Thesis
Reference
Study of the response to anoxia in Caenorhabditis elegans
GENTINA, Sébastien
Abstract
Oxygen is mandatory for most organisms to survive. It is used to produce energy in the form of adenosine triphosphate (ATP) during oxidative phosphorylation. While most organisms fail upon oxygen deprivation (anoxia), the Nematode Caenorhabditis elegans is able to face this stress for 48 hours. This work describes the involvement of the sphingolipid metabolism in the resistance to anoxia in this Nematode. It suggests that the accumulation of some species of sphingolipid (glucosylceramides and sphingomyelins) may have a toxic effect that could weaken the nematode in anoxic condition. It reveals that the tetratricopeptide protein (TTC-1) has an important role in maintaining the sphingolipid homeostasis and is essential to counteract the anoxia hypersensitivity found in a mutant that is depleted for its ceramide synthase (HYL-2). The depletion of TTC-1 restores a normal resistance to 48 hours of anoxia in HYL-2 mutants by avoiding the accumulation of C24/C25 glucosylceramides and C24/C25/C26 sphingomyelins.
GENTINA, Sébastien. Study of the response to anoxia in Caenorhabditis elegans. Thèse de doctorat : Univ. Genève, 2015, no. Sc. 4755
URN : urn:nbn:ch:unige-465712
DOI : 10.13097/archive-ouverte/unige:46571
Available at:
UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Département de biologie cellulaire Professeur Jean-Claude Martinou
Study of the Response to Anoxia in Caenorhabditis elegans
THÈSE
Présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie
par
Sébastien Gentina
de
Lancy (GE)
Thèse N° 4755
GENÈVE
Atelier de reproduction de l’Université de Genève 2015
I. Remerciements
J’adresse en premier lieu mes remerciements à mon superviseur de thèse, le Professeur Jean-Claude Martinou qui m’a donné l’opportunité d’accomplir ce travail de recherche dans son laboratoire, m’a fait confiance et m’a laissé un maximum de liberté dans l’accomplissement de mon doctorat. Sa curiosité scientifique ainsi que ses conseils avisés furent pour moi source d’inspiration. Je lui en suis reconnaissant.
Un grand merci aux Professeurs Barbara Conradt et Howard Riezman d’avoir accepté de faire partie de mon jury de thèse et d’avoir pris le temps de lire ce travail.
Je voudrais également remercier les Professeurs Monica Gotta et Howard Riezman également pour m’avoir suivi tout au long de mon doctorat en participant à mon comité de thèse. Les meetings que nous avons eus ensemble m’ont toujours été très utiles et m’ont permis de progresser.
Merci Audrey pour tout ce que tu m’as apporté tout au long de mon doctorat. Ton soutien, ton aide ainsi que les nombreuses discussions que nous avons eues au sujet du ver m’ont été très précieux. Merci beaucoup pour tout le temps que tu as pris pour corriger ce mémoire ainsi que pour tous les moments que nous avons partagés au quotidien au labo aussi bien qu’en dehors de celui-ci.
Merci Monique pour ton aide technique, tes conseils, les services rendus ainsi que pour tous les agréables moments passés ensemble durant toutes ces années.
Merci Minkyoung, pour tout le travail que tu as fourni et ton aptitude à changer de sujet en poursuivant le projet sur la mouche Drosophila melanogaster. Merci pour ton sérieux et tous les services rendus au quotidien.
Merci Thomas, pour ta collaboration ainsi que pour les discussions constructives que nous avons eues sur le sujet des sphingolipides de même que pour tes corrections.
Merci au Professeur François Karch pour son expertise et pour avoir rendu réalisables les expériences sur la mouche Drosophile. Merci à Dragan pour son aide et ses conseils sur le sujet ainsi qu’aux autres membres du laboratoire pour leurs services et leur accueil chaleureux.
Merci également au Professeur David Morton pour sa collaboration sur ce même sujet.
Mes remerciements s’adressent à tous les membres du laboratoire que j’ai eu la chance de côtoyer durant cette période. Andrés, Alexis, Audrey, Benoit, Chris, Erik, Etienne, Gabrielle, Kinsey, Kristina, Minkyoung, Mirko, Monique, Oihana, Sandra, Sébastien, Sylvie, Thomas, Tom, Vincent, Yann et Zeinab, nous avons énormément partagé et vous côtoyer au quotidien m’a beaucoup apporté. J’ai eu bien du plaisir à travailler à vos côtés.
Mes remerciements tous particuliers à Alexis Jourdain et Erik Boehm pour leur précieuse et efficace aide dans la correction de ce travail.
Merci à tous les membres du département de biologie cellulaire pour tous les échanges scientifiques et les moments d’échanges.
Je voudrais remercier tous mes amis et les personnes qui me sont très proches et qui ont toujours été à mes côtés durant cette entreprise. Elise, Julien, Ratheesh, Grégoire, Guillaume, Alexis, Lena, Achu, Nicolas, Léo, Raph, Antoine ainsi que mon Grand-Père et tous les membres de ma famille, mille mercis !
Finalement, un immense MERCI à mes parents pour leur précieux et indéfectible soutien tout au long de mon travail.
II. Table of content
I. Remerciements ... 3
II. Table of content ... 9
III. Abstract ... 11
IV. Résumé ... 17
V. Introduction ... 23
1. Oxygen and life ... 25
1.1 Aerobic organisms ... 26
1.1.1 Respiration in mammals ... 27
1.1.2 Respiration in other organisms ... 29
1.2 Aerobic respiration and energy production ... 30
1.3 Variation of oxygen supply ... 32
1.3.1 Ischemia and reperfusion-related damage ... 33
1.3.2 Hypoxia and the Hif-1 pathway ... 36
1.3.3 Anoxia ... 38
1.3.3.1 Strategies to survive anoxia ... 39
1.3.4 Examples of oxygenation deficits in human ... 41
1.3.4.1 Cancer cells and hypoxia ... 42
2. Sphingolipids ... 42
2.1 Basic structures of sphingolipids ... 43
2.2 Implication of sphingolipids in cancer, related diseases and inflammation ... 45
2.3 Sphingolipids in C. elegans ... 47
2.4 Implication of sphingolipids in anoxia or in hypoxia ... 48
3. Caenorhabditis elegans: a model to study the response to anoxia ... 49
3.1 How does C. elegans sense oxygen? ... 49
3.1.1 C. elegans and the pathways regulating hypoxia and anoxia responses ... 50
3.1.2 Behaviour of C. elegans in anoxia ... 53
4. Background of the thesis projects ... 55
4.1 Project 1: ... 56
4.1.1 Aim 1: ... 56
4.1.2 Aim 2: ... 56
4.2 Side Projects ... 57
4.2.1 Side project 1: ... 57
4.2.2 Side project 2: ... 57
VI. Results ... 59
Mutations in ttc-1 restore resistance to anoxia in hyl-2 deficient mutant in the nematode Caenorhabditis elegans ... 61
Abstract ... 63
Introduction ... 64
Results ... 67
Isolation of a suppressor that restores normal resistance to anoxia in hyl-2(gnv1) mutant ... 67
ttc-1 is expressed in the pharynx, the tail, the gut and in the developing vulva ... 68
Lipid profile of hyl-2(gnv1);ttc-1(gnv3) mutants ... 69
Discussion ... 71
Materials and Methods ... 74
Figure legends ... 79
Supplemental Figure legends ... 87
VII. Discussion ... 93
1. Response to anoxia in C. elegans ... 95
2. hyl-2(gnv1) and the sensitivity to anoxia ... 95
2.1 Implication of sphingolipid homeostasis in the development of C. elegans and in the protection to anoxia ... 97
2.1.1 Complex sphingolipid accumulation might be the cause of the sensitivity to anoxia ... 99
3. Characterization of ttc-1 ... 100
3.1 Expression of ttc-1 ... 101
3.2 Implication of ttc-1(gnv3) in the sphingolipids homeostasis ... 102
3.2.1 Interaction with SR protein kinase homologue spk-1 ... 103
4. Sphingolipids and their role in triggering protection to anoxia ... 104
4.1 hyl-2 expression and the resistance to anoxia ... 105
4.1.1 Ceramides feeding, a possible rescue for hyl-2(gnv1)? ... 106
5. Characterisation of rta-1 and cng-2 mutants ... 106
6. Final conclusion ... 108
VIII. Appendix 1 ... 109
Protection of C. elegans from Anoxia by HYL-2 Ceramide Synthase ... 113
IX. Appendix 2 ... 149
Study of the lifespan of C. elegans ... 153
rta-1, a mutant with an extended lifespan ... 153
Results ... 154
Conclusion ... 157
Material and Methods of Appendix 2 ... 158
X. Appendix 3 ... 161
Description of cng-2(gnv2); hyl-2(gnv1) ... 165
The Cyclic Nucleotide Gated channel family ... 167
Regulation of CNGs ... 167
Downregulation of CNGs homologs in Drosophila melanogaster allow the fly to recover faster from a period of anoxia ... 169
Conclusion ... 172
Material and Methods of Appendix 3 ... 173
XI. References ... 175
III. Abstract
Oxygen is mandatory for most organisms to survive. It is used to produce energy in the form of adenosine triphosphate (ATP) during oxidative phosphorylation. Thus, a strong decrease of the oxygen in the environment can lead to dramatic consequences. The normal concentration in the atmosphere is about 21%, whereas the concentration that reaches cells in mammals varies between 1.3 and 2.5%.
