Epithelial autophagy and longevity of Hydra oligactis, a new model for aging research

Thesis

Reference

Epithelial autophagy and longevity of Hydra oligactis, a new model for aging research

TOMCZYK, Szymon

Abstract

Hydra, a freshwater cnidarian possesses high regenerative potential and negligible aging.

However, the Cold Sensitive H. oligactis strain (Ho_CS) induces aging upon the loss of interstitial stem cell lineage. Aging in Ho_CS is characterized by the loss of budding, regeneration and degeneration followed by death of animals within three months. This result is surprising as epithelial cells of most Hydra strains adapt to the loss of i-cells and sustain the animal. We measured proliferation, autophagy and protein aggregation in epithelial cells and found all three processes modified upon aging induction. Our cellular and molecular analysis provides evidence for deficient epithelial autophagy driving aging in Ho_CS. Chronic exposure to Rapamycin positively impacts the lifespan and fitness by modifying engulfment behavior, lipid metabolism and self-renewal of epithelial stem cells in autophagy independent fashion.

We demonstrate that H. oligactis provides potent model system to study the relationship between autophagy, stem cells and aging.

TOMCZYK, Szymon. Epithelial autophagy and longevity of Hydra oligactis, a new model for aging research. Thèse de doctorat : Univ. Genève, 2017, no. Sc. 5047

URN : urn:nbn:ch:unige-925147

DOI : 10.13097/archive-ouverte/unige:92514

Available at:

http://archive-ouverte.unige.ch/unige:92514

UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES

Département de Génétique & Évolution Professeure Brigitte GALLIOT

Epithelial autophagy and longevity of Hydra oligactis, a new model for aging research

THÈSE

présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Szymon TOMCZYK de

Varsovie (Pologne)

Thèse n° 5047 Genève

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Acknowledgments

First of all, I would like to thank my PhD advisor, Prof. Brigitte Galliot for giving me the opportunity to join her group and to work on this exciting project. I am grateful for her continuous trust and support as well as constant stimulation to develop my skills as a scientist.

Secondly, I would like to thank Prof. Robbie Loewith and Prof. Trond Lamark for their interest in my work and for accepting to be in my PhD thesis committee.

My deep gratitude goes also to Prof. Steven Austad for the collaboration on this project, to Dr. Hiroshi Shimizu for providing us with the “correct” Hydra oligactis strain, Christoph Bauer for fruitful collaboration on the electron microscopy of Hydra tissues, Prof. Thierry Soldati, Prof. Robbie Loewith, Prof. Ana Maria Cuervo for useful comments and stimulating discussions.

Next, I would like to thank all the present and past members of the Galliot group for creating amazing atmosphere for research and for your friendship inside and outside of the university. A special warm thanks goes to Chrystelle Perruchoud, Nenad Suknovic, Sarah Al Haddad, Ana Paula Catunda, Matthias Vogg, Pedro Machado, Laura Bocanegra, Laura Iglesias, Quentin Schenkelaars, Wanda Buzgariu, Yvan Wenger and Valérie Mino. Moreover, I would like to thank my master students Kazadi Ekundayo and Asli Akin for great times working together. I would like to thank Denis Benoni, Marie-Laure Curchod, Joana Cruz, Julien Candrian and Lisbeth Muster for their extraordinary technical help throughout the project.

To Dr. Joanna Krwawicz and late Dr. Marek Keller warm thank you for showing me passion for science and for guidance during my university years.

To Kamila Kowa special thanks for never ending supplies of support, love and friendship over all these years.

To my family, especially my dad Andrzej and my sister Agata for always believing in me and going along with my crazy ideas. “Dziękuję!”

Warmest thank you to all friends I met in Geneva especially Adrien Vallino, Carlos Rivera, Krisztian Katona, Adriana Arongaus, Vinicius Galvao, Alejandro Melero, Isabel Guerreiro, Valentina Galli and to those I left in Poland but are always with me, to Michał Kwapiński, Michał Płachta and Dawid Ślaski-Sawicki.

The research conducted in this thesis would not be possible without the support of the National Institute of Health (grant NIH-R01AG037962), the Swiss National Science Foundation (SNF 31003A_149630) and the canton of Geneva.

ACKNOWLEDGMENTS ... 5

LIST OF FIGURES ... 9

LIST OF ABBREVIATIONS ... 10

R^ÉSUMÉ ... 13

SUMMARY ... 15

INTRODUCTION ... 17

1.INTRODUCTION TO AGING ... 17

1.1 The theories of aging ... 17

1.1.1 Programmed aging theories ... 18

1.1.2 The “wear and tear” or damage theories of aging ... 18

1.2 The hallmarks of aging ... 20

1.2.1 Genomic instability ... 20

1.2.2 Telomere shortening ... 21

1.2.3 Epigenetic alterations ... 22

1.2.4 Loss of protein homeostasis (proteostasis) ... 22

1.2.5 Altered nutrient sensing ... 24

1.2.6 Mitochondrial dysfunction ... 26

1.2.7 Cellular senescence ... 27

1.2.8 Stem cell exhaustion ... 27

1.2.9 Altered intercellular signaling ... 28

1.3 Autophagy, a key response to environmental stresses ... 28

1.3.1 Regulation of macroautophagy ... 29

1.3.2 Molecular mechanism of macroautophagy ... 30

1.3.3 Crosstalk between macroautophagy and ubiquitin-proteasome system ... 30

1.4 Necessity for new model organisms for aging research ... 31

2.INTRODUCTION TO THEHYDRA MODEL SYSTEM ... 33

2.1 Basic characteristics of Hydra ... 33

2.2 Tissue organization and homeostasis of Hydra ... 33

2.3 Research tools available for Hydra ... 35

3 AGING IN HYDRA ... 36

3.1 Negligible aging in Hydra vulgaris ... 36

3.2 Inducible aging in Hydra oligactis ... 37

3.3 H. oligactis as a model for aging research ... 37

4 AIMS OF THE PROJECT ... 39

RESULTS ... 41

CHAPTER 1:HYDRA OLIGACTIS AS A NEW MODEL SYSTEM FOR AGING ... 41

CHAPTER 2:IDENTIFICATION OF PROCESSES DRIVING AGING IN HO_CSAND RAPAMYCIN MEDIATED MODULATION OF LIFESPAN OF HYDRA. ... 49

DISCUSSION ... 89

1.SUMMARY OF THE PRESENTED DATA ... 89

2.A DEFICIENT AUTOPHAGY FLUX CHARACTERIZES AGING IN HO_CS ... 91

2.1 ULK1 dependent regulation of autophagy in aging Hydra ... 92

2.2 Role and regulation of p62/SQSTM1 in aging Hydra ... 93

2.3 Loss of self renewal in epithelial stem cells in aging Hydra ... 95

3.RAPAMYCIN-INDUCED RESCUE OF AGING ... 95

3.1 Autophagy-independent rescue of longevity in Ho_CS ... 96

3.2 Is the Rapamycin-induced engulfment effect related to entosis, LC3-associated phagocytosis (LAP) or phagoptosis? ... 96

3.3 Rapamycin-induced rescue of epithelial cell cycling ... 98

3.4 Rapamycin-induced formation of lipid droplets in epithelial cells ... 98

4. ROLE OF PROTEIN HOMEOSTASIS AND AGGREGATE FORMATION IN HYDRA AGING ... 99

5. EVOLUTIONARY CONSERVATION OF AGING IN H. OLIGACTIS AN IMMORTAL ANIMAL WITH INDUCIBLE AGING. ... 100

5.1 Autophagy and longevity, a link conserved across animal evolution? ... 101

6.CONCLUSIONS AND PERSPECTIVES ... 102

6.1 Further strategies to dissect autophagy in aging Hydra ... 102

BIBLIOGRAPHY ... 105

APPENDIX ... 123

1.VECTOR MAPS ... 123

2.PROTEIN AND DNA SEQUENCE ALIGNMENTS ... 126

CURRICULUM VITAE ... 135

List of figures

Figure 1 Nine hallmarks of aging process.

