Christoph Ufer

Redox Control in Mammalian Embryo Development

The development of an embryo constitutes a complex choreography of regulatory events that underlies precise temporal and spatial control. Throughout this process the embryo encounters ever changing environments, which challenge its metabolism. Oxygen is required for embryogenesis but it also poses a potential hazard via formation of reactive oxygen and reactive nitrogen species (ROS/RNS). These metabolites are capable of modifying macromolecules (lipids, proteins, nucleic acids) and altering their biological functions. On one hand, such modifications may have deleterious consequences and must be counteracted by antioxidant defense systems. On the other hand, ROS/RNS function as essential signal transducers regulating the cellular phenotype. In this context the combined maternal/embryonic redox homeostasis is of major importance and dysregulations in the equilibrium of pro- and antioxidative processes retard embryo development, leading to organ malformation and embryo lethality. Silencing the in vivo expression of pro- and antioxidative enzymes provided deeper insights into the role of the embryonic redox equilibrium. Moreover, novel mechanisms linking the cellular redox homeostasis to gene expression regulation have recently been discovered (oxygen sensing DNA demethylases and protein phosphatases, redox-sensitive microRNAs and transcription factors, moonlighting enzymes of the cellular redox homeostasis) and their contribution to embryo development is critically reviewed.

Molecular biology of glutathione peroxidase 4: from genomic structure to developmental expression and neural function.

Abstract Selenoproteins have been recognized as modulators of brain function and signaling. Phospholipid hydroperoxide glutathione peroxidase (GPx4/PHGPx) is a unique member of the selenium-dependent glutathione peroxidases in mammals with a pivotal role in brain development and function. GPx4 exists as a cytosolic, mitochondrial, and nuclear isoform derived from a single gene. In mice, the GPx4 gene is located on chromosome 10 in close proximity to a functional retrotransposome that is expressed under the control of captured regulatory elements. Elucidation of crystallographic data uncovered structural peculiarities of GPx4 that provide the molecular basis for its unique enzymatic properties and substrate specificity. Monomeric GPx4 is multifunctional: it acts as a reducing enzyme of peroxidized phospholipids and thiols and as a structural protein. Transcriptional regulation of the different GPx4 isoforms requires several isoform-specific cis-regulatory sequences and trans-activating factors. Cytosolic and mitochondrial GPx4 are the major isoforms exclusively expressed by neurons in the developing brain. In stark contrast, following brain trauma, GPx4 is specifically upregulated in non-neuronal cells, i.e., reactive astrocytes. Molecular approaches to genetic modification in mice have revealed an essential and isoform-specific function for GPx4 in development and disease. Here we review recent findings on GPx4 with emphasis on its molecular structure and function and consider potential mechanisms that underlie neural development and neuropathological conditions.

The roles of glutathione peroxidases during embryo development

Embryo development relies on the complex interplay of the basic cellular processes including proliferation, differentiation, and apoptotic cell death. Precise regulation of these events is the basis for the establishment of embryonic structures and the organ development. Beginning with fertilization of the oocyte until delivery the developing embryo encounters changing environmental conditions such as varying levels of oxygen, which can give rise to reactive oxygen species (ROS). These challenges are met by the embryo with metabolic adaptations and by an array of anti-oxidative mechanisms. ROS can be deleterious by modifying biological molecules including lipids, proteins, and nucleic acids and may induce abnormal development or even embryonic lethality. On the other hand ROS are vital players of various signaling cascades that affect the balance between cell growth, differentiation, and death. An imbalance or dysregulation of these biological processes may generate cells with abnormal growth and is therefore potentially teratogenic and tumorigenic. Thus, a precise balance between processes generating ROS and those decomposing ROS is critical for normal embryo development. One tier of the cellular protective system against ROS constitutes the family of selenium-dependent glutathione peroxidases (GPx). These enzymes reduce hydroperoxides to the corresponding alcohols at the expense of reduced glutathione. Of special interest within this protein family is the moonlighting enzyme glutathione peroxidase 4 (Gpx4). This enzyme is a scavenger of lipophilic hydroperoxides on one hand, but on the other hand can be transformed into an enzymatically inactive cellular structural component. GPx4 deficiency – in contrast to all other GPx family members – leads to abnormal embryo development and finally produces a lethal phenotype in mice. This review is aimed at summarizing the current knowledge on GPx isoforms during embryo development and tumor development with an emphasis on GPx4.