Under this concentration cells are undergoing hypoxia, and when the oxygen is totally absent we talk about anoxia. The Hif-1 factor pathway has been revealed to be essential for metazoans to manage decreases of oxygen. In fact, once activated, Hif- 1 induces the transcription of target genes that help facing such a stress. However, studies on the nematode Caenorhabditis elegans suggest that the response to anoxia does not involve the Hif-1 factor (Menuz et al., 2009; Padilla et al., 2002). It is interesting to note that several organisms, such as the fresh water turtle, zebra fish embryos, the fly Drosophila melanogaster and also the nematode C. elegans have developed strategies that allow them to survive to periods of anoxia. Thus, their study could allow us better understand the mechanism of resistance to oxygen deprivation.
Since C. elegans can resist at least 48 hours of anoxia, we use it as an animal model in our laboratory to try to better understand the mechanism that underlies the resistance to anoxia. Loss of function mutants in the insulin-like daf-2 pathway in C.
elegans have already been well characterized and are known to display increased resistance to anoxia. In our laboratory, the work of Vincent Menuz, a former PhD student, revealed the importance of ceramides for C. elegans to survive in anoxia. In fact, the loss of function mutation of the ceramide synthase HYL-2 modifies the sphingolipid composition of the nematode, which becomes hypersensitive to anoxia.
In this study, the results showed that hyl-2(gnv1) mutants contained less ceramides
and sphingomyelins with C20 to C22 fatty acyl chains and more with C24 to C26 fatty acyl chains compared to the wild type.
This work pointed out a protective role of ceramides in the resistance to anoxia. In addition, it revealed that this protective effect did not involve daf-2.
My thesis work aimed at better understanding how ceramides could participate in protection of C. elegans against anoxia. To assess this question, we performed a suppressor screen in the hyl-2(gnv1) mutant which is hypersensitive to anoxia, and isolated a suppressor mutant that displayed a normal resistance to anoxia. We mapped this mutation using in parallel two complementary techniques, Rapid Single Nucleotide Mapping and Whole Genome Sequencing. We identified two mutations lying in the gene ttc-1. While one of them generates a premature stop codon in the protein, the other changed the property of an amino acid at the beginning of the protein. To confirm that these mutations in ttc-1 were restoring resistance of hyl- 2(gnv1) to anoxia, we generated transgenic worms by inserting a wild type copy of ttc-1 in the suppressor mutant.
ttc-1 encodes the poorly characterized tetratricopeptid protein TTC-1. It is known that tetratricopeptid domains are involved in scaffolding of protein complexes and protein- protein interactions. Expression of ttc-1 fused to GFP allowed us to observe that ttc-1 is essentially expressed in the pharynx, in the gut, in the tail and in the vulva during development of the nematode. Interestingly, ttc-1 and hyl-2 expression patterns are partially overlapping. Most importantly, lipidomic analysis of hyl-2(gnv1) and of the suppressor mutant allowed us to confirm the previous results of Vincent Menuz, and revealed that certain glucosylceramides and sphingomyelin found to be at elevated
accumulation of these sphingolipids may have a toxic effect that could weaken the nematode in anoxic condition and that TTC-1 has an important role in maintaining the sphingolipid homeostasis, which is essential to counteract the anoxia hypersensitivity of hyl-2(gnv1).
IV. Résumé
L’oxygène est indispensable pour la survie de la plupart des organismes vivants. Il leur est nécessaire pour produire de l’énergie en quantité suffisante sous forme d’adénosine triphosphate (ATP) lors de la phosphorylation oxydative. C’est pourquoi, une forte diminution de l’apport d’oxygène aux cellules peut avoir des conséquences délétères. La concentration en oxygène considérée normale dans l’atmosphère est d’environ 21%. Cette concentration diffère en fonction des tissus et varie entre 1.3 et 2.5% dans les cellules. Lorsqu’elle est inférieure le milieu est dit hypoxique et lorsque qu’elle est égale à 0% on parle d’anoxie. Chez les métazoaires, l’importance du facteur Hif-1 induit par l’hypoxie et de tous les événements cellulaires qui en découlent est désormais bien établie. En effet, une fois activé, Hif-1 induit la transcription de gènes essentiels pour faire face à une diminution d’oxygène. En ce qui concerne l’anoxie, différentes études avec le nématode Caenorhabditis elegans suggèrent que la réponse face à un tel stress est différente de celle en situation hypoxique, n’impliquant pas le facteur Hif-1 (Menuz et al., 2009; Padilla et al., 2002).
Différents organismes vivants comme certaines tortues d’eau douce, les embryons de poisson zébré, la mouche Drosophila melanogaster ainsi que C. elegans ont développé des stratégies leur permettant de survivre à des périodes d’anoxie.
Etudier ces organismes pourrait donc nous permettre de mieux comprendre le fonctionnement de la résistance à un tel stress.