Figure 2 Scheme of the cellular proteostasis systems.

Figure 3 Scheme representing the key nutrient singling in the cell.

Figure 4 Scheme representing different possible fate of the stem cells during aging.

Figure 5 Scheme representing an overview of the macroautophagy.

Figure 6 Phylogenetic position, tissue organization and stem cell activity of Hydra.

Figure 7 Summary of the experiments on the reproduction and survival of three species of Hydra performed by Brien (1953).

Figure 8 Loss of ISCs uncovers the deficiency of ESC of Ho_CS to adapt to stress and promote survival.

Figure 9 Summary of the changes observed upon the rapamycin exposure in Ho_CS and Ho_CR.

Figure 10 Summary model of the possible molecular mechanisms of the Rapamycin induced rescue of the aging phenotype in Ho_CS.

List of abbreviations

AMP - Adenosine monophosphate aPKC – Atypical protein kinase C ARE – Antioxidant response element ATP - Adenosine triphosphate BECN1 - Beclin-1

CMA - Chaperone-mediated autophagy CR – Caloric restriction

DNA - Deoxyribonucleic acid

EcESCs – Ectodermal epithelial stem cells EnESCs – Endodermal epithelial stem cells ESCs - Epithelial stem cells

HDAC6 - Histone deacetylases 6 Ho – Hydra oligactis

Ho_CR – Hydra oligactis Cold Resistant Ho_CS – Hydra oligactis Cold Sensitive HS – Heat shock

HSP – Heat shock proteins HU - Hydroxyurea

Hv – Hydra vulgaris

IGF-1 – Insulin-like Growth Factor 1 ISCs – Interstitial stem cells

KIR – Keap1 interacting region LAP – LC3 associated phagocytosis LIR – LC3 interacting motif

mTOR – Mechanistic target of Rapamycin

mtUPS – Mitochondrial unfolded protein response NER – Nucleotide excision repair

NF-κB - Nuclear factor kappa-light-chain-enhancer of activated B cells

NIH - National Institutes of Health NOX2 - NADPH oxidase 2

Nrf2 - Nuclear factor (erythroid-derived 2)-like 2 PE - Phosphatidylethanolamine

PP2A - Protein phosphatase 2A RNA - Ribonucleic acid

RNAi - RNA interference ROS – Reactive oxygen species

SASP – Senescence-associated secretory phenotype SIRT1 – Sirtuin 1

SOD – Superoxide dismutase SQSTM1 – Sequestosome 1

TEM - Transmission electron microscopy TLR – Toll-like receptors

TRAF6 - TNF receptor associated factor 6 Ulk1 - Unc-51-like kinase 1

UPR – Unfolded protein response UPS – Ubiquitin-proteasome system

Vps34 - Vacuolar protein sorting-associated protein 34

Résumé

Le but principal de mon travail de thèse a été d’identifier les bases du phénotype de vieillissement induit par la gamétogénèse découvert en 1950 chez Hydra oligactis par Paul Brien. L’effort initial a porté sur la caractérisation de ce vieillissement aux niveaux phénotypique, cellulaire et moléculaire dans le but de préparer une analyse plus approfondie des mécanismes qui en sont responsables.

Durant la première année du projet, nous avons découvert que ce vieillissement inductible n’est pas observé chez toutes les souches d’Hydra oligactis. En effet, nous avons identifié deux souches proches, que nous avons nommées sensible au froid (Ho_CS) et résistante au froid (Ho_CR), dont la réponse à la gamétogénèse induite par un abaissement de la température à 10°C, est extrêmement différente : les animaux de la souche Ho_CS montrent des signes progressifs de dégénération aboutissant à leur mort en trois à quatre mois, tandis que les animaux de la souche Ho_CR retournent à un état asexué et ne manifestent ensuite aucun signe de vieillissement sur une période d’au moins un an.

Le vieillissement chez l’Hydre et chez les vertébrés partage de nombreuses caractéristiques telles que la perte précoce de la capacité à se régénérer, un déclin de la prolifération cellulaire, la perte de cellules souches, et une détérioration du système nerveux, qui combinée avec une désorganisation des fibres musculaires aboutit à l’altération des comportements actifs de l’animal tels que sa capacité à s’alimenter, et finalement à sa mort. De manière intéressante, le phénotype de vieillissement peut aussi être induit chez Ho_CS en l’absence de gamétogénèse, en exposant les animaux de cette souche à de l’hydroxyurée. Les deux traitements, exposition au froid ou inhibiteur de la prolifération, aboutissent à la perte rapide des cellules de la lignée interstitielle dans les deux souches.

Contrairement à Ho_CR, les cellules épithéliales de Ho_CS ne sont apparemment pas capables de s’adapter à la perte des cellules interstitielles. En plus d’un renouvellement cellulaire déficient, les cellules épithéliales de Ho_CS échouent à augmenter le flux d’autophagie en réponse au jeûne, à l’exposition au froid, à l’inhibition de TORC1 ou du protéasome, comme le démontrent la quantification de vacuoles LC3 positives ou l’analyse du flux d’autophagie in-vivo. Nous avons identifié un blocage de la formation de phagophores en notant l’absence de sur- expression d’AMBRA1 et de Beclin1 chez les animaux mis à 10°C ainsi qu’une inhibition de l’activité de la kinase ULK1. Nous avons aussi mis en évidence une accumulation de la protéine cargo p62/SQSTM1 aux niveaux protéiques et transcriptomiques. Nous avons découvert que les cellules épithéliales Ho_CS sont incapables de former des agrégats de protéines, tandis que celles de la souche Ho_CR le font de manière transitoire. De ces observations, nous avons conclu

qu’une autophagie déficiente est la cause la plus probable du vieillissement induit par la gamétogénèse chez Ho_CS.

