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Jiménez-Hidalgo, María et al. ; Thioredoxins are a class of evolutionarily conserved proteins that have been demonstrated to play a key role in many cellular processes involving redox reactions. We report here the genetic and biochemical characterization of Caenorhabditis elegans TRX-3, the first metazoan thioredoxin with an intestine-specific expression pattern. By using green fluorescent protein reporters we have found that TRX-3 is expressed in both the cytoplasm and the nucleus of intestinal cells, with a prominent localization at the apical membrane. Although intestinal function, reproductive capacity, longevity, and resistance of trx-3 loss-of-function mutants to many stresses are indistinguishable from those of wild-type animals, we have observed a slight reduction in size and a minor reduction in the defecation cycle timing of trx-3 mutants. Interestingly, trx-3 is induced upon infection by Photorhabdus luminescens and Candida albicans, and TRX-3 overexpression provides a modest protection against these pathogens. Together, our data indicate that TRX-3 function in the intestine is dispensable for C. elegans development but may be important to fight specific bacterial and fungal infections. © 2013 Elsevier Inc. ; Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440) and the Japanese National Bioresource Project of the MEXT, Japan. We thank Howard Baylis, Keith Nehrke, Bart Braeckman, and Jim McGhee for sharing worm strains and plasmids. We are grateful to María Jesús Rodriguez-Palero and Fernando Calahorro for excellent technical assistance, to Cristina Méndez-Vidal for help with qPCR, to Helen M. Crook-McMahon for help with PRDX-2 blots, and to Britta Spanier for critical reading of the manuscript. A.M.-V. was supported by the Instituto de Salud Carlos III (Projects PI050065 and PI080557, cofinanced by the Fondo Social Europeo) and Junta de Andalucía (Projects P07-CVI-02697 and P08-CVI-03629), Spain. The work in the laboratory of P.S., a member of the NordForsk Nordic C. elegans Network, was supported by a grant from the Swedish Research Council. C.L.K. was supported by the INSERM, the CNRS, and the French Ministry of Research. E.L.B. and E.A.V. were supported by the MRC. J.R.P. was supported by the Plan de Apoyo a la Investigación, Desarrollo Tecnológico e Investigación de la Universidad de Jaén (Project UJA2011/12/55). J.C. was funded by the Spanish Ministry of Science and Innovation (Grant BFU2010-21794) and the Rioja Salud Foundation. E.L. and A.G.S. were supported by grants from the Fondo de Investigaciones Sanitarias (PI080642 and PI110120) and Ramón y Cajal Program to E.L. (Spanish government). ; Peer Reviewed

[Aim]: Functional in vivo studies on the mitochondrial thioredoxin system are hampered by the embryonic or larval lethal phenotypes displayed by murine or Drosophila knock-out models. Thus, the access to alternative metazoan knock-out models for the mitochondrial thioredoxin system is of critical importance. [Results]: We report here the characterization of the mitochondrial thioredoxin system of Caenorhabditis elegans that is composed of the genes trx-2 and trxr-2. We demonstrate that the proteins thioredoxin 2 (TRX-2) and thioredoxin reductase 2 (TRXR-2) localize to the mitochondria of several cells and tissues of the nematode and that trx-2 and trxr-2 are upregulated upon induction of the mitochondrial unfolded protein response. Surprisingly, C. elegans trx-2 (lof ) and trxr-2 (null) single and double mutants are viable and display similar growth rates as wild-type controls. Moreover, the lack of the mitochondrial thioredoxin system does not affect longevity, reactive oxygen species production or the apoptotic program. Interestingly, we found a protective role of TRXR-2 in a transgenic nematode model of Alzheimer's disease (AD) that expresses human β-amyloid peptide and causes an age-dependent progressive paralysis. Hence, trxr-2 downregulation enhanced the paralysis phenotype, while a strong decrease of β-amyloid peptide and amyloid deposits occurred when TRXR-2 was overexpressed. [Innovation]: C. elegans provides the first viable metazoan knock-out model for the mitochondrial thioredoxin system and identifies a novel role of this system in β-amyloid peptide toxicity and AD. [Conclusion]: The nematode strains characterized in this work make C. elegans an ideal model organism to study the pathophysiology of the mitochondrial thioredoxin system at the level of a complete organism. ; A.M.-V. was supported by the Instituto de Salud Carlos III [Projects PI050065 and PI080557, co-financed by the Fondo Social Europeo, FEDER] and Junta de Andalucía [Projects P07-CVI-02697 and P08-CVI-03629], Spain. B.C.-V. was supported by a fellowship from the Consejo Nacional de Ciencia y Tecnología (CONACYT) from the Government of Mexico. P.N. was supported by the Instituto de Salud Carlos III [project PI080500, co-financed by the Fondo Social Europeo, FEDER], and Junta de Andalucía [project P08-CTS-03988]. Work in the laboratory of P.S., a member of the NordForsk Nordic C. elegans network, was supported by a grant from the Swedish Research Council. J.C. was supported by the Spanish Ministry of Education and Science Grant BFU2010-21794 and the RiojaSalud Foundation. ; Peer reviewed

Cisplatin and derivatives are commonly used as chemotherapeutic agents. Although the cytotoxic action of cisplatin on cancer cells is very efficient, clinical oncologists need to deal with two major difficulties, namely the onset of resistance to the drug and the cytotoxic effect in patients. Here, we used Caenorhabditis elegans to investigate factors influencing the response to cisplatin in multicellular organisms. In this hermaphroditic model organism, we observed that sperm failure is a major cause of cisplatin-induced infertility. RNA sequencing data indicate that cisplatin triggers a systemic stress response, in which DAF-16/FOXO and SKN-1/NRF2, two conserved transcription factors, are key regulators. We determined that inhibition of the DNA damage-induced apoptotic pathway does not confer cisplatin protection to the animal. However, mutants for the pro-apoptotic BH3-only gene ced-13 are sensitive to cisplatin, suggesting a protective role of the intrinsic apoptotic pathway. Finally, we demonstrated that our system can also be used to identify mutations providing resistance to cisplatin and therefore potential biomarkers of innate cisplatin-refractory patients. We show that mutants for the redox regulator trxr-1, ortholog of the mammalian thioredoxin reductase 1 TRXR1, display cisplatin resistance. By CRISPR/Cas9, we determined that such resistance relies on the presence of the single selenocysteine residue in TRXR-1. ; This study was supported by grants from the Instituto de Salud Carlos III (ISCIII; PI15/00895 to J.C., PI16/01898 to A.V.), which is co-funded by the European Regional Development Fund/FEDER. The A.V. laboratory was also supported by funding from Ministerio de Economía y Competitividad (BFU2007-67123). M.B. was supported by a Netherlands Organization for Scientific Research (NWO) chemical sciences ECHO grant (711.014.005). E.N. was supported a Sociedad Española de Oncología Médica (SEOM) grant for emerging research groups. F.J.G.-R. was the recipient of a PFIS predoctoral fellowship from ISCIII. A.M.-V. was supported by a grant from Ministerio de Economía y Competitividad (BFU2015-64408-P, co-financed by the European Social Fund) and is a member of the EU-ROS Cost Action of the European Union. J.C., M.B., S.H. and A.M.-V are members of the GENIE EU Cost action. D.B. was recipient of a Consejo Nacional de Ciencia y Tecnología 'al extranjero' fellowship (523048). ; Peer reviewed

© 2015 by the Genetics Society of America. The identity of a given cell type is determined by the expression of a set of genes sharing common cis-regulatory motifs and being regulated by shared transcription factors. Here, we identify cis and trans regulatory elements that drive gene expression in the bilateral sensory neuron ASJ, located in the head of the nematode Caenorhabditis elegans. For this purpose, we have dissected the promoters of the only two genes so far reported to be exclusively expressed in ASJ, trx-1 and ssu-1. We hereby identify the ASJ motif, a functional cis-regulatory bipartite promoter region composed of two individual 6 bp elements separated by a 3 bp linker. The first element is a 6 bp CG-rich sequence that presumably binds the Sp family member zinc-finger transcription factor SPTF-1. Interestingly, within the C. elegans nervous system SPTF-1 is also found to be expressed only in ASJ neurons where it regulates expression of other genes in these neurons and ASJ cell fate. The second element of the bipartite motif is a 6 bp AT-rich sequence that is predicted to potentially bind a transcription factor of the homeobox family. Together, our findings identify a specific promoter signature and SPTF-1 as a transcription factor that functions as a terminal selector gene to regulate gene expression in C. elegans ASJ sensory neurons. ; Some C. elegans strains were provided by the CGC, which is funded by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440), and by the Japanese National Bioresource Project, which is funded by the Japanese Ministry of Education, Culture, Sport, Science and Technology. We thank Nuria Flames for advice and support and María Jesús Rodríguez-Palero and Francisco José Naranjo-Galindo for excellent technical assistance. This work was financed by grants to A.M.-V. from the Junta de Andalucía (Projects P07-CVI-02697 and P08-CVI-03629). Work in the laboratory of P.S. was supported by grants from the Swedish Research Council and the Torsten Söderberg Foundation. E.K. was supported by a grant from the European Union FP6 Marie Curie Research Training Network "EUrythron" MRTN-CT-2004-005499. ; Peer Reviewed

