies have provided insight into the function ofRONS regulatory systems in neuromuscular ageing. This review summarizes almost 30 years of research in the field of redoxhomeostasis and muscle ageing, providing a detailed discussion of the experimental approaches that have been undertaken inmurine models to examine the role of redox regulation in age-related muscle atrophy and weakness