The Role of Phospholipid Hydroperoxide Glutathione Peroxidase Isoforms in Murine Embryogenesis

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a selenocysteine-containing enzyme, and three different isoforms (cytosolic, mitochondrial, and nuclear) originate from the GPx4 gene. Homozygous GPx4-deficient mice die in utero at midgestation, since they fail to initiate gastrulation and do not develop embryonic cavities. To investigate the biological basis for embryonic lethality, we first explored expression of the GPx4 in adult murine brain and found expression of the protein in cerebral neurons. Next, we profiled mRNA expression during the time course of embryogenesis (embryonic days 6.5-17.5 (E6.5-17.5)) and detected mitochondrial and cytosolic mRNA species at high concentrations. In contrast, the nuclear isoform was only expressed in small amounts. Cytosolic GPx4 mRNA was present at constant levels (about 100 copies per 1000 copies of glyceraldehyde-3-phosphate dehydrogenase mRNA), whereas nuclear and mitochondrial isoforms were down-regulated between E14.5 and E17.5. In situ hybridization indicated expression of GPx4 isoforms in all developing germ layers during gastrulation and in the somite stage in the developing central nervous system and in the heart. When we silenced expression of GPx4 isoforms during in vitro embryogenesis using short interfering RNA technology, we observed that knockdown of mitochondrial GPx4 strongly impaired segmentation of rhombomeres 5 and 6 during hindbrain development and induced cerebral apoptosis. In contrast, silencing expression of the nuclear isoform led to retardations in atrium formation. Taken together, our data indicate specific expression of GPx4 isoforms in embryonic brain and heart and strongly suggest a role of this enzyme in organogenesis. These findings may explain in part intrauterine lethality of GPx4 knock-out mice.

Monoamine oxidase-A modulates apoptotic cell death induced by staurosporine in human neuroblastoma cells

Monoamine oxidases (MAOs) are mitochondrial enzymes which control the levels of neurotransmitters in the brain and dietary amines in peripheral tissues via oxidative deamination. MAO has also been implicated in cell signalling. In this study, we describe the MAO‐A isoform as functional in apoptosis induced by staurosporine (STS) in human dopaminergic neuroblastoma cells (SH‐SY5Y). Increased levels of MAO‐A activity were induced by STS, accompanied by increased MAO‐A protein and activation of the initiator of the intrinsic pathway, caspase 9, and the executioner caspase 3. MAO‐A mRNA levels were unaffected by STS, suggesting that changes in MAO‐A protein are due to post‐transcriptional events. Two unrelated MAO‐A inhibitors reduced caspase activation. STS treatment resulted in sustained activation of the mitogen‐activated protein kinase pathway enzymes extracellular regulated kinase, c‐jun terminal kinase and p38, and depletion of the anti‐apoptotic protein Bcl‐2. These changes were significantly reversed by MAO inhibition. Production of reactive oxygen species was increased following STS exposure, which was blocked by both MAO inhibition and the antioxidant N‐acetylcysteine. Therefore our data provide evidence that MAO‐A, through its production of reactive oxygen species as a by‐product of its catalytic activity on the mitochondrial surface, is recruited by the cell to enhance apoptotic signalling.

Monoamine oxidases in development.

Monoamine oxidases (MAOs) are flavoproteins of the outer mitochondrial membrane that catalyze the oxidative deamination of biogenic and xenobiotic amines. In mammals there are two isoforms (MAO-A and MAO-B) that can be distinguished on the basis of their substrate specificity and their sensitivity towards specific inhibitors. Both isoforms are expressed in most tissues, but their expression in the central nervous system and their ability to metabolize monoaminergic neurotransmitters have focused MAO research on the functionality of the mature brain. MAO activities have been related to neurodegenerative diseases as well as to neurological and psychiatric disorders. More recently evidence has been accumulating indicating that MAO isoforms are expressed not only in adult mammals, but also before birth, and that defective MAO expression induces developmental abnormalities in particular of the brain. This review is aimed at summarizing and critically evaluating the new findings on the developmental functions of MAO isoforms during embryogenesis.

Monoamine oxidase-A knockdown in human neuroblastoma cells reveals protection against mitochondrial toxins

The study examined how the mitochondrial enzyme monoamine oxidase‐A (MAO‐A), which produces hydrogen peroxide as a catalytic by‐product, influences death and survival mechanisms. Targeted microRNA (miRNA) was used to stably knock down MAO‐A mRNA, protein, and catalytic activity by 60–70% in SH‐SY5Y human neuroblastoma cells. The effects of MAO‐A knockdown (KD) on ATP, oxidative stress, electron transport chain, and survival following exposure to mitochondrial toxins were assessed. In control cells, complex I inhibition resulted in caspasemediated cell death linked with ROS production and reduced ATP, followed by up‐regulation of MAO‐A mRNA, protein, and enzyme activity levels. Inhibition of complex III and IV resulted in a similar increase in MAO‐A expression, while up‐regulation of MAO‐A was lower following complex II inhibition. MAO‐A KD decreased basal reactive oxygen species levels by 50% and increased levels of ATP and reduced glutathione and Bcl‐2. MAO‐A KD specifically increased the activity of complex I but had no effect on complex II‐IV activities. Furthermore, MAO‐A KD protected against inhibitors of complex I, III, and IV. In summary, endogenous MAO‐A levels influence mitochondrial function, notably complex I activity, and MAO‐A may be a target for protection against neurodegenerative conditions that involve oxidative stress and mitochondrial dysfunction as underlying pathogenic factors.—Fitzgerald, J. C., Ugun‐Klusek, A., Allen, G., De Girolamo, L. A., Hargreaves, I., Ufer, C., Abramov, A. Y., Billett, E. E. Monoamine oxidase‐A knockdown in human neuroblastoma cells reveals protection against mitochondrial toxins. FASEB J. 28, 218–229 (2014). www.fasebj.org

Monoamine Oxidase A Expression Is Vital for Embryonic Brain Development by Modulating Developmental Apoptosis

Monoamine oxidases (MAO-A, MAO-B) metabolize biogenic amines and have been implicated in neuronal apoptosis. Although apoptosis is an important process in embryo development, the role of MAO isoenzymes has not been investigated in detail. We found that expression of MAO-A and MAO-B can be detected early on during embryo development. Expression levels remained constant until around midgestation but then dropped to almost undetectable levels toward birth. Similar expression kinetics were observed in the brain. Isoform-specific expression silencing of MAO-A mediated by siRNA during in vitro embryogenesis induced developmental defects, as indicated by a reduction of the crown rump length and impaired cerebral development. These alterations were paralleled by elevated serotonin levels. Similar abnormalities were observed when embryos were cultured in the presence of the MAO-A inhibitor clorgyline or when the transcriptional inhibitor of MAO-A expression R1 was overexpressed. In contrast, no such alterations were detected when expression of MAO-B was knocked down. To explore the underlying mechanisms for the developmental abnormalities in MAO-A knockdown embryos, we quantified the degree of developmental apoptosis in the developing brain. MAO-A knockdown reduced the number of apoptotic cells in the neuroepithelium, which coincided with impaired activation of caspases 3 and 9. Moreover, we observed reduced cyclin D1 levels as an indicator of impaired cell proliferation in MAO-A knockdown embryos. This data highlights MAO-A as a vital regulator of embryonic brain development.

A link between monoamine oxidase-A and apoptosis in serum deprived human SH-SY5Y neuroblastoma cells

SummaryIncreased monoamine oxidase (MAO) activity was recently shown to accompany apoptotic cell death of various neuronal cells following growth factor deprivation. Here we show that in serum deprived SH-SY5Y cells, MAO-A mRNA levels and catalytic activities are increased, linked with activation of the apoptotic executioner caspase-3. Importantly, specific inhibition of MAO-A activity resulted in loss of apoptotic cell morphology. Our study indicates that MAO catalytic activity is involved in apoptotic signalling in response to serum withdrawal in neuronal cells.

Cross-regulation between β1- and β3-adrenoceptors following chronic β-adrenergic stimulation in neonatal rat cardiomyocytes

Background and purpose:  We have previously shown that β‐adrenoceptors continuously stimulated with noradrenaline induces an increase in β3‐adrenoceptors (GαiPCRs) and a decrease in β1‐adrenoceptors (GαsPCRs) at functional, genomic and protein levels. This compensatory modification induced by noradrenaline is probably one of the consequences of cardiac depression observed in heart disease. Therefore, we investigated further the interaction between β1‐ and β3‐adrenoceptors in neonatal rat cardiomyocytes.