Afin de tenter d’inverser le processus de vieillissement, nous avons exposé de manière chronique des animaux en condition de vieillissement avec de la Rapamycine. Ce traitement rétablit la prolifération des cellules épithéliales, la régénération de la tête, retarde le vieillissement et allonge la durée de vie des Hydres Ho_CS pour une période d’environ trois semaines. Le traitement par la Rapamycine ne semble pas agir via une augmentation du flux d’autophagie mais bien plutôt grâce à une augmentation massive de la capacité des cellules épithéliales à phagocyter les petites cellules environnantes, et à produire des gouttelettes lipidiques ainsi qu’à former des agrégats protéiques. Au final ce travail établit un lien entre une autophagie épithéliale efficace et la longévité de l’Hydre. Il révèle aussi un effet inédit de la Rapamycine sur la phagocytose par les cellules épithéliales qui, chez l’Hydre,contirbue à retarder le vieillissement. Ces différents résultats permettent de proposer l’Hydre comme nouveau modèle pour la recherche sur le vieillissement.

Summary

The aim of my PhD was to identify the basis of the inducible aging phenotype discovered in Hydra oligactis by Paul Brien in 1950. The initial effort of the project was to characterize aging in H. oligactis on the phenotypical, cellular and molecular levels to provide the groundwork for a more mechanistic analysis.

During the first year of the project we discovered that inducible aging is not a property shared by all H. oligactis strains. Indeed we identified two closely related strains, named cold sensitive (Ho_CS) and cold resistant (Ho_CR), that exhibit dramatically different responses to cold exposure: Ho_CS animals show signs of degeneration and die within 3-4 months, while Ho_CR animals revert to the asexual state without exhibiting any signs of aging over at least one year.

Hydra and vertebrate aging share many characteristics, such as the rapid loss of regeneration, a continuous decrease in cell proliferation, the loss of stem cells, the deterioration of the nervous system that together with the disorganization of the muscular fibers irreversibly alter the active behaviors including feeding, and finally lead to animal death. Interestingly, the aging phenotype can also be induced in Ho_CS in the absence of gametogenesis, by transiently exposing the animals to hydroxyurea. Both procedures, cold and anti-proliferative drugs, lead to a dramatic depletion in the somatic interstitial cells in both strains.

However, in contrast to Ho_CR, Ho_CS epithelial cells appear unable to adapt to this loss. Beside deficient self-renewal, Ho_CS epithelial cells fail to properly up- regulate the autophagy flux upon starvation, cold exposure, TORC1 or proteasome inhibition as deduced from the quantification of LC3 positive vacuoles and from the in vivo monitoring of the autophagy flux. We identified a blockade in phagophore formation with a lack of AMBRA1 and Beclin1 up- regulation, and a repressed ULK1 activity. We also evidenced an accumulation of the p62/SQSTM1 cargo protein detected at the transcriptomic and proteomic levels. We discovered that epithelial cells of aging Ho_CS are unable to form protein aggregates while those from Ho_CR transiently do. We concluded that deficient autophagy is the most plausible driver of aging in H. oligactis.

To reverse the aging process, we chronically aging animals with Rapamycin, a treatment that rescues epithelial proliferation and head regeneration, delays aging and extends the lifespan by about three weeks. Rapamycin treatment does not seem to enhance the autophagy flux but instead to promote the phagocytic behavior of Ho_CS epithelial cells that engulf and digest surrounding small cells.

In addition, Rapamycin treatment induces the formation of lipid droplets, and in Ho_CS the formation of protein aggregates. This work points to a link between efficient epithelial autophagy and sustained longevity in Hydra. It also reveals a previously unknown effect of Rapamycin on epithelial phagocytosis, which in Hydra likely contributes to delay aging. All these features support Hydra as a powerful new model for aging research.

INTRODUCTION

1. Introduction to aging

Aging is a process that all of us will inevitably experience in our lives. Initially by observing aging of those around us and later on we will go through it ourselves.

This inevitability fascinated great minds around the world for centuries and people were always trying to answer “How and why aging happens? Is there anything that we can do about it?”. Ancient philosopher Aristotle in his treatise

“On Longevity and Shortness of Life” made some interesting observations about the differences in lifespan among various animal species, pointing that it would be interesting to know the factors that are responsible for these differences. In the modern times aging became more intensively studied four decades ago but it is only since few years with the development of advanced molecular and genetic techniques that we observed a rapid increase in the research on the molecular basis of aging (Guarente 2014).

1.1 The theories of aging

Aging is a complex and still quite poorly understood process classically defined as accumulation of negative changes over time that leads to decreased fitness and ultimately to death of the organism. Over the years scientist formulated hundreds of hypotheses to explain the mechanism of aging (Medvedev 1990) but all the available theories explain the process only fragmentarily. The difficulty to formulate one unified theory of aging comes from the fact that the large amount of data gathered by the researchers over the years indicates that aging is extremely complex and multilayered process (Rattan 2006). With time it became accepted that aging rate and characteristics are very different not only between species but also between individuals of the same species and even between tissues and cells of the same individual. This conclusion implies that a more global approach needs to be taken to understand the contribution of different components to the phenotypical outcome of aging in different biological systems (Kirkwood 2011). The current understanding of aging comes mostly from research on four major model organisms: budding yeast, fruit fly, nematode and mice (Martin 2011).

Current aging theories fall in two categories: the programmed theories state that aging is a continuation of the developmental program and it is regulated by genes, hormones etc. and the “wear and tear” or damage theories claim that aging is simply an outcome of accumulation of unrepaired damage to macromolecules (Jin 2010). Both views have their supporters trying to advocate

for one of the two theories whenever new discoveries are published (Blagosklonny 2013, Goldsmith 2014).

1.1.1 Programmed aging theories

Programmed aging theories are based on the idea that the aging process can present an evolutionary advantage to the species by preventing overpopulation, removing the individuals no longer fit for reproduction and ensuring the generation turnover (Longo, Mitteldorf et al. 2005, Kirkwood and Melov 2011).

This mechanism would require an active selection during evolution for genes and pathways that regulate the aging process in individuals. Indeed, there have been many reports on mutations that extend the lifespan of model organisms (Fontana, Partridge et al. 2010). However, there are several facts that make the programmed theories of aging unlikely or at least not sufficient to explain aging.

Firstly, in the wild animals mostly die as a consequence of environmental challenges, predation or diseases long before significant signs of aging can be observed (Finch 1990). Secondly, the programmed aging is not a process benefiting the individual but rather a population as a whole. Indeed mutations that allow an individual to escape aging, would most likely provide advantages to mutated individuals over the aging population, therefore they would propagate and be further selected by natural selection (Kirkwood 2005). Thirdly, in contrast to expectations, a complete suppression of aging in animals harboring a combination of mutations that each separately extend lifespan, was never observed.

1.1.2 The “wear and tear” or damage theories of aging

The damage theories of aging suggest that organisms age due to the lack of selection for pro survival maintenance mechanisms in the post reproductive stage of life (Johnson, Sinclair et al. 1999). In this case aging would be an outcome of accumulation of lesions in the absence of proper repair. All macromolecules in the cells are susceptible to deterioration. Damage to genomic and/or mitochondrial DNA, protein aggregation and protein misfolding, advanced glycation end-products (AGEs) and lipid peroxidation has been all described to contribute to aging (Dukic-Stefanovic, Schinzel et al. 2001, Niki, Yoshida et al. 2005, Freitas and de Magalhaes 2011, Koga, Kaushik et al. 2011).

