FACULTY OF SCIENCES
DEPARTMENT OF BIOLOGY
RESEARCH GROUP AGING PHYSIOLOGY AND MOLECULAR EVOLUTION

During the past 12 months this research overwhelmed my daily thoughts while worms were figuring in many dreams. More than once I was told I was cruel because I liquidated ‘mother worms bearing babies’. However, I argued doing so for a higher purpose: delivering a thesis and hopefully contributing to the knowledge about aging
Caenorhabditis elegans
. Nonetheless, I had a remarkable time at the lab, I learned lots of things, I got to know nice people and had frustrating ánd proud moments sitting behind my computer. After all it was an unforgettable year (in the good way)!
Obviously, you would not be reading this without the opportunity given to me by several scientists.
First of all I am very grateful to my supervisor, Prof. Dr. Bart P. Braeckman , for giving me the possibility and the resources to conduct fascinating research at his lab. He inspired me considerably during the last three years with his enthusiastic way of teaching, interesting topics, exciting stories, challenging examinations and his motivating personality.
The lab and therefore also this thesis would not exist without the decades of efforts in research and building up of this well equipped lab by
Prof. Dr. Jacques Vanfleteren
, to whom I therefore express my gratitude.
Obviously, my thesis would be nothing alike without my outstanding mentor Dr. Filip Matthijssens .
He ‘masterminded’ the topic and goals, explained me how each instrument that crossed our sight operates, guided me intensively, encouraged me to think of and to perform experiments autonomously, assisted me in interpreting data, corrected and improved my thesis draft versions, astonished me with his seemingly endless theoretical and practical knowledge and pleasured me by jointly reasoning, discussing and thinking of solutions, possibilities and opportunities. And all this taking place with a great sense of humour. Thank you Filip!
Further I would like to thank (in random order) all the persons that contributed to this master thesis while working in a pleasant atmosphere (surrounded by millions of worms, as my two old aunts imagine the place).
Renata : For possibly saving my life a few times by making me aware of the hidden dangers of many chemicals, for executing a lot of preparatory work for my experiments, for learning me the importance of working absolutely sterile, for the nice chats and of course for the delicious ice cream with strawberries and chocolate sauce!
Patricia : For teaching me, with her experience in biosensors, to process the images of Perceval transgenic worms, giving me tips and tricks in experimental as well as data-managing techniques, rescuing experiments in weekends and of course the pleasant conversations.

Geert : I really got to know Geert when he sabotaged my first experiments by throwing my wild-type juveniles in the trash after he begged to use a few of them. Or, more likely, this ‘sad’ incident was just a consequence of his laborynthic mind full of hypotheses. After all, he really rectified this by feeding my worms several weekends, by giving me interesting insights and hints and by having amusing discussions.
Ineke
: For teaching me the possibilities of confocal laser scanning microscopy, for showing me how to process the resulting images and sharing her experience to deliver a good thesis.
Sasha : For giving me a lot of practical advice concerning nematode phenotypes and culturing, helping me out with experiments during weekends when I could not make it to the lab and assisting me in planning the time schedule for the delivery of the thesis.
Nilesh : For teaching me a regional Indian language (episode 1.1.1), improving my spoken English at many occasions, learning me interesting intercultural differences and having funny talks.
Natascha
: For giving me tips and tricks about culturing techniques, for sharing ‘metabolic’ knowledge and alerting me for the dangers of working with cyanide.
Andy
: For sequencing the expression plasmids, troubleshooting me during technical and computerrelated issues and, of course, joking around.
Caroline
: For making it possible for me to get and find everything I needed for my experiments, for keeping an eye on the autoclave and for organizing a nice quiz (with some dubious arbitral decisions), a dinner and an ice skating trip with the ‘aging’ group which facilitated my integration in the team.
Marjolein : For executing the microinjection several times successfully.
Kristel : For introducing me in the fabulous world of
Caenorhabditis elegans
and its aging during my bachelor thesis and teaching me a dozen of techniques that gave me a significant advantage during this master thesis.
I especially want to thank my mom and dad for giving me the opportunity to start and finish these higher studies in the best circumstances. They supported and motivated me endlessly, while encouraging me to explain the world of aging
C. elegans
in easy words to a non-scientific audience.
Thanks to the many interesting talks and discussions with my parents and my brother Siemen I am now rewarded with a questioning and critical mind which is indispensable in this area of expertise.
Moreover, I am very grateful for the many fine moments we shared this year.
I also want to express my gratitude to both my lovely aunts (Charline and Alice) for their limitless support and encouragements and for all the good care I received my entire life.

Ik wil ook mijn dank uiten aan mijn fantastische “tantjes”, tante Charline en tante Alice , die me altijd gesteund en aangemoedigd hebben. Bedankt voor de verwennerij, goede zorgen en vele leuke en gezellige momenten dit en de voorbije 20 jaar.
I also want to thank someone very special to me, my girlfriend
Nathalie
. She really motivated and stimulated me this year. When experiments failed she was there to support me and encourage me to be resilient. She was able to get my head out of the world of worms and gave me mental rest when I needed it most. I could also share my happiness with her when I discovered something interesting. At last, she was responsible for most of the (non-research-related) pretty and funny moments the past year. Thank you Nathalie!
Finally, thank you, dear reader , for your interest in this master thesis.
Arne

1 Samenvatting ................................................................................................................................. 1
2 Introduction ................................................................................................................................... 7
2.1
Aging ....................................................................................................................................... 7
2.2
The model organism
Caenorhabditis elegans
....................................................................... 11
2.2.1
Ecology of C. elegans .................................................................................................... 13
2.2.2
Morphology and Anatomy of
C. elegans
...................................................................... 13
2.2.3
Life cycle of C. elegans ................................................................................................. 17
2.2.4
Insulin/IGF-1 pathway in C. elegans ............................................................................. 20
2.3
Adenine nucleotides .............................................................................................................. 28
2.3.1
ADP/ATP and AMP/ATP ratios ................................................................................... 28
2.3.2
Control of energy level .................................................................................................. 29
2.3.3
Energy charge ................................................................................................................ 29
2.3.4
Aging and adenine nucleotides ...................................................................................... 30
2.3.5
AMPK as energy sensor ................................................................................................ 32
2.4
Perceval: a fluorescent reporter of ADP/ATP ratio ............................................................... 39
2.4.1
ATP detection ................................................................................................................ 39
2.4.2
Perceval ......................................................................................................................... 39
3 Goals and experimental outline .................................................................................................. 43
4 Materials and methods
................................................................................................................
44
4.1
C. elegans culture .................................................................................................................. 44
4.1.1
Used strains ................................................................................................................... 44
4.1.2
Freezing and thawing strains ......................................................................................... 45
4.1.3
Agar plates ..................................................................................................................... 46
4.1.4
Escherichia coli ............................................................................................................. 47
4.1.5
Setting up synchronized aging
C. elegans
cohorts ........................................................ 48
4.1.6
Sampling ........................................................................................................................ 50
4.2
HPLC-fluorometric assay ...................................................................................................... 51
4.2.1
Formation of fluorescent derivatives ............................................................................. 51
4.2.2
RP-HPLC analysis ......................................................................................................... 52
4.2.3
BCA assay for protein quantification ............................................................................ 55
4.3
Perceval: proof of concept ..................................................................................................... 56
4.3.1
Molecular cloning techniques ........................................................................................ 56
4.3.2
Transformation of C . elegans by microinjection .......................................................... 64
4.3.3
Biolistic transformation of
C. elegans ........................................................................... 66
4.3.4
Measuring ATP/ADP ratios in vivo with Perceval upon sodium azide treatment ......... 73
5 Results
.......................................................................................................................................... 75
5.1
HPLC-fluorometric assay ...................................................................................................... 75
5.1.1
Chromatograms ............................................................................................................. 75
5.1.2
Absolute concentrations of adenine nucleotides ........................................................... 79
5.1.3
Indexes of cellular energy ............................................................................................. 84
5.1.4
Effect of culture and sampling method on energy indexes ........................................... 87

5.2
Proof of concept: Perceval reports ADP/ATP ratio in C. elegans ......................................... 88
5.2.1
Obtaining Perceval transgenic
C. elegans
..................................................................... 88
5.2.2
Sodium azide treatment of Perceval transgenic worms ................................................. 93
6 Discussion
.....................................................................................................................................
98
6.1
HPLC-fluorometric assay ...................................................................................................... 98
6.1.1
Evaluation of the assay .................................................................................................. 98
6.1.2
Aging daf2 mutants maintain high energy levels ....................................................... 100
6.2
Proof of concept: Perceval reports ADP/ATP ratio in
C. elegans
....................................... 104
6.2.1
Obtaining Perceval transgenic C. elegans ................................................................... 104
6.2.2
Sodium azide treatment of Perceval transgenic
C. elegans
......................................... 105
7 Conclusion .................................................................................................................................. 107
8 References .................................................................................................................................. 108

Summary
Veroudering heeft een belangrijke impact op onze samenleving. De voorbije decennia is de wetenschappelijke wereld geïntrigeerd geraakt door dit zeer complexe biologisch fenomeen. Een belangrijk doel bij het onderzoek naar veroudering is het vertragen van dit proces. Zo zou men ouderdomsgerelateerde aandoeningen kunnen uitstellen en dus de levenskwaliteit op hogere leeftijd significant verbeteren. Vanuit economisch standpunt zou dit een belangrijke stap zijn bij het oplossen van de vergrijzingsproblematiek.
De voorbije 20 jaar lag moleculaire genetica aan de basis van enkele belangrijke doorbraken. Eén daarvan is de ontdekking dat de levensduur sterk verlengd kan worden door één enkel specifiek gen uit te schakelen. In het modelorganisme
Caenorhabditis elegans
vond men dat de levensduur verdubbeld kan worden via een mutatie in het daf-2 gen dat codeert voor de insuline/IGF-1 receptor. De daaruitvolgende verminderde activiteit van de insuline/IFG-1 signaalcascade is via verschillende, nog niet volledig gekende mechanismen verantwoordelijk voor de levensduurverlenging. Opmerkelijk is dat dit eerder gepaard gaat met een langere vitale periode dan met een langere aftakeling. Een veranderd energiemetabolisme zou hierbij een niet te onderschatten rol kunnen spelen.
Het doel van dit onderzoek is tweeledig en omvat een toename in de kennis omtrend de relatie tussen energiemetabolisme en veroudering in Caenorhabditis elegans .
1.
Er werd getracht om een completer beeld te verkrijgen van de dynamiek van ATP, ADP en
AMP in zowel verouderende wildtype wormen als in langlevende daf-2 mutanten. Door verschillende indicatoren van cellulaire energieniveaus te bepalen waren we in staat een mogelijk mechanisme voor te stellen dat de energietoestand koppelt aan het verouderingsproces.
2.
De genetisch gecodeerde fluorescente sensor ‘Perceval’ maakt het mogelijk om intracellulaire
ADP/ATP ratios in vivo en in real time te detecteren in zoogdiercellen. In dit deel van het onderzoek werd Perceval tot expressie gