Elsevier

Neuroscience

Volume 425, 15 January 2020, Pages 235-250
Neuroscience

Research Article
Thioredoxin 1 Plays a Protective Role in Retinas Exposed to Perinatal Hypoxia–Ischemia

https://doi.org/10.1016/j.neuroscience.2019.11.011Get rights and content

Highlights

  • Hypoxia-ischemia produces changes in Trx1 protein levels in rat retinas.

  • Hypoxia-reoxygenation produces changes in Trx1 protein levels in ARPE-19 cells.

  • Trx1 affects ARPE-19 cell morphology and differentiation.

  • Changes in Trx1 levels affect ARPE-19 cell differentiation.

Abstract

Thioredoxin family proteins are key modulators of cellular redox regulation and have been linked to several physiological functions, including the cellular response to hypoxia–ischemia. During perinatal hypoxia–ischemia (PHI), the central nervous system is subjected to a fast decrease in O2 and nutrients with a subsequent reoxygenation that ultimately leads to the production of reactive species impairing physiological redox signaling. Particularly, the retina is one of the most affected tissues, due to its high oxygen consumption and exposure to light. One of the main consequences of PHI is retinopathy of prematurity, comprising changes in retinal neural and vascular development, with further compensatory mechanisms that can ultimately lead to retinal detachment and blindness. In this study, we have analyzed long-term changes that occur in the retina using two well established in vivo rat PHI models (perinatal asphyxia and carotid ligation model), as well as the ARPE-19 cell line that was exposed to hypoxia and reoxygenation. We observed significant changes in the protein levels of the cytosolic oxidoreductase thioredoxin 1 (Trx1) in both animal models and a cell model. Knock-down of Trx1 in ARPE-19 cells affected cell morphology, proliferation and the levels of specific differentiation markers. Administration of recombinant Trx1 decreased astrogliosis and improved delayed neurodevelopment in animals exposed to PHI. Taken together, our results suggest therapeutical implications for Trx1 in retinal damage induced by hypoxia–ischemia during birth.

Introduction

Thioredoxin (Trx) family proteins are key modulators of cellular redox regulation by controlling the activity of different target proteins through thiol/disulfide exchange reactions (Lee et al., 2013, Meyer et al., 2009) and other oxidative modifications at Cys residues. They depend on specific active site motifs containing one or two cysteine residues; in the case of Trx the conserved Cys-Gly-Pro-Cys motif. Trx was first described by Peter Reichard in Escherichia coli (Laurent et al., 1964). The Trx system includes Trx1 and Trx2 that are localized in the cytosol and in mitochondria, respectively (Meyer et al., 2009, Lee et al., 2013, Hanschmann et al., 2013, Lu and Holmgren, 2014). Trx1 and Trx2 are reduced by Thioredoxin reductase (TrxR) 1 and 2, respectively, with electrons donated from NADPH (Lu and Holmgren, 2014). Trx proteins have been linked to several physiological functions such as regulation of gene expression, proliferation, metabolism and cell death. Via the regulation of specific substrates within signaling circuits, Trx proteins are key regulators of redox signaling. They have also been implicated in various pathological conditions and diseases, including disorders related to inflammation and hypoxia–ischemia (Powis et al., 2000, Iwata et al., 2010, Hanschmann et al., 2013, Zhang et al., 2017). During perinatal hypoxia–ischemia the central nervous system (CNS) is subjected to changes in oxygen levels that lead to the production of reactive species (de Groot and Rauen, 2007, Johnston et al., 2000). In brief, the decrease in the levels of O2 in astrocytes produces a reduction in ATP production that alters membrane polarization due to a reduced activity of the Na+/K+ ATPase pump. Meanwhile, a massive production of glutamate (Glu) in neurons causes the activation of Glu-dependent Ca2+ channels, generating postsynaptic membrane polarization changes. Due to hypoxia, the system consumes part of its antioxidant capacity, and therefore cannot cope with the new influx of O2 during reoxygenation and the generation of different reactive species in the CNS, such as NO*, O2* and H2O2 (de Groot and Rauen, 2007, Ferreiro et al., 2001, Johnston et al., 2000, Johnston et al., 2001, Kalogeris et al., 2012, Uria-Avellanal and Robertson, 2014). We have previously shown that PHI is accompanied with an increase in the protein levels of Trx1 and glutaredoxin (Grx2) in the hippocampus (Romero et al., 2015, Romero et al., 2017). The neural retina is exposed to different levels of reactive species under physiological conditions (Tanito et al., 2002, Lillig and Holmgren, 2007). However, the increase in reactive species induced by a PHI event followed by reperfusion and reoxygenation can lead to neuronal loss and ultimately blindness (Wang et al., 2015). One of the main consequences of PHI in the retina is retinopathy of prematurity (ROP). In places where the neonatal unit has advanced techniques, regarding intensive care, most cases of ROP occur in gestational ages of less than 28 weeks (Holmstrom et al., 2014). ROP comprises changes in retinal neural and vascular development with further compensatory mechanisms (such as an increase in VEGF and vessel growth) that induce aberrant vascularization and can later lead to retinal detachment and blindness (Tsui and Chu, 2017, Zhang et al., 2018). In the present study, we investigated long-term damage of the retina induced by the exposure of new born rats to perinatal hypoxia–ischemia and analyzed the underlying impact of Trx1 in the ARPE-19 cell model. We were able to evidence changes in Trx1 protein levels both in the animal models i.e. perinatal asphyxia model and carotid ligation model, and in ARPE-19 cells due to hypoxia-reoxygenation. Administration of recombinant Trx1 to animals exposed to perinatal hypoxia–ischemia, decreased astrogliosis and improved the delayed neurodevelopment.