The damage to macromolecules come predominantly from reactive oxygen species (ROS) and free radicals, but also from other cellular metabolites, as well as from errors in cellular process like DNA replication, transcription, etc.

Therefore, three distinct theories can be distinguished, i) the genomic instability theory, which is further detailed in the next section, ii) the free radical theory, and iii) the protein error theory.

The free radical theory of aging suggests that it is the accumulation of damage caused by ROS that leads to aging (Harman 2006). ROS are partially reduced oxygen containing molecules produced mainly as byproduct of mitochondrial respiration, as well as by membranal NADPH oxidases (Schieber and Chandel 2014). High levels of ROS produce deleterious effects on cellular macromolecules (Finkel and Holbrook 2000) and overexpressing proteins involved in antioxidant defense like superoxide dismutase (SOD) and catalase has a positive impact on the lifespan in Drosophila and mice (Orr and Sohal 1994, Schriner, Linford et al.

2005). However, in C. elegans knocking down SOD has very little effect on the lifespan of the animal (Doonan, McElwee et al. 2008). In summary, increasing the intake of antioxidants may increase, decrease or show no effect on the lifespan depending on the studied organism (Sadowska-Bartosz and Bartosz 2014).

In addition, the ROS impact is likely not only linked to their direct toxicity but also to effects linked to ROS signaling. For example C. elegans worms overexpressing SOD do not exhibit lower oxidative damage but live longer due to the activation of proteome maintenance mechanisms (Cabreiro, Ackerman et al.

2011). In fact ROS are not only a source of damage but also important signaling molecules in many biological processes (D'Autreaux and Toledano 2007). The complex relationship between the level of ROS, antioxidants and the lifespan suggests that besides cellular damage, modulations of ROS-dependent signaling could positively contribute to aging.

The protein error theory of aging states that the progressive increase in the level of aberrant proteins involved in transcription and translation compromises cellular fitness and leads to aging (Holliday 1996). This theory is widely disputed but some evidences support the involvement of protein errors in aging:

increasing the frequency of protein errors negatively impact the lifespan of cultured cells and bacteria, while improving the fidelity of protein production extends the lifespan in fungi (Rattan 2006).

1.2 The hallmarks of aging

Over the past decades a tremendous amount of data was gathered to characterize age-related changes in many different organisms at all levels of organization. To help organize the available knowledge and design the most appropriate future studies, López-Otin, Blasco et al. (2013) attempted to systematize the age-related changes proposing nine hallmarks of aging present in mammals during physiological aging (Fig. 1). These processes can be grouped in three main groups that correspond to three successive steps leading to reduction in animal lifespan when aggravated and in most cases prolong the lifespan and/or the health span when counteracted.

1.2.1 Genomic instability

The genomic instability theory of aging argues that the accumulation of DNA damage and the loss of telomere repeats are the major driving forces behind aging (Vijg and Suh 2013). As an evidence, several genetic diseases called progeroid syndromes characterized by an accelerated aging either in human patients or in mouse models, are associated with mutations in DNA repair genes (Hasty, Campisi et al. 2003). Also increasing DNA damage or down-regulating certain DNA repair mechanisms contributes to aging (reviewed in Freitas and de Magalhaes 2011). Indeed various forms of DNA damage accumulate in aging cells (Moskalev, Shaposhnikov et al. 2013) while the efficiency of DNA repair

Fig. 1 Nine hallmarks of aging process proposed by López-Otin, Blasco et al. (2013) divided into three hierarchical categories. Reproduced from López-Otin, Blasco et al. (2013).

pathways decline with age (Freitas and de Magalhaes 2011). Impairment of specific DNA repair genes especially those belonging to the nucleotide excision repair pathway (NER) is associated with accelerated aging phenotypes in human and model organisms (Hoeijmakers 2009). A recent extensive study where DNA repair genes were overexpressed in the fruit fly demonstrated a complex relationship between DNA repair and longevity (Shaposhnikov, Proshkina et al.

2015). Indeed the lifespan of the fly can be negatively or positively modulated depending not only on the overexpressed gene but also on the sex of the animal, the period of expression and the tissue where it is expressed. Mitochondrial DNA, which possesses its own DNA repair mechanisms, is also susceptible to damage (Wisnovsky, Jean et al. 2016). Despite the multiplicity of mitochondria in each cell, those harboring mutations can get selected and, as a consequence, variants of mutated mitochondrial genomes can become prevalent in the cells (Ameur, Stewart et al. 2011). Additionally, mutations in the mitochondrial DNA polymerase gamma lead to accelerated aging in mice (Trifunovic, Wredenberg et al. 2004). Mutations in nuclear and mitochondrial DNA more specifically affect the stem cells due to their high metabolic rate and proliferative potential. These mutations can cause malignant transformation, senescence and apoptosis of the stem cells depleting their pool and leading to deficient tissue homeostasis (Jones and Rando 2011). Maintaining a proper nuclear architecture is another factor critical for genomic stability (Oberdoerffer and Sinclair 2007), as aged human cells produce a defective form of the nuclear scaffold protein lamin A, which is associated with premature aging disorders (Vlcek and Foisner 2007).

1.2.2 Telomere shortening

During the genome replication the DNA polymerase is not able to replicate the ends of the linear chromosomes and with each round of replication a small portion of the terminal DNA is lost. To buffer this loss the ends of the chromosomes are protected by a repetitive DNA sequence named telomeres.

The telomeric repeats are bound by multiprotein complex called shelterin that forms a loop and stabilizes the telomere (Xin, Liu et al. 2008). This structure prevents the recognition of telomeres by DNA repair machinery. The telomeres can be synthetized by the telomerase complex, however, most of the cells do not express the telomerase. The loss of telomeric repeats is associated with the replicative lifespan of the cultured cells (Hayflick and Moorhead 1961). The shortening of telomeric repeats is a source of aberrant DNA repair and leads to genomic instability and aging (Aubert and Lansdorp 2008). Moreover, overexpressing the telomerase in mouse increases its lifespan (Bernardes de Jesus, Vera et al. 2012).

1.2.3 Epigenetic alterations

In addition to the changes in DNA sequence and chromosome structure, the epigenome of aging animals undergoes dramatic alterations. The changes include decrease in number of histones, alteration to the histone modification levels and patterns, changes in the level of cytosine methylation in the genome and chromatin remodeling (Sen, Shah et al. 2016). These changes lead to the deregulation of transcription together with an increase in the transcriptional noise. It is not completely clear how the age-related epigenetic changes are triggered or what is the mechanism through which they impact aging. A study performed in nematode indicates that epigenetic changes could affect the expression levels of crucial lifespan regulating pathways like insulin growth factor pathway (Jin, Li et al. 2011).

1.2.4 Loss of protein homeostasis (proteostasis)

The proper folding of newly produced proteins, the removal of proteins that are damaged and the disposal of protein aggregates are all together defined as proteostasis (Balch, Morimoto et al. 2008). Proteostasis is a crucial process for the maintenance of cell homeostasis. The progressive loss of efficiency of the mechanisms that maintain proteome integrity plays a significant role in physiological aging and is involved in numerous degenerative diseases (Kaushik and Cuervo 2015). Cells possess a highly complex proteostasis network responsible for maintaining the proper folding and solubility of proteins in homeostasis as well as in stress conditions (Fig. 2).

Chaperones and Heat Shock Proteins (HSP): Key actors for repairing, refolding proteins or targeting damaged proteins for degradation are chaperones that mediate de novo folding and conformational changes of proteins (Saibil 2013). Chaperones also regulate the transport of proteins for degradation and promote the formation of protein complexes. Chaperones recognize damaged or unfolded proteins and target them for repair or destruction based on the energetic state of the cell and on inputs from signaling pathways. Function of many chaperones is ATP dependent, however in aged cells the efficiency of ATP production is compromised, as a consequence the activity of chaperones get altered upon aging (Brehme, Voisine et al. 2014). Moreover, aged cells contain

Fig. 2 Scheme of the cellular proteostasis systems. Each newly synthesized protein needs assistance of chaperones to facilitate proper folding. If a protein unfolds or gets damaged it can be refolded/repaired or targeted to the degradation by ubiquitin-proteasome system (UPS), chaperon mediated autophagy (CMA) or selective macroautophagy. The yellow, grey and blue circles represent chaperones. The aging related changes in each of the proteostasis components were listed in the box.

The arrows indicate the direction of the age related change. Reproduced from Kaushik and Cuervo (2015).

more damaged proteins that need to be sequestered into larger protein aggregates to prevent their toxicity (Walther, Kasturi et al. 2015). These aggregates are formed with the assistance of proteins from the small heat shock protein family, which are crucial for preventing the toxic effects of damage proteins. Finally chronic stress associated with aging causes the depletion of chaperones, an additional way to lose proteostasis (Yu, Shibata et al. 2014).

Degradation pathways: When the protein is irreversibly damaged or when the energetic status of the cell is unfavorable for protein repair, chaperones target it for degradation. There are two major mechanisms that degrade proteins within the cell: autophagy and proteasome degradation. Both processes are highly efficient with majority of damaged and unfolded proteins being targeted to the proteasome. For proteasome degradation, the protein needs to be polyubiquitinated, i.e. tagged by a chain of small peptides named ubiquitin. This modification is carried out by a cascade of three enzymes, with the last one named E3 ubiquitin ligase attaching the ubiquitin peptide to the target protein (Lecker, Goldberg et al. 2006). Polyubiquitinated proteins are then recognized by 26S proteasome, a large multimeric protease complex that degrades the protein to small peptides. Autophagy (described in section 1.3) provides a complementary degradation pathway as it is able to remove more bulky substrates like protein aggregates (Lamark and Johansen 2012). The efficiency of both proteostasis pathways progressively decreases with aging (Rubinsztein, Marino et al. 2011).

1.2.5 Altered nutrient sensing

The nutrient sensing pathways are responsible for probing the energetic state of the cell, adjusting the cellular metabolism and activating the proper stress response in case of starvation (Fig. 3). Moreover, in almost all model organisms limiting the caloric intake (caloric restriction, CR) positively impacts the lifespan and health span (Bordone and Guarente 2005). However the efficiency of CR in primates and potentially human is still under dispute and it was suggested that CR can have different impact according to genotypes (Sohal and Forster 2014).

There are four major partially overlapping components of the metabolic sensory network that integrate the information about the energetic state of the cell (Efeyan, Comb et al. 2015):

1. The insulin and insulin-like growth factor 1 pathway (IGF-1) share the downstream signal transduction pathway and sense the glucose and growth hormone levels respectively (Siddle 2011). The downstream effectors of the IGF-1 pathway are mainly the mTOR complex and the forkhead box transcription factor family (FOXO). Two components of the IGF-1 pathway were the first genes identified to modulate the lifespan of an organism. Loss- of-function mutations in daf-2, the C. elegans ortholog of IGF-1 receptor extends the lifespan of worm while loss-of-function mutation of daf-16, the FOXO homolog abolishes the positive effect of daf-2 mutation (Kenyon, Chang et al. 1993). Since the first discovery, the positive effect of down- regulation of the IGF-1 signaling on the lifespan was observed in almost all tested organism (Fontana, Partridge et al. 2010). In addition, the beneficial effects of the CR are at least partially mediated by IGF-1 signaling pathway.

2. The mTOR kinase is a member of two enzymatic complexes (TORC1 and TORC2) responsible for sensing the level of amino acids and integration of the signals from many other pathways to regulate the cellular metabolism (Laplante and Sabatini 2012). Down-regulating TORC1 activity genetically or pharmacologically (Rapamycin) extends lifespan in model organism (Johnson, Rabinovitch et al. 2013). Moreover, the CR and inhibition of TORC1

Fig. 3 Scheme representing the key nutrient singling in the cell. The mTOR, insulin/IGF-1 and AMPK pathways are the key regulators of metabolism responding to levels of glucose, amino acids, ATP and growth hormones. The pathways interact with each other to fine tune cellular metabolism in response to the available resources. Reproduced from Richardson, Schalm et al.

(2004).

do not have additive effect suggesting that the beneficial effects of the diet are mTOR dependent. Rapamycin has been used as an immunosuppressant in transplantology for years. However, serious side effects associated with chronic exposure limit the application of rapamycin as a potential lifespan modulator in human (Wilkinson, Burmeister et al. 2012).

3. The AMPK pathway responds to the low energy levels by sensing the AMP.

The activity of AMPK declines with aging and the increase of the kinase activity extends lifespan in model organism (Salminen, Kaarniranta et al.

2013). The up-regulation of the AMPK signaling induces Nrf2 and FOXO mediated stress response.

4. Sirtuins are NAD-dependent protein deacetylases that play a role in controlling the metabolism and stress response by deacetylating target proteins. The deacetylase activity level is directly related to the NAD⁺ content that reflects low energy state of the cell (Guarente 2011). Sirtuin 1 (Sirt1) mediates the CR effects and when overexpressed increases the lifespan of model organism (Haigis and Sinclair 2010). The impact of Sirtuins on the regulation of lifespan in mammals was called into question after the mouse overexpressing Sirt1 showed improved health span but not lifespan (Herranz, Munoz-Martin et al. 2010). However, in a recent publication brain specific overexpression of Sirt1 increased lifespan and improved the neural activity in aging mice (Satoh, Brace et al. 2013).

1.2.6 Mitochondrial dysfunction

As mentioned previously, mitochondria play an important role in the aging process. In addition to the aging dependent increase in ROS production and mutations to mitochondrial genome the efficiency of ATP production in aged mitochondria drops dramatically (Drew, Phaneuf et al. 2003). This drop is likely associated with the age dependent decline in mitochondrial quality control.

Fusion and fission of the mitochondria are necessary to ensure proper removal of the damaged mitochondria, survival under stress conditions and biogenesis of the new organelles (Youle and van der Bliek 2012). De-regulation of the mitochondrial fusion/fission dynamics has a negative impact on the lifespan of budding yeast (Bernhardt, Muller et al. 2015). Mitochondria posses their own chaperon system maintaining the protein homeostasis in the organelle called mitochondrial unfolded protein response (mtUPR) (Haynes and Ron 2010). The proper function of the mtUPR is a mediator of many life extending manipulations i.e. cytochrome C oxidase down regulation in nematode (Durieux, Wolff et al.

2011). Additionally, mitophagy becomes less efficient in aging animals leading to

impaired removal, aberrant fission and accumulation of dysfunctional mitochondria (Palikaras, Lionaki et al. 2015, Diot, Morten et al. 2016).

1.2.7 Cellular senescence

To prevent the oncogenic transformation and promote repair upon insult, cells can enter a state of permanent cycling arrest called cellular senescence (Campisi and d'Adda di Fagagna 2007). Cellular senescence can be caused by telomere attrition, DNA damage, deregulation of mitogenic signaling and activation of tumor suppressor genes like p16^INK4a (Campisi 2013). In addition to arrest of cell cycle senescent cells are characterized by altered gene expression patterns and secretion of an array of signaling proteins, proteases and extracellular matrix proteins (Coppe, Desprez et al. 2010). Together this phenomenon is known as the senescence associated secretory phenotype (SASP) and allows the senescent cells to alter homeostasis of the surrounding tissue. Normally the senescent cells are cleared by immune system but with age the immunological response becomes compromised and the senescent cells accumulate in certain tissues (van Deursen 2014). The contribution of senescent cells to aging on the organismal level is still a matter of debate. Recent studies show that improved clearance of the senescence cells can improve the function of hematopoietic stem cells in aged mice and contribute the health span (Chang, Wang et al. 2016).

1.2.8 Stem cell exhaustion

The activity of the stem cells is very important for tissue homeostasis and repair throughout the life of the organism. The age related changes include impaired self-renewal, over-proliferation and altered differentiation behavior (Fig. 4) (Liu and Rando 2011). Loss of stem cell proliferation is associated with decline in hematopoiesis and senescence of the immune system (Shaw, Joshi et al. 2010). In the aging muscle the altered wnt signaling causes aberrant differentiation and lineage conversion of the satellite cells (Brack, Conboy et al. 2007). Over proliferation can also cause premature stem cell exhaustion and malignant transformation.

1.2.9 Altered intercellular signaling

The changes in intercellular signaling lead to a chronic low-grade inflammation response that is associated with aging in mammals (Franceschi and Campisi 2014). Among the causes of inflammation are accumulation of tissue damage, secretion of pro-inflammatory factors by senescent cells and age related decline in the function of the immune system (immunosenescence). All of these changes result in an over activation of inflammatory pathways that contribute to development of aging related diseases like diabetes or arthrosclerosis (Licastro, Candore et al. 2005).

1.3 Autophagy, a key response to environmental stresses

Autophagy is a process that cells use to degrade different components of the cytoplasm (Mizushima 2007). There are three major types of autophagy:

macroautophagy, chaperon mediated autophagy and microautophagy. In microautophagy and chaperon mediated autophagy the cargo is recognized and directly delivered to the lysosome for degradation. The macroautophagy is more

Fig. 4 Scheme representing different possible fate of the stem cells during aging. Loss of genome stability and protein homeostasis in aged stem cells leads to loss of self-renewal capabilities and can cause changes in lineage specificity. Accumulation of unrepaired DNA damage can lead to cell cycle arrest and senescence or to malignant transformation.

Reproduced from Liu and Rando (2011).

complex and involves formation of a specific double membrane vesicle called autophagosome (Fig. 5). Macroautophagy can degrade various cellular components in bulk (non-selective macroautophagy) or by targeting them specifically (selective macroautophagy) (Feng, He et al. 2014). The core components of macroautophagy are common between the two subtypes. The link between decrease in efficient autophagy and aging is well established and the available knowledge was recently reviewed by Rubinsztein, Marino et al.

(2011). Perturbations of autophagy in model organisms have negative impact on the lifespan. Moreover, lack of efficient autophagy abolishes the positive effect of caloric restriction.

1.3.1 Regulation of macroautophagy

The upstream regulators of autophagy are nutrient sensing pathways and stress response pathways (He and Klionsky 2009). The Atg1/Ulk1 is a serine/threonine-protein kinase that forms a complex with ATG13 and FIP200 that serves as a hub for integrating autophagic signals (Wong, Puente et al.

2013). In the absence of amino acids MTORC1 becomes inactivated and no

Fig. 5 Scheme representing an overview of the macroautophagy. (I) Inactive TORC1 de-represses Ulk1 complex that phosphorylates components of the Beclin 1 complex (II) initiating formation of the autophagosome. (III) LC3 protein is conjugated to phosphatydyloethanolamine and is incorporated into the forming phagophore allowing elongation and maturation of he autophagosome. (IV) In the final step autophagosome fuses with the lysosome allowing digestion of its cargo. Proteins critical for each step were represented on the scheme. Reproduced with modifications from Yang and Liang (2015).

longer represses the activity of Ulk1 complex. Active Ulk1 complex phosphorylates Serine15 of Beclin 1 activating BECN1-Vps34 complex that initiates autophagosome formation (Russell, Tian et al. 2013). Other pathways like AMPK or insulin/IGF-1 pathways become activated in response to low levels of energy or growth hormones and can repress the activity of TORC1 initiating autophagy. Interestingly, Ulk1 itself can regulate the activity of TORC1 and AMPK by inhibiting them through a positive feedback loop with the first and negative feedback loop with the second (Russell, Yuan et al. 2014).

Activation of response to stress like hypoxia or oxidation also up-regulates autophagy. Induction of stress dependent transcription factors like FoxO or c-jun upregulates expression of many components of autophagic machinery (Wu, Wang et al. 2009). Hypoxia and unfolded protein response can also activate expression of autophagy genes or prevent interaction between Bcl2 and BECN1 that inhibit the activity of the Beclin 1 complex (Senft and Ronai 2015).

1.3.2 Molecular mechanism of macroautophagy

Phosphorylation of BECN1-Vps34 complex leads to its recruitment to the phagophore where it phosphorylates phosphatidylinositol stabilizing the forming autophagosome (Itakura and Mizushima 2010). To allow elongation and maturation of forming autophagosome the ATG8/LC3 protein must be conjugated to phosphatydyloinositol (PE) and anchored in the autophagosome membranes. Initially the C-terminal arginine of ATG8/LC3 is removed by ATG4 and subsequently a lipidation is carried out by a ATG12-ATG5-ATG16L complex in an ubiquitin like manner (Klionsky and Schulman 2014). The mature autophagosome fuses with the lysosome forming an autolysosome in which the cargo is degraded. The ATG8/LC3 incorporated in the membrane of autophagosome provides the platform for interaction with selective autophagy receptors like p62/SQSTM1 or NBR1 (Birgisdottir, Lamark et al. 2013). These autophagy receptors are able to bind ubiquitinated protein aggregates or damaged organelles and bring them to autophagosome for degradation by interacting with the ATG8/LC3. This interaction is mediated by a conserved LC3 interacting motif (LIR).

1.3.3 Crosstalk between macroautophagy and ubiquitin-proteasome system The ubiquitin-proteasome system (UPS) is a multi protein protease complex that degrades most of the proteins in the cell in ATP dependent fashion. Autophagy presents an alternative pathway for degradation of damaged proteins that can be up-regulated when proteasome is overwhelmed or inhibited (Lilienbaum 2013).

The p62/SQSTM1 is a multifunctional protein that can bind cytoplasmic and

nuclear damaged proteins incorporate them into aggregates and mediate their degradation by selective autophagy (aggrephagy) (Pankiv, Lamark et al. 2010).

p62/SQSTM1 recognizes mainly ubiquitnated cargo but can also mediate degradation of non-ubiquitinated proteins (Kirkin, Lamark et al. 2009, Watanabe and Tanaka 2011). When bound to cargo p62/SQSTM1 multimerizes and is incorporated into the forming autophagosome by direct integration with LC3 through the LIR motif. The p62/SQSTM1 and its cargo remain bound and become degraded when autophagosome fuses with the lysosome. Therefore accumulation of p62/SQSTM1 protein is a well accepted hallmark of inefficient autophagy flux. p62/SQSTM1 is linked with protein aggregates forming in age related neurodegenerative disease like Parkinson and Alzheimer diseases (Bitto, Lerner et al. 2014).

1.4 Necessity for new model organisms for aging research

To the disappointment of many scientists after decades of research, we still fail to formulate one unified theory of aging that would explain all aspects of the process. The picture emerging from decades of research in classical model organisms is that aging is a complex multilayered process highly heterogeneous between species and individuals. The heterogeneity of aging in different species indicates that the field may greatly benefit from new model systems that exhibit different life histories. This would help get a more global view of the aging phenotypical traits across evolution. Also a more systematic analysis of the cellular and molecular changes that occur during aging would help identify the evolutionarily conserved basis of aging.

All the models used so far in aging studies possess critical advantages for genetic and biochemical approaches and therefore will continue to be important tools for aging research (Buffenstein, Edrey et al. 2008). However, these models have also substantial shortcomings that call for new model organisms to be adopted by the aging field (Austad 2009). The two animal models that provided most data about aging are the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans. These two species belong to the superphylum Ecdysozoa (Fig. 6A)that according to genomic and transcriptomic analyses lost up to 11%

of the human orthologs that were identified in the last common ancestor of eumetazoans, namely in cnidarians (Kortschak, Samuel et al. 2003, Wenger and Galliot 2013). As a second limitation, both the fly and the nematode can enter an alternative developmental phase in response to harsh environmental conditions (Larsen, Albert et al. 1995, Tatar, Chien et al. 2001). This phenomenon has no counterpart in developing vertebrates and could be involved in the regulation of lifespan in these models. Additionally, as adults, flies and nematodes have very

limited cell proliferation and tissue repair, while these processes are highly altered in aging vertebrates. All these issues have pushed the National Institute of Health (NIH) in the United States to launch in 2010 a research program to establish novel robust invertebrate model systems that could greatly benefit the aging research field (Murthy and Ram 2015). The selected species for this new program were the freshwater hydrozoan polyp Hydra (Bellantuono, Bridge et al.

2015, Tomczyk, Fischer et al. 2015), the monogonont rotifer Brachionus manjavacas (Snell, Johnston et al. 2015), the planktonic crustacean Daphnia (Schumpert, Handy et al. 2014), sea urchin species that are either long- (Strongylocentrotus franciscanus), intermediate- (S. purpuatus) or short-lived (Lytechinus variegatus) (Bodnar 2015), two urochordate species, the colonial ascidian Botryllus schlosseri (Munday, Rodriguez et al. 2015, Voskoboynik and Weissman 2015) andthe tunicate Ciona intestinalis (Jeffery 2015). Thanks to this NIH grant obtained with Prof. Steven Austad located at that time at the Barshop Institute for Longevity and Aging Studies, University of Texas San Antonio (Texas), we could initiate a new program of research on Hydra aging in 2011.

2. Introduction to the Hydra model system

2.1 Basic characteristics of Hydra

Hydra is a freshwater polyp used in biological research for more than two centuries (Galliot 2012). Hydra belong to Cnidaria, a sister group to Bilateria. The genus Hydra contains four distinct species: Hydra vulgaris (Hv; brown Hydra) Hydra oligactis (Ho), Hydra braueri and the symbiotic H. viridissima that possesses an algae endosymbiont (Fig. 6A). Hydra has a radial symmetry and a simple tube shaped body plan with oral and aboral pole. The unique opening, named mouth, located at the oral pole, is surrounded by a ring of tentacles and used to capture preys. On the aboral pole the body column becomes thinner, a region named peduncle and terminates with a basal disc that secretes mucus and allows for the attachment of the animal to substrates (leaves, wooden sticks etc.).

Hydra exhibits an astonishing ability to regenerate any missing structure after bisection of its body column, and to re-aggregate when tissues are disrupted to a single cell suspension. Under favorable condition Hydra reproduces asexually by budding, a process where the excess of cells is incorporated into a new clone of the parent, which develops on the lower third of the parental polyp. Under stressful conditions Hydra can undergo gametogenesis and reproduce sexually.

2.2 Tissue organization and homeostasis of Hydra

Hydra tissue is organized in two layers: epidermis and gastrodermis separated by an extracellular matrix layer named mesoglea (Fig. 6B). Hydra possesses three stem cell populations: endodermal epithelial stem cells (EnESCs), ectodermal epithelial stem cells (EcESCs) and interstitial stem cells (ISCs) that continuously cycle throughout the life of the animal and all together give rise to 12 distinct cell types cite (Bode 1996, Galliot, Miljkovic-Licina et al. 2006, Siebert, Anton-Erxleben et al. 2008, Hobmayer, Jenewein et al. 2012). Epithelial stem cells are predominantly restricted to the gastric region of the animal and with time become displaced towards extremities where they undergo terminal differentiation (Steele 2002). The interstitial stem cells are located in-between the epithelial cells of the epidermis and can differentiate into somatic cells, i.e.

neurons and nematocytes (stinging cells) that form the nervous systems, gland cells in the gastrodermis, but also into germ cells when sexual reproduction is induced (Fig. 6C).

Interestingly, Hydra completely depleted of ISCs (thus named “epithelial” Hydra) no longer capture preys but if forced fed are able to maintain their developmental functions i.e. budding or regeneration (Marcum and Campbell 1978, Sugiyama and Fujisawa 1978). The Galliot group recently showed that in

such epithelial Hydra, ESCs actually adapt by modifying their genetic program, up-regulating genes annotated as neurogenic, neurotransmission or reprogramming in human (Wenger, Buzgariu et al. 2016). In fact epithelial cells in Hydra are multifunctional (Buzgariu, Al Haddad et al. 2015), they not only provide the necessary protection from the environment for the outer layer, the digestive function for the inner layer but are also responsible for the contractility of the animal as they posess myofibrils (thus named myoepithelial). Moreover, upon starvation epithelial cells of both layers rapidly induce autophagy to promote the survival of the animal (Buzgariu, Chera et al. 2008, Chera, Buzgariu et al. 2009). Indeed an increase in the mature form of LC3 and the formation of the autophagosomes can be detected in Hydra after three days of starvation.

Analysis shows that the autophagic machinery and its upstream regulators are very well conserved and are ubiquitously expressed in Hydra tissue (Buzgariu, Al Haddad et al. 2015). Autophagy in Hydra can be readily induced pharmacologically by Rapamycin or Wortmannin treatment. Proper regulation of autophagy is also critical for the response to tissue damage. In the Kazal1 knocked-down animals amputation leads to over activation of autophagy and massive cell death of gland cells (Chera, de Rosa et al. 2006). Epithelial cells of

Fig. 6 Phylogenetic position, tissue organization and stem cell activity of Hydra. (A) Hydra oligactis within the Cnidaria phylum and the phylogenetic relationship with other major aging model organisms. (B) Tissue layers and cell type organization within the Hydra polyp, (C) Three distinct stem cell populations of Hydra, their cycling properties and basic functions. Reproduced from Schenkelaars, Tomczyk et al. (2017).

Hydra remain in contact with the environment and provide a platform for colonization by microbiota and for defense against pathogens (Franzenburg, Walter et al. 2013). Pathogens are recognized by Toll-like receptors (TLR) that recruit adaptor proteins like MyD88 that in turn activate transcription factors inducing transcription of antimicrobial peptides (Franzenburg, Fraune et al.

2012). In fact the epithelial cells of Hydra induce expression of many immunity related genes immediately after bisection providing a protective function in response to injury (Wenger, Buzgariu et al. 2014).

2.3 Research tools available for Hydra

In recent years many new resources became available for Hydra model system.

The genome of Hydra magnipapillata was published (Chapman, Kirkness et al.

2010) and the new high throughput sequencing techniques yielded extensive transciptomic data (Boehm, Khalturin et al. 2012, Hemmrich, Khalturin et al.

2012, Wenger and Galliot 2013). The gene expression data from many different experimental contexts will soon be available as an online database HydrATLAS (Wenger et al., in preparation). Moreover, a proteomic approach was recently used in attempt to elucidate the basis of Hydra regeneration (Petersen, Hoger et al. 2015). A battery of technics that allows for visualization and manipulation of molecular processes in Hydra has been developed. Transgenic animals can be successfully produced by microinjecting fertilized Hydra eggs (Wittlieb, Khalturin et al. 2006), genes can be transiently expressed in Hydra tissue (Bottger, Alexandrova et al. 2002, Miljkovic, Mazet et al. 2002) and feeding or electroporation of RNAi can be used to knock down expression of the genes of interest (Chera, de Rosa et al. 2006, Watanabe, Schmidt et al. 2014). Besides regeneration and development Hydra has been successfully used to study evolutionary context of many biological processes like immunity (Wenger, Buzgariu et al. 2014), stem cell biology (Hobmayer, Jenewein et al. 2012), apoptosis (Lasi, David et al. 2010, Reiter, Crescenzi et al. 2012), autophagy (Buzgariu, Chera et al. 2008) and symbiosis (Kovacevic 2012).

Due to the high tissue turnover and regenerative potential Hydra is considered as species that has no or negligible aging. From the mathematical modeling of the available demographic data, it was proposed that Hydra could live more than one thousand years under laboratory conditions (Jones, Scheuerlein et al. 2014).

Interestingly, in one group of Hydra species aging can be induced. This phenomenon presents a unique opportunity for model system in which aging and non-aging phenotypes can be directly compared.

3 Aging in Hydra

3.1 Negligible aging in Hydra vulgaris

The first person that became interested in the longevity of Hydra was Belgian biologist Paul Brien. He followed cohorts of Hydra vulgaris (Hv), Hydra viridissima and Hydra oligactis (Ho) over several years to investigate the reproduction and mortality rate of these animals (Fig. 7)(Brien 1953). He observed that when maintained at 18°C all three species continue to reproduce asexually by budding and do not show any signs of aging or mortality. Moreover, during the recorded period H. vulgaris and H. viridissima went through several rounds of sexual reproduction maintaining the fitness of the animals. More recently, these finding were confirmed by two studies that recorded no mortality for Hydra maintained at 18°C for three or eight years confirming that Hydra vulgaris indeed exhibits negligible senescence in laboratory conditions (Martinez 1998, Schaible, Scheuerlein et al. 2015).

Fig. 7 Summary of the experiments on the reproduction and survival of three species of Hydra performed by Brien (1953). Hydra oligactis undergo sexual differentiation when moved to 10°C, rapidly stop budding and become exhausted within several weeks. H. viridis and H. vulgaris do not show any signs of aging. Reproduced from Schenkelaars, Tomczyk et al. (2017).

3.2 Inducible aging in Hydra oligactis

Interestingly, in the study of Paul Brien H. oligactis exhibited dramatically different behavior than H. vulgaris. When maintained at 18° C in asexual state H. oligactis polyps did not show any signs of decline. However, when culture temperature was changed to 10°C the Ho polyps rapidly stopped budding and started developing sexual traits (Fig. 7). They reached sexual maturity in about three weeks and then started to show signs of aging-like degeneration that Brien described as “exhaustion”. Later on, the same phenomenon was briefly described by Noda (1982) for sexual Pelmatohydra robusta, a species closely related to H.

oligactis.

More recently, Yoshida, Fujisawa et al. (2006) characterized the inducible aging phenomenon in H. oligactis. They observed that shortly after reaching sexual maturity Ho polyps undergo adverse morphological changes and start to show first signs of mortality after about two months post transfer to 10°C with the maximum lifespan of about 5 months in 10°C. The observed mortality rates fit the Gomperzian function commonly used to characterize aging populations (Finch 1990). The few characteristics of aging in Ho described by Yoshida et al.

were progressive decrease of contractility and prey capture potential together with loss of interstitial stem cells lineage and disorganization of the actin muscle fibers. Both exhaustion of stem cells and muscle degeneration are well-accepted hallmarks of aging.

Careful investigation of inducible aging in different Ho strains revealed two closely related strains with different response to the sexual differentiation (Tomczyk, Fischer et al. 2015). The first strain called Cold Sensitive (Ho_CS) upon transfer to 10°C behaves the same as the strain used by Yoshida and Brien. The second called Cold Resistant (Ho_CR) upon transfer to 10°C goes through one round of sexual reproduction, loses sexual traits and does not show any signs of aging over more than one year. This finding shows that inducible aging is not an innate property of all H. oligactis strains and Ho_CS and Ho_CR can be used to compare the cellular and molecular processes in aging and non-aging setting.

3.3 H. oligactis as a model for aging research

As mentioned before the existing invertebrate model systems for aging have substantial drawbacks that the new model should be able to ameliorate. Deep analysis of Hydra transciptome revealed that indeed many human genes that were lost in fruit fly and nematode are conserved and expressed in Hydra (Wenger and Galliot 2013). Out of 305 human genes associated with aging (Human Ageing Genomic Resources) 83% are present in H. vulgaris but only