The identity of a given cell type is determined by the expression of a set of genes sharing common cis-regulatory motifs and being regulated by shared transcription factors. Here, we identify cis and trans regulatory elements that drive gene expression in the bilateral sensory neuron ASJ, located in the head of the nematode Caenorhabditis elegans. For this purpose, we have dissected the promoters of the only two genes so far reported to be exclusively expressed in ASJ, trx-1 and ssu-1. We hereby identify the ASJ motif, a functional cis-regulatory bipartite promoter region composed of two individual 6 bp elements separated by a 3 bp linker. The first element is a 6 bp CG-rich sequence that presumably binds the Sp family member zinc-finger transcription factor SPTF-1. Interestingly, within the C. elegans nervous system SPTF-1 is also found to be expressed only in ASJ neurons where it regulates expression of other genes in these neurons and ASJ cell fate. The second element of the bipartite motif is a 6 bp AT-rich sequence that is predicted to potentially bind a transcription factor of the homeobox family. Together, our findings identify a specific promoter signature and SPTF-1 as a transcription factor that functions as a terminal selector gene to regulate gene expression in C. elegans ASJ sensory neurons. ; Some C. elegans strains were provided by the CGC, which is funded by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440), and by the Japanese National Bioresource Project, which is funded by the Japanese Ministry of Education, Culture, Sport, Science and Technology. We thank Nuria Flames for advice and support and María Jesús Rodríguez-Palero and Francisco José Naranjo-Galindo for excellent technical assistance. This work was financed by grants to A.M.-V. from the Junta de Andalucía (Projects P07-CVI-02697 and P08-CVI-03629). Work in the laboratory of P.S. was supported by grants from the Swedish Research Council and the Torsten Söderberg Foundation. E.K. was supported by a grant from the European Union FP6 Marie Curie Research Training Network "EUrythron" MRTN-CT-2004-005499. ; Peer reviewed

Glutathione is the most abundant thiol in the vast majority of organisms and is maintained in its reduced form by the flavoenzyme glutathione reductase. In this work, we describe the genetic and functional analysis of the Caenorhabditis elegans gsr-1 gene that encodes the only glutathione reductase protein in this model organism. By using green fluorescent protein reporters we demonstrate that gsr-1 produces two GSR-1 isoforms, one located in the cytoplasm and one in the mitochondria. gsr-1 loss of function mutants display a fully penetrant embryonic lethal phenotype characterized by a progressive and robust cell division delay accompanied by an aberrant distribution of interphasic chromatin in the periphery of the cell nucleus. Maternally expressed GSR-1 is sufficient to support embryonic development but these animals are short-lived, sensitized to chemical stress and have increased mitochondrial fragmentation and lower mitochondrial DNA content. Furthermore, the embryonic lethality of gsr-1 worms is prevented by restoring GSR-1 activity in the cytoplasm but not in mitochondria. Given the fact that the thioredoxin redox systems are dispensable in C. elegans, our data support a prominent role of the glutathione reductase/glutathione pathway in maintaining redox homeostasis in the nematode. ; Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440) and by the Japanese National Bioresource Project. We thank Cristina Cecchi, Amir Shapir, Paul Sternberg, Bart Braeckman, Chris Link, Simon Tuck, Keith Blackwell, José López-Barneo and LivOn Labs for strains and chemicals, Katie McCallum and Danielle Garsin for their help with skn-1 experiments and Elizabeth Veal and Michel Toledano for critical reading of the manuscript. Prof. Rafael Fernández-Chacón is deeply acknowledged for his continuous support. AMV was supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2015-64408-P) and the Instituto de Salud Carlos III (PI11/00072, cofinanced by the Fondo Social Europeo) and is a member of the GENIE and EU-ROS Cost Action of the European Union. NJS was supported by a grant from the US National Institutes of Health National Institute for Arthritis and Musculoskeletal and Skin Diseases (AR-054342). CJG was funded by a Doctoral Training Studentship provided by the University of Nottingham. PA was supported by the Spanish Ministry of Economy and Competitiveness (BFU2013-42709P). JC is a member of the GENIE Cost action and was funded by Rioja Salud Foundation (Onco-2-2015). ; Peer Reviewed

In the presence of aggregation-prone proteins, the cytosol and endoplasmic reticulum (ER) undergo a dramatic shift in their respective redox status, with the cytosol becoming more oxidized and the ER more reducing. However, whether and how changes in the cellular redox status may affect protein aggregation is unknown. Here, we show that C. elegans loss-of-function mutants for the glutathione reductase gsr-1 gene enhance the deleterious phenotypes of heterologous human, as well as endogenous worm aggregation-prone proteins. These effects are phenocopied by the GSH-depleting agent diethyl maleate. Additionally, gsr-1 mutants abolish the nuclear translocation of HLH-30/TFEB transcription factor, a key inducer of autophagy, and strongly impair the degradation of the autophagy substrate p62/SQST-1::GFP, revealing glutathione reductase may have a role in the clearance of protein aggregates by autophagy. Blocking autophagy in gsr-1 worms expressing aggregation-prone proteins results in strong synthetic developmental phenotypes and lethality, supporting the physiological importance of glutathione reductase in the regulation of misfolded protein clearance. Furthermore, impairing redox homeostasis in both yeast and mammalian cells induces toxicity phenotypes associated with protein aggregation. Together, our data reveal that glutathione redox homeostasis may be central to proteostasis maintenance through autophagy regulation. ; . The Spanish Ministry of Economy and Competitiveness supported EF-S and VG (BFU2016–78265-P), PA (BFU2016– 79313-P and MDM-2016–0687), and AM-V (BFU2015–64408-P). AM-V was also supported by the Instituto de Salud Carlos III (PI11/ 00072) and RPV-M (CPII16/00004, PI14/00949 and PI17/00011). All projects were cofinanced by the Fondo Social Europeo (FEDER). AM-V is a member of the GENIE and EU-ROS Cost Actions of the European Union and RPV-M is a Marie Curie Fellow (CIG322034, EU).