Section snippets

Chemicals and reagents

Chemicals and enzymes used in the present work were of analytical grade or better. Unless otherwise noted, reagents were acquired from Sigma-Aldrich (Munich, Germany). SDS-PAGE were run using precasted stain-free gradient (4–20%) gels (Mini PROTEAN TGX, Biorad, Munich, Germany), and the Turbo RTA Transfer Kit with PVDF membranes (Biorad, Munich, Germany) was used for protein transfer. Recombinant Trx1 was expressed and purified via the IMAC principle, as described before (Godoy et al. 2011).

Ethical statement

Perinatal hypoxia–ischemia induces changes in retinal layer thickness, reactive gliosis, and neuronal death and changes in retinal layer thickness

To study perinatal hypoxia–ischemia in vivo, we chose two in vivo rat models, the PA and the carotid ligation (CAR), that constitute two well established and studied models. PHI affects retinal morphology (Rey-Funes et al., 2013) particularly producing changes in layer thickness. We analysed these changes in order to determine the damage caused in our two models. Retinal tissue was subjected to hematoxilin-eosin staining in order to properly preserve and visualize each layer and thus, compare

Perinatal hypoxia–ischemia induced long-term damage to the retina

The CNS is particularly susceptible to hypoxia–ischemia; among the changes caused by the disruption in oxygen and blood flow are neuronal loss and astrogliosis (Bernert et al., 2003, Saraceno et al., 2010). The impact of perinatal hypoxia–ischemia over the CNS is not homogeneous, different areas and cell populations are particularly affected (Laptook et al., 1994, Thompson, 1995). When compared to other tissues in the CNS, it is clear that the retina represents the highest rates in metabolism

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (SFB593-N01, LI984/3-1, and GRK1947-A1) to CHL, the Federal Ministry for Science and Education (BMBF: 01DN13023-PAREDOX) and MINCYT to CHL and FC, the German Academic Exchange Service DAAD and MINCYT (PROALAR program) to CHL and FC, the (PIP1142010010019, CONICET, Argentina) to FC, and the University of Buenos Aires (UBACYT 20020090100118) to FC. MIH is a fellowship holder from the National Scientific and Technical ResearchCouncil

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