Le nématode C. elegans est capable de bien résister à une période de 48 heures d’anoxie et nous l’utilisons dans notre laboratoire comme animal modèle pour essayer de mieux comprendre quels mécanismes induisent cette résistance. Un mécanisme déjà bien décrit est la perte de fonction de l’homologue du récepteur de l’insuline, daf-2, qui augmente considérablement la résistance de C. elegans à différents stress dont l’anoxie. Dans notre laboratoire, le travail de Vincent Menuz,
ancien étudiant en thèse, a montré l’importance des céramides chez C. elegans pour survivre en anoxie. En effet, la perte de fonction de la céramide synthase HYL-2 a pour conséquence de perturber la composition sphingolipidique du nématode le rendant hypersensible à l’anoxie. Les résultats de l’analyse lipidomique ont montré une diminution de céramides et de sphingomyélines pourvus de chaines d’acides gras de longueur C20 à C22 et une augmentation de céramides et de sphingomyélines pourvus de chaines d’acides gras de longueur C24 à C26 dans le mutant hyl-2(gnv1) en comparaison avec le type sauvage.
Ce travail a mis en exergue l’implication des céramides dans la résistance à l’anoxie.
Il a également permis de démontrer que cette implication des céramides fonctionnait de manière indépendante à daf-2.
Durant mon travail de thèse, dans le but de mieux comprendre l’implication des céramides dans la protection à l’anoxie, nous avons effectué un crible suppresseur du mutant hypersensible à l’anoxie hyl-2(gnv1) et avons isolé un mutant dont la résistance à l’anoxie était complètement rétablie. Nous avons ensuite cartographié cette mutation en utilisant deux techniques en parallèle : la cartographie rapide d’un polymorphisme pour un seul nucléotide et le séquençage complet du génome. La première approche nous a permis de restreindre nos recherches à une région définie sur le chromosome V et la deuxième nous a permis d’identifier deux mutations sur le gène ttc-1. L’une d’entre elle génère un codon stop prématuré à la fin de la protéine et l’autre change la propriété d’un acide aminé au début de celle-ci. Nous avons ensuite généré des animaux transgéniques ré-exprimant le gène sauvage ttc-1 et restauré leur sensibilité à l’anoxie.
ttc-1 code pour une protéine, TTC-1, peu caractérisée. Nous savons qu’elle contient un domaine tetratricopeptidique connu pour favoriser l’interaction entre protéines et la formation de complexes. En générant une protéine de fusion TTC-1-GFP nous avons pu montrer que ttc-1 est exprimé essentiellement au niveau du pharynx, de l’intestin, de la queue ainsi que dans la vulve en formation du nématode. Il est intéressant de noter que son profil d’expression est partiellement similaire à celui de hyl-2. Plus important, l’analyse lipidomique du mutant hyl-2(gnv1) et du suppresseur nous a permis de confirmer les résultats obtenus précédemment par Vincent Menuz et de constater que les niveaux de certains glucosylceramides et de sphingomyelines qui étaient considérablement augmentés dans le mutant hyl-2(gnv1) étaient à nouveau normaux dans le suppresseur. Ceci suggère que l’accumulation de certains sphingolipides pourrait avoir un effet toxique chez le nématode en anoxie. TTC-1 aurait un rôle important dans la maintenance de l’homéostasie sphingolipidique, qui serait donc altérée chez hyl-2(gnv1) en condition anoxique.
V. Introduction
1. Oxygen and life
Oxygen is one of the most abundant elements in the universe with hydrogen and helium (Dole, 1965). We are continuously breathing it in the form of dioxygen (O2) without even noticing this colourless, odourless and tasteless gas. However, O2 was not available on Earth from the beginning. In fact, the atmosphere was first free of O2
and it is estimated to have appeared around 2.3 billion years ago with the Great Oxygenation Event (GOE) (Holland, 2002). Different theories have tried to explain this big change and one of them supports that oxygen appeared as a by-product of proliferating cyanobacteria (Flannery and Walter, 2012), which are believed to be the first prokaryotic microorganisms capable of producing O2 by oxygenic photosynthesis (Sessions et al., 2009; Summons et al., 1999). The atmosphere was hypoxic at first after the GOE event and fluctuated between 10 and 23% from the Jurassic (200 Ma) to present to reach the current concentration of 21% (Falkowski et al., 2005).
Oxygenic photosynthesis by plants and eukaryotic algae allows maintenance of the current 21% concentration of O2 in the atmosphere (Figure 1).
Figure 1: The equation of photosynthesis. Using CO2, H2O and light as a source of energy, plants release oxygen in the atmosphere, which is used by aerobic organisms.
Nevertheless oxygen and all oxidizing events that it generates are potentially poisonous since almost all unicellular obligate anaerobic organisms disappeared after the GOE (Webster, 2007). Other less strict anaerobes, called aerotolerant
microorganisms, could live in the presence of oxygen although they did not require it (Sutart, 2005).
1.1 Aerobic organisms
Aerobic organisms use oxygen molecules from the environment to survive. Basically, mitochondria inside cells use oxygen to produce energy in a bio-available form known as adenosine triphosphate (ATP) through oxidative phosphorylation (Bonora et al., 2012; Habersetzer et al., 2013). Fundamentally, this is the inverse reaction to the photosynthesis (Figure 2).
Figure 2: The equation of the anaerobic respiration. Oxygen is used together with glucose, leading to the production of energy in the form of ATP.
To make this reaction possible, organisms have evolved different circulatory systems that allow them to deliver molecules of oxygen to tissues (Brainerd, 1997). The mechanism of aerobic respiration will be discussed later in the introduction.
1.1.1 Respiration in mammals
In lungs of mammals, oxygen is loaded on the heme group of haemoglobin in erythrocytes and is driven to the organs by the cardiovascular system (Brainerd, 1997), which ensures the proper distribution throughout the body in order to supply all the cells with a required amount of oxygen, used for the production of energy in the form ATP (Hardison, 1998; Mohanty et al., 2014). Oxygen is distributed to tissues by gas exchange (Figure 3). This is made possible thanks to a gradient of partial pressure of oxygen (pO2) throughout the body from 150 mmHg in the upper respiratory tract to pO2 that vary according to tissues. For instance it is around 34 mmHg in the brain and around 72 mmHg in kidneys (Carreau et al., 2011). Finally inside cells, O2 is mainly utilized for oxidative phosphorylation within mitochondria.
Figure 3: Modified from tooloop human anatomy picture
Once blood is enriched with O2, it will distribute it throughout the body. Its consumption results in the release of CO2 that will be driven back to the lungs from where it will be released in the air. The pulmonary circuit ensures the interface between gas and blood. The systemic circuit ensures the distribution of oxygen rich blood throughout the body. The heart, working as a double pump, and movements of the body ensure good blood circulation.
Sensing O2 is a key process of cell metabolism and cell adaptation to environmental conditions. For instance, the pO2 of atmosphere decreases with altitude, mimicking a hypoxia event, which is called hypobaric hypoxia (Li et al., 2013). Consequently, this decrease leads to a reduced pO2 at the alveoli level, which finally leads to a reduced amount of oxygen available for the tissues. The organism reacts by increasing pulmonary ventilation improving the transport of O2 in the blood (Frisancho, 2013) through an increase in haemoglobin concentration. A risk is high altitude polycythemia leading to high blood viscosity, which impairs oxygen delivery to tissues (Lorenzo et al., 2014; Reeves and Leon-Velarde, 2004; Sahota and Panwar, 2013).
living in high altitude that leads to the avoidance of the haemoglobin increase, which prevents them from suffering from polycythemia (Lorenzo et al., 2014). In this study, Lorenzo et al. reported a missense mutation, affecting a prolyl hydroxylase, which leads to abolishment of erythropoiesis mediated by hypoxia.
1.1.2 Respiration in other organisms
Throughout vertebrates, the blood circulates in a closed circuit, despite a few differences in fishes, amphibians, reptiles and birds. For instance, the heart of air- breathing fishes consists in a single pump working in a single circulatory system and although turtles and squamates have arterial and venous blood flows that are separated, they often mix the flows because of a connection between heart ventricles (Brainerd, 1997). Other organisms, such as Drosophila melanogaster, have an open circulatory system in which the fluid that brings oxygen and nutrients to the organs is called hemolymph (Wolf and Rockman, 2011). Nematodes, such as Caenhorabditis elegans, which are even simpler organisms, lack a respiratory system and gas enter into cells by simple diffusion (Van Voorhies and Ward, 2000).
1.2 Aerobic respiration and energy production
For all aerobic organisms, oxygen is required for the synthesis of ATP. In aerobic conditions, more than 90% of consumed oxygen is used within the mitochondria as the last electron acceptor of the oxidative phosphorylation reaction (Goda, 2012;
Goda and Kanai, 2012; Lu et al., 1999; Saraste, 1999). The theory that Rich proposes is that oxidation of one molecule of glucose through oxidative phosphorylation in mitochondria generates more than 30 ATP molecules, which is consequently 15 times more than the amount of ATP produced by glycolysis (i.e.
anaerobic glycolysis produces 2 ATP from one glucose molecule) (Rich, 2003).
Briefly, cells break down food molecules such as glucose. Glycolysis is conserved from bacteria to metazoans and its last step ends with the production of pyruvate, which will be imported into the mitochondria, then converted into acetyl CoA, which enters into the Krebs cycle (citric acid cycle). This cycle will release CO2 and reduced form of nicotinamide adenine dinucleotide (NADH), the main source of electrons for the electron transport chain. O2 molecules participate in the last step of this process by accepting electrons and protons to form H2O molecules (Alberts B., 2008) (Figure 4).
Figure 4: Modified from Openstax CNX:
From complex I to complex IV, the electron transport chain ensures the formation of a gradient of H+ between the intermembrane space and the matrix of the mitochondria. This electro-chemical force is used by the ATP synthase to generate ATP from ADP and Pi. Water molecules are produced at the end of the electron transport chain.
Hence, oxidative respiration is how aerobic cells produce the majority of the energy consumed by the whole organism.
In the absence of oxygen, production of energy is possible via anaerobic glycolysis, which, as mentioned above, produces 2 ATP per molecule of glucose. However, a prolonged period without oxygen in aerobic organisms leads to irreversible damage, impairing the metabolism, ultimately leading to death of the organism.
1.3 Variation of oxygen supply
As mentioned before, under normal conditions the oxygen level in the environment is at 21% with a pressure of 150 mmHg. This condition is called normoxia. Oxygen in the environment can vary from this 21% level. In cells, the normal concentration of oxygen is in between 1.3 and 2.5% and hypoxia is defined when the concentration goes below this level (Carreau et al., 2011). Since cell culture is performed at 21%
oxygen, it means that these cells undergo hyperoxia (when oxygen concentration exceeds the levels considered normal). Finally, anoxia defines certain extreme cases, when oxygen is completely absent (Nystul and Roth, 2004).
The consequence of strong variations of the cellular concentration of oxygen can be the production of oxygen species (ROS), which can lead to cellular dysfunction, diseases and even death (Gore et al., 2010; Guzy and Schumacker, 2006). Thus any oxygen variations will require organisms to adjust and develop specific strategies in order to maintain their metabolism with minimal disruption. It has been shown that most animals are able to sense variations of external and internal oxygen levels and to specifically respond by looking for the best environmental oxygen concentration and/or changing their physiology to maintain the most proper oxygen supply to the tissues (Cheung et al., 2005; Hetz and Bradley, 2005; Sundin et al., 2007;
Vermehren et al., 2006). Sensing oxygen variations is for instance ensured by specialized cells such as the O2-sensitive glomus cells of the carotid body in mammals or specific sensory neurons in C. elegans (Busch et al., 2012; Gray et al., 2004; Prabhakar, 2012; Sundin et al., 2007).
The following sections will only focus on the decrease of the oxygen availability. First, ischemia and ischemia/reperfusion damage will be treated and then hypoxia and anoxia together with the mechanisms of defence that they trigger will be discussed in more details.
1.3.1 Ischemia and reperfusion-related damage
Ischemia occurs when the blood supply to a tissue is reduced. Consequently, the cells that are affected are not provided with enough nutrients including glucose, amino acids, lipids and O2,necessary to maintain a normal metabolism. Thus these cells are not properly oxygenated, leading to hypoxic or anoxic situations (see next sections of the introduction).
Ischemia accompanies myocardial infarction, stroke, or peripheral vascular diseases (Kalogeris et al., 2012). Studies of the myocardium helped to better understand the consequences of an ischemia due to its high-energy requirements. In the case of a prolonged ischemia, because of lactate accumulation due to anaerobic glycolysis, the pH of the cells will strongly decrease and ATP production will drop. As a consequence, ion exchange/transport will be disturbed and will lead to the release of Ca2+ into the cytosol from the endoplasmic reticulum and to an increased entry of Ca2+ into the mitochondria (Sanada et al., 2011).
These changes can lead to the death of the cells and the extent of the damage caused on the tissues will be logically correlated with the duration of the ischemia (Bulkley, 1987).
Contrary to what could be expected, the reperfusion after a period of ischemia is generally not beneficial and can be dangerous for the cell, the tissue and the whole organism. The negative effect, whose cause is reperfusion, is called reperfusion injury. In fact, although oxygen influx and nutrients are again provided to the cells, reperfusion has harmful effects that can lead, as with ischemia, to cell death (Logue et al., 2005; Zhao et al., 2000). The mechanisms occurring upon reperfusion injury are diverse and complex. They include reactive oxygen species (ROS) generation, calcium overload, opening of the mitochondrial permeability transition pore (MPT), high inflammatory response and also endothelial dysfunction and thrombotic events (Kalogeris et al., 2012; Raedschelders et al., 2012; Sanada et al., 2011).
ROS generated by reperfusion can disturb the steady state between cellular level of antioxidant and prooxidant molecules (Betteridge, 2000; Cadenas and Sies, 1985).
Superoxide (O2-), hydrogen peroxide (H2O2), hydroperoxyl (HOO•), hydroxyl (•OH), peroxiynitrous acid (ONOOH), are all extremely reactive and can have negative effects for the cell in damaging lipids, proteins, DNA, in disturbing signalling pathways and in reducing the availability of nitric oxide (NO) (Raedschelders et al., 2012).
Calcium overload is a consequence of the attempt of the cell to re-establish a normal pH, release of Ca2+ from the ER and the mitochondria (following the MPT). This can lead to the activation of Ca2+/calmodulin-dependant protein kinases (CaMKs) and calpains. CaMKs can be implicated in cell death and calpains can degrade many proteins including mitochondrial proteins (Croall and Ersfeld, 2007; Goll et al., 2003;
Vila-Petroff et al., 2007).
A high inflammatory response is a consequence of an increase of the production of cytokines upon ischemia/reperfusion. Since there is no pathogen to fight against, this response, considered as a sterile inflammation, can be highly deleterious because of collateral damage induced by the presence of neutrophils within re-perfused tissues (Garcia et al., 1997; Kvietys and Granger, 2012; van Golen et al., 2013; van Golen et al., 2012).
Mitochondria also suffer from ischemia/reperfusion injury: inhibition of mitochondrial metabolism due to lack of oxygen, production of mitochondrial ROS, opening of the MTP and imbalance of mitochondrial fission/fusion are responsible for mitochondria dysfunction (Di Lisa et al., 2007; Ong and Gustafsson, 2012; Solaini and Harris, 2005; Zorov et al., 2000).
Altogether, these modifications affecting the metabolism of the cells after a reperfusion lead to irreversible states that cells are not always able to face, which causes brain and heart cells, for example, to die via either apoptosis, autophagy, necrosis or necroptosis (Broughton et al., 2009; Gottlieb and Mentzer, 2010; Kroemer et al., 2007; Levine and Kroemer, 2008; Smith and Yellon, 2011; Whelan et al., 2010).
Recent studies performed with rats revealed the protective effect of isoflurane and puerarin from reperfusion injury opening therapy perspectives (Taheri et al., 2014;
Tang et al., 2014).
1.3.2 Hypoxia and the Hif-1 pathway
From C. elegans to mammals, upon hypoxia, the conserved Hypoxia Inducible Factor 1 (HIF-1) pathway is activated, which promotes the transcription of target genes required to respond to the decrease in oxygen (Bruegge et al., 2007). Two subunits interact to trigger the transcription of target genes: HIF-1α (O2 responsive subunit) and HIF-1ß (constitutively expressed subunit) (Semenza, 2003; Wang et al., 1995).
Since the second one does not depend on O2 variations, stabilization of HIF-1α directly depends on the partial pressure of O2 that will influence HIF-1α degradation (Huang et al., 1996; Jiang et al., 1996; Salceda and Caro, 1997). In normoxia, the factor HIF-1α is hydroxylated at P402 and/or P564 by prolyl hydroxylases (PHD1, 2 or 3). This modification mediates its recognition by the tumour suppressor protein pVHL, which leads to ubiquitination of HIF-1α and its degradation by the proteasome.
Upon hypoxia, hydroxylases, which need O2, are inactive and HIF-1α can translocate into the nucleus where it dimerises with HIF-1ß (Berra et al., 2003; Bruick and McKnight, 2001; Ivan et al., 2001; Jiang et al., 1997). This active complex thus binds gene regulatory sequences called hypoxia responsive elements (HRE) together with co-activators, which activates various target genes involved in the response to hypoxia (Arany et al., 1996; Ebert and Bunn, 1998; Maurer et al., 2012; Semenza and Wang, 1992) (Figure 5). This means that the HIF-1 pathway is responsible for triggering the activation of different cascades that cooperate to respond to an oxygen decrease.
Studies of tumours, in which centre cells undergo hypoxia, helped reveal that this mainly leads to the stimulation of genes that have been implicated in erythropoiesis
such as the vascular endothelial growth factor (VEGF) (Forsythe et al., 1996; Hicklin and Ellis, 2005; Lin et al., 2004) were also stimulated, as well as genes involved in glycolysis favouring energy production through the anaerobic pathway (Ziello et al., 2007). In contrast, studies performed with rodent, monkey and human cells showed that HIF-1α was implicated in cell cycle arrest, through the regulation of cyclin kinase inhibitors (CKIs), and in apoptosis via induction of proapoptotic genes (Bruick, 2000;
Goda et al., 2003; Kothari et al., 2003; Sowter et al., 2001).
Figure 5: Modified from (Bruegge et al., 2007):
In blue, the way leading to HIF-1α degradation in normoxia: after being hydroxylated, recognised by VHL and ubiquitinated, HIF-1α is degraded. In red, the translocation of HIF-1α into the nucleus upon hypoxia: the decrease of O2 induces the inactivation of PHD1,2 and 3, this stabilizes HIF-1α, which allows it to dimerise with HIF-1ß in the nucleus leading to the transcription of target genes.
The HIF-1 response pathway, which is well conserved in mammals (Wenger et al., 1996), in Drosophila melanogaster and C. elegans (Cheng et al., 1998; Jiang et al.,
2001), is quickly activated. For instance in normoxia or upon reoxygenation it has been shown in human cells (Hep 3B) that HIF-1α was rapidly degraded, with a half- life of 5-10 minutes (Salceda and Caro, 1997; Wang et al., 1995). This rapidity, which ensures a fast adaptation to an oxygen supply problem, is indicative of a stress response pathway.
Despite the fact that hif-1 is essential for C. elegans embryos to survive periods of hypoxia, it is dispensable in anoxic conditions (Padilla et al., 2002). In addition, Vincent Menuz, a former PhD student in our laboratory, showed that hif-1 deficient young adults still resist 48 hours of anoxia (Menuz et al., 2009). Thus, these observations strongly suggest that hypoxia and anoxia activate distinct response pathways.
1.3.3 Anoxia
Anoxia is defined as a complete lack of oxygen in the environment (O2 pressure <
0.001 kPa) (Nystul and Roth, 2004). The response of organisms to such a stress still remains poorly understood. Since it needs a large amount of oxygen to function, the brain of vertebrates is one of the first organs to fail upon anoxia (Nilsson and Lutz, 2004; P.L. Lutz, 2003). For instance a cerebrovascular accident (CVA) can lead to the formation of a clot, resulting in an oxygen-deprived area of cells (Sims and Muyderman, 2010). As previously discussed, even though reperfusion (i.e. blood supply returning oxygen to a tissue after a period of oxygen deprivation) is needed for the organs to recover after anoxia, it is known that reperfusion can trigger cell
In contrast, several animals such as the fruit fly, zebrafish embryos and the fresh water turtle, have adopted strategies to survive periods of anoxia. For instance, the fresh water turtle is able to survive for months under anoxic conditions, entering a comatose-like state, reducing its ATP consumption and its metabolism (Haddad et al., 1997; Padilla and Roth, 2001; Willmore and Storey, 1997). Even more surprising, is the ability of populations of loriciferans to live within anoxic sediments of the L’Atalante Basin. In fact, these metazoans have developed obligate anaerobic metabolism that remains a mystery (Danovaro et al., 2010).
1.3.3.1 Strategies to survive anoxia
The response to anoxia is still poorly understood. Nevertheless, as previously mentioned, some anoxia tolerant-turtles are able to survive this stress and their study could help us to better understand how a brain could function in the absence of oxygen. Their adaptations could be explained by the fact that turtles are one of the oldest living vertebrates that colonized marine, freshwater and terrestrial environments (Li et al., 2008; Reisz and Head, 2008). The fresh water turtles Trachemys scripta and Chrysemys picta have this ability to endure anoxia (Lutz, 1992; Ultsch, 1985). Basically, researchers propose that what allows these turtles to manage to survive period of anoxia is their ability to decrease their general metabolic rate, to tolerate increased metabolic by-products and to be able to avoid damage after re-oxygenation (Bickler and Buck, 2007). For instance, at low temperatures, the reduction of ATP consumption in Chrysemys picta extends its survival in anoxia (Jackson, 2002) and the downregulation of glutamatergic signalling together with a concommitant upregulation of GABAergic signalling decrease electrical activity,
It has been recently shown that high levels of Bcl-2 and Heat shock protein 72 (Hsp72) in Trachemis scripta led to neuroprotection during anoxia and re- oxygenation, respectively preventing apoptosis pathways and maintaining low level of ROS preventing cell death in re-oxygenation (Kesaraju et al., 2014). This could be an explanation of how the turtle brain is protected. In addition, it was previously reported that overexpression of Hsp72 prevented the production of ROS in mammalian astrocytes and increased superoxide dismutase (SOD) activity, which protects mitochondria from re-oxygenation injury and also tends to reduce mitochondria-related apoptosis (Ouyang et al., 2006; Suzuki et al., 2002; Voloboueva et al., 2008; Xu et al., 2011). It was also shown that this protein could inhibit Schwann cell apoptosis by inhibiting activation of caspase-3 and -9 induced by H2O2, accompanied by an increase of Bcl-2 (Luo et al., 2012). Thus Hsp72 seems to be a key protein involved in the protection against anoxia byproduct-induced death. Going in the same direction, Akt1 (protein kinase B) overexpression in rat cardiomyocytes was shown to have a protective effect against re-oxygenation injury after anoxia, possibly via upregulation of Bcl-2 (Du et al., 2014). In fact, Akt1 was reported to be implicated in different biological responses such as survival, acting in the phosphoinositide 3-kinase pathway (Brazil et al., 2002; Cantley, 2002).
1.3.4 Examples of oxygenation deficits in human
We can find different cases in humans in which oxygen concentration can be strongly decreased, for example in cancer and in ischemia. When a tumour develops, since it is abnormally vascularized, an oxygen gradient appears from the peripheral cells to the central cells of the tumour. Thus, tumour cells will be differently supplied with oxygen: while the peripheral cells of the tumour will be properly oxygenated, the central cells will be poorly oxygenated (Figure 6A). In the brain, ischemia can occur after formation of a blood vessel clot, which accounts for 80% of strokes (Flynn et al., 2008). This occlusion deprives cells of blood supply, and thus of oxygen (Figure 6B).
Improving our understanding of the mechanisms underlying the behaviour of the cells upon decrease of oxygen supply could help us to find solutions in cancer therapy and limit damages after reperfusion of patient that suffered a stroke, and any other events that triggered cells-deprived area.
Figure 6: Modified from (Semenza, 2008) and from http://www.anticoagulant-drugs.com
In A, cells in the centre of a solid tumour are poorly oxygenated in contrast to the external solid tumour cells. This sets up an oxygen gradient throughout the tumor. In B, after a stroke, the formation of a blood clot in vessel is very frequent and leads to area of cells, which will be deprived of blood, and consequently of nutrients and oxygen. This is called an ischemia.
1.3.4.1 Cancer cells and hypoxia
Hypoxia occurs in most solid tumours and leads to the activation of Hif-1 pathway.
Although it helps healthy cells to respond to oxygen supply deficiency, this pathway, highly active in cancer, unfortunately also supports tumour cell growth and proliferation within the organism (Keith et al., 2012; Semenza, 2012). In fact, different studies revealed that HIF-1α is overexpressed in several cancers such as gastric, breast, prostate and colon cancers (Gort et al., 2006; Imamura et al., 2009; Maxwell et al., 2007; Stoeltzing et al., 2004). HIF-1α activity in turn allows the overexpression of genes implicated in tumour cell proliferation and angiogenesis (Chouaib et al., 2012). Although this helps to increase the tumour blood vessel network, the network is often chaotically organised and leaky. Thus it unreliably and inconsistently provides cells with nutrients and oxygen (Brown and Giaccia, 1998; Goel et al., 2011). Consequently, this often maintains high hypoxia in solid tumour microenvironments (Balkwill et al., 2012).
2. Sphingolipids
In 1884, because of their enigmatic function and multifaceted characteristics, Sphingolipids (SLs) were named as a way of alluding to the mythological sphinx by the physician and biochemist J.L.W. Thudichum (Merrill et al., 1997). SLs were found in mammalian cells and are considered to be present mainly in eukaryotes (Breslow, 2013; Hannich et al., 2011; Kovacik et al., 2014). It has been shown that they could be essential in cell signalling, in the regulation of different events such as histone
complexity and diversity they appear to be the central players of many different processes like cell growth, metabolism, aging and programmed cell death (Bartke and Hannun, 2009; Hannun and Obeid, 2008; Pettus et al., 2002; Taha et al., 2006).
In addition to these signalling characteristics, SLs can also have more structural functions related to the fluidity and the shape of membranes (Chrast et al., 2011;
Feingold, 2009) and can also be implicated, together with sterols and proteins, in the formation and organization of membrane microdomains called rafts (Lingwood and Simons, 2010).
2.1 Basic structures of sphingolipids
A ceramide is a basic sphingolipid that consists of a sphingosine whose primary amino group is acylated by a fatty acyl chain that can vary in length from C14 to C26
(Mencarelli and Martinez-Martinez, 2013; Saddoughi and Ogretmen, 2013). Then diverse and more complex SLs can be produced through changes that occur at the primary hydroxyl group of the ceramide. For example, this can lead to the production of sphingomyelin (SM) or glucosylceramide (GlcCer). The phosphorylation of the primary hydroxyl group of Sphingosine and Ceramide leads to Sphingosine-1- phosphate (S1P) and ceramide-1-phosphate respectively. The production of ceramide can also be achieved by the breakdown of the complex sphingolipids. All these reactions are catalysed by number of specific enzymes such as ceramide Synthase (CerS), Cer-1-phosphate phosphatase, sphingomyelinase (SMase) or cerebrosidase (Hannun and Obeid, 2011; Kovacik et al., 2014; Marchesini and Hannun, 2004; Saddoughi and Ogretmen, 2013) (Figure 7). Briefly, in mammals, endogenous ceramides are produced via the de novo pathway that starts at the
endoplasmic reticulum with the condensation of serine with palmitoyl-CoA, which leads to the formation of 3-ketosphinganine and after a reduction step to sphinganine (Gault et al., 2010). Ceramide synthases catalyse the formation of dihydroceramide (DHCers). Then, the desaturation of DHCers produces ceramides (Causeret et al., 2000) that are transported to the Golgi apparatus where headgroup modifications will lead to sphingomyelin and glucosylceramide (and other gylcosphingolipids) (Hannun and Obeid, 2008; Ichikawa et al., 1996; Tettamanti, 2004). Finally sphingolipids are directed to the plasma membrane and subcellular organelles (Kitatani et al., 2008).
Another way to produce endogenous ceramides is via the salvage pathway, in which ceramides are regenerated by CerS that reacylates sphingosine, which is produced by the breakdown of complex sphingolipids mostly in the lysosome (Hannun and Obeid, 2008; Kitatani et al., 2008).
Figure 7: Simplified sphingolipids metabolism, created with Adobe Illustrator CS5
Ceramide (Cer) is a basic sphingolipid that can give rise, through enzymatic reactions, to more complex sphingolipids such as Sphingomyelin (SM), Glucosylceramide (GlcCer) and Ceramide-1- phosphate (Cer-1-phosphate). The de novo pathway starts from serine + palmitoyl-CoA. The salvage pathway involves the reacetylation of Sphingosine by the ceramide synthases.
2.2 Implication of sphingolipids in cancer, related diseases and inflammation
Because ceramide is the centre molecule that gives rise to complex sphingolipids, its stability and metabolism are especially important and implicated in different cellular responses in the organism. It has been shown that ceramides could have diverse implications in cancer and cell death (Karahatay et al., 2007; Mullen et al., 2011).
Studies demonstrated the ambivalence of ceramide variations. In fact, while C18-
and neck cancer (Karahatay et al., 2007; Senkal et al., 2007), experiments with mice reported that C16-ceramides induced tumour proliferation by protecting cells from death induced by the ER-stress (Senkal et al., 2010; Senkal et al., 2011). Thus, these observations demonstrated that, depending on the ceramides species that are expressed or accumulating, the consequences may be completely opposite.
Since the brain synthesizes lipids at high concentrations, abnormal ceramide metabolism is also the cause of many neuronal related disorders like Farber’s disease (Levade et al., 1995; Sugita et al., 1972), Krabbe’s and Gaucher’s diseases (Brady et al., 1966a; Orvisky et al., 2002; Suzuki and Suzuki, 1985) and Niemann Pick’s disease (Brady et al., 1966b), and also the cause of neurodegenerative dementia such as Alzheimer’s, and Parkinson’s diseases (Fabelo et al., 2011; Han et al., 2002; He et al., 2010; Satoi et al., 2005).
Farfel-Becker et al. recently showed that the accumulation of GlcSer reaching a certain threshold could trigger neuroinflammation and neurodegeneration in specific brain areas in a mouse model of neuropathic Gaucher disease (Farfel-Becker et al., 2014). Sphingolipids metabolites have been pointed out to have important roles in inflammation in regulating the function of immune cells and their trafficking (Cyster and Schwab, 2012; Rivera et al., 2008). For instance, sphingosine-1-phosphate can promote the release of lymphocytes into the blood and is also implicated in maintaining the endothelial barrier integrity (Cyster and Schwab, 2012; Garcia et al., 2001; Lee et al., 1999; Natarajan et al., 2013). Consequently, changes in sphingolipid homeostasis have impact on the inflammatory response, and S1P metabolism is a drug target for many inflammatory diseases (Kunkel et al., 2013).
2.3 Sphingolipids in C. elegans
C. elegans contains three genes encoding ceramide synthases, hyl-1 and hyl-2 (homolog of yeast longevity gene 1 and 2) and lagr-1 (LAG1 (yeast longevity- assurance gene) related), which synthetize different ceramide species (Ashrafi et al., 2003; Menuz et al., 2009). They belong to the longevity assurance gene family (Lass genes), first discovered in yeast, and share a similar lag motif needed for enzyme activity (Benson et al., 2008; Kageyama-Yahara and Riezman, 2006; Menuz et al., 2009; Teufel et al., 2009). The sphingolipids produced in the nematode are a bit different from other eukaryotes because they are built from long chain sphingoid bases that are entirely iso-branched (Chitwood et al., 1995; Hannich et al., 2011).
In C. elegans, sphingolipids participate in different processes. For instance, studies have revealed an essential role of sphingolipids for intestinal cells and for the developing intestine (Marza et al., 2009; Zhang et al., 2011). Deng et al.
demonstrated an involvement of ceramides in radiation induced-apoptosis in the germ line of C. elegans. Indeed, they showed that radiation induced-apoptosis was suppressed in hyl-1 and lagr-1 deletion mutants (Deng et al., 2008). More recently, ceramides have been shown to be involved in mitochondrial surveillance and morphology in the nematode, involving hyl-1, lagr-1 and a serine palmitoyl transferase (sptl-1) that also plays a role in sphingolipid biosynthesis (Liu et al., 2014). Interestingly, the group of Aroian showed that the absence or the strong decrease of ceramide-based glycolipids was correlated with the resistance to Bacillus thuringiensis Crystal Toxin (Griffitts et al., 2005). Finally, as discussed in the next section 2.4, sphingolipids also play a role in the response to anoxia (Devlin et al., 2011; Menuz et al., 2009).
Altogether, these studies reveal the involvement of sphingolipid homeostasis in various processes from development to stress response in C. elegans.
2.4 Implication of sphingolipids in anoxia or in hypoxia
Different stress signals have been revealed to influence the sphingolipid metabolism, inducing most of the time the accumulation of ceramides in eukaryotic cells (Hannun and Luberto, 2000), which suggest that sphingolipids are implicated in the stress response. Interestingly, recent studies have provided evidence that lipids could be invovled in stress responses generated by anoxia or by hypoxia. In fact, as previously described in our laboratory (Menuz et al., 2009) ceramides are essential for the protection of C. elegans in anoxic conditions. In 2011 Devlin et al. reveal that DHCers seemed to be associated with the regulation of cell proliferation in hypoxic conditions (Devlin et al., 2011). Moreover, studies on foetal asphyxia in rat brain revealed significant changes in mRNA levels of ceramide synthase, Lass1, Lass2 and Lass5, enzymes, which are all involved in ceramide metabolism, emphasizing the importance of this class of sphingolipids in the response to changes in oxygen availability (Vlassaks et al., 2013).
Altogether these recent studies confirm that ceramides could be essential in facing oxygen variations. Study of novel ceramide-protein interactions can reveal how ceramide metabolism and signalling could be regulated.
3. Caenorhabditis elegans: a model to study the response to anoxia
C. elegans has been known as a very useful genetic model to study various fundamental cellular processes including apoptosis and RNA interference (Conradt, 2009; Fire et al., 1998). One of the successes of this animal model lies in the following properties: transparency, simplicity, short lifespan and lifecycle, freezability, genetic homology with humans, hermaphrodite and male sexes. In addition, C.
elegans is a powerful tool to study the response to anoxia since it is one of the animals able to face this stress. Wild-type C. elegans naturally resists up to 48 hours of anoxia and some mutants resist up to 72 hours of anoxia (Mendenhall et al., 2006;
Paul et al., 2000; Van Voorhies and Ward, 2000).
3.1 How does C. elegans sense oxygen?
As mentioned before, C. elegans, as several small and simple organisms, does not possess any circulatory system. No lungs, no heart, no blood is present to distribute O2 molecules throughout the body (Figure 8). Gas exchange is ensured by simple diffusion (Van Voorhies and Ward, 2000). Similar to other animals, C. elegans also has a sensory system, which allows it to sense the most appropriate oxygen concentration to its metabolism. This concentration is between 5% and 12% (Gray et al., 2004). Signalling mediated by specific guanylate cyclases, which are expressed in URX and BAG sensory neurons, ensures the oxygen sensing of the worm. An increase in O2 levels activates URX neurons whereas a decrease in O2 levels activates BAG neurons (Gray et al., 2004; Zimmer et al., 2009).
Figure 8: Modified from K.D. Schroeder, Wikipedia commons
Schema representing an hermaphrodite adult of C. elegans. The simplicity of this animal makes it a powerful tool for genetic studies. Its transparency makes it advantageous to perform various microscopic experiments.
3.1.1 C. elegans and the pathways regulating hypoxia and anoxia responses
Despite its preference for a poorly oxygenated environment, C. elegans manages to resist hypoxia through the HIF-1 pathway in a similar manner to humans, controlling expression of hypoxia response genes via hif-1 (Epstein et al., 2001; Jiang et al., 2001; Powell-Coffman et al., 1998; Shen et al., 2005). In addition, it has been reported in different studies that hif-1 in C. elegans was also required for aging and coping with different stresses such as protection against pathogenic bacteria or in heat acclimation (Bellier et al., 2009; Budde and Roth, 2010; Chen et al., 2009;
Mehta et al., 2009; Treinin et al., 2003; Zhang et al., 2009). Thus, this protein appears essential and multifunctional in C. elegans. However, in the past decades several studies have shown that the HIF-1 pathway was not required by C. elegans to face anoxic stress, underlining differences between hypoxia and anoxia
not need HIF-1 to survive in anoxic conditions (Menuz et al., 2009; Padilla et al., 2002).
Several pathways are involved in protection of C. elegans in anoxia. For instance, the insulin-like pathway has been revealed to be implicated in the resistance to anoxia (Mendenhall et al., 2006). When the insulin-like receptor daf-2 is not functional, it mimics an absence of insulin, which can be comparable to a dietary restriction. In normal conditions, the activation by insulin of the receptor daf-2 leads to the phosphorylation by AKT protein kinase B of the FOXO protein daf-16, which is thus sequestered in the cytosol (Figure 9). In contrast, when daf-2 is not activated due to a dietary restriction or because of a mutation, non-phosphorylated daf-16 is free to go to the nucleus and to trigger the transcription of genes responsible for the resistance to different stresses such as heat-shock and anoxia. This also results in doubling of the lifespan of daf-2 mutants compared to wild type (Kenyon et al., 1993; Kimura et al., 1997; Lin et al., 2001).
Figure 9: Simplified insulin-like pathway adapted from (Nemoto and Finkel, 2004).
When the insulin-like receptor DAF-2 is active, it leads to the phosphorylation of the protein DAF-16, which will be sequestered in the nucleus. Inactivation of DAF-2, by mutation or dietary restriction, releases DAF-16 to the nucleus, which triggers the transcription of genes implicated in stress defence and in the extension of the lifespan of C. elegans.
Recent studies revealed that loss of function of several other genes was also able to increase the resistance to anoxia, independently of the insulin-like pathway. This is the case of nsy-1 and glp-1. nsy-1 encodes the conserved protein Apoptosis Signalling-Regulating (ASK), which interestingly provides a strong resistance to anoxia when mutated, while mutants are more sensitive than wild type to other stresses such as oxidative stress induced by paraquat (Hayakawa et al., 2011). The way by which nsy-1 is activated and triggers increased resistance to anoxia still remains unclear. glp-1, a member of LIN-12/Notch family protein, encodes a N-
anoxia in an insulin-like pathway independent manner (Mendenhall et al., 2009). In addition, in our lab we demonstrated that proper ceramide production was necessary for a normal resistance to anoxia and that this is also unrelated to the insulin-like pathway (Menuz et al., 2009) (see results).
Altogether, these studies clearly show that we cannot restrict anoxia resistance to only one pathway, but that C. elegans has acquired multiple pathways to combat anoxic conditions. This does not exclude the possibility that these pathways share many common actors at converging points. This is the case for the daf-2 and glp-1 related pathways which both involve the AMP-activated protein kinase (AMPK) under certain conditions (LaRue and Padilla, 2011). This indicates, that, even if anoxia response pathways are diverse, they might sometimes share common processes.
In addition to mutations, environmental conditions are also crucial for the resistance to oxygen variation such as anoxia. In fact, temperature and food can precondition the worms, helping them to better manage the anoxic conditions (LaRue and Padilla, 2011). Hypoxic preconditioning was also shown to increase the resistance of C.
elegans to a subsequent and longer hypoxia period (Dasgupta et al., 2007).
3.1.2 Behaviour of C. elegans in anoxia
From the anaerobic biobag to the glove box chamber, there are different procedures to generate an anoxic environment in a laboratory (Padilla, 2012). In ours, we do it in a glove-chamber using nitrogen gas to flush oxygen out. Basically the way it works is similar to an ischemia and a reperfusion. Ischemia starts as soon as the animals are
in the box and reperfusion takes place when the worms are removed from the box and put back to normoxia environment. This means that the worm undergoes two stresses that could be potentially deleterious: first, the absence of oxygen, then re- oxygenation. We have observed that some worms die before being re-oxygenated because they are disintegrated on the plate as soon as it is taken out from anoxia.
Most worms are inanimate but look morphologically in good shape and die within 24 hours of re-oxygenation. It is difficult to assess when they precisely die during re- oxygenation.
The behaviour of C. elegans upon anoxia can be described as follows: after a period of a few hours in anoxia, wild type worms arrest all observable biological processes, such as movement, eating and cell division and it takes several hours for them to recover and to move normally upon reoxygenation (Padilla et al., 2002; Van Voorhies and Ward, 2000). This reversible state is called suspended animation (Padilla et al., 2002; Pamela A. Padilla, 2012). Preliminary results allowed us to observe the same behaviour after an anoxic period of 48 hours and a recovery time of 24 hours in normoxia. In addition, we observed that when put in anoxic conditions the worms did not enter suspended animation straight away, but that it took in between 2 and 3 hours for the worms to strongly decrease their activity. Performing time-lapse microscopy, Mendhall et al. observed that wild type worms were able to continue moving for 8 to 16 hours after incubation in anoxia and even more for daf-2 mutants (Mendenhall et al., 2006).
4. Background of the thesis projects
As discussed in the introduction, oxygen is required for most organisms to survive. In our lab, we used Caenorhabditis elegans to try to have a better understanding of the mechanisms by which this worm can resist anoxia for 48 hours. Vincent Menuz initiated this work and mapped a mutation promoting sensitivity to anoxia in a gene called hyl-2. The hyl-2 loss of function mutants cannot survive more than 24 hours in anoxic conditions. hyl-2 encodes a ceramide synthase that has been shown to protect worms from anoxia (Menuz et al., 2009) (for full publication, see Appendix 1).
In order to better understand the mechanism by which the nematode is protected against anoxia, Audrey Bellier, a postdoctoral fellow in our lab, performed a mutagenesis suppressor screen of the hyl-2 loss of function mutants in an attempt to rescue resistance to 48h anoxia. This screen provided several suppressors. Among them, we selected the two most potent suppressors, gnv2 and gnv3, with the idea to identify the specific mutations that, when combined with the hyl-2 loss of function, can restore the wild type phenotype. The gnv2 mutation affects a cyclic nucleotide gated channel, cng-2, and gnv3 affects a putative uncharacterized protein, T19A5.1 (renamed TTC-1). While gnv3 completely restored resistance to anoxia in hyl-2(gnv1) mutants, gnv2 only achieved partial resistance. After segregation of the two mutations, we found that gnv2 promoted survival in anoxia independently of hyl-2 loss of function. Indeed, gnv2 single mutants resisted anoxia better (up to 72 hours) than wild type worms. In contrast, segregation of the gnv3 mutation from hyl-2(gnv1) revealed that gnv3 mutant worms were not more resistant than wild type worms. The two alleles (gnv2 and gnv3) that we identified have not been previously reported to be involved in stress resistance.
4.1 Project 1:
4.1.1 Aim 1:
The main project of my thesis was first to confirm the suppressor phenotype of gnv3 and then to identify gnv3 mutation using in parallel two complementary approaches:
Rapid Single Nucleotide Polymorphism mapping and Whole Genome Sequencing (WGS).
4.1.2 Aim 2:
The goal was to understand how the suppressor gene that we identified contributed to sensitivity of hyl-2(gnv1) to anoxia. This answer to this question was mainly obtained through profiling of the lipidome of the mutant worms.
All the results that we obtained on this project are reported in the manuscript in preparation, which consists of the results part of this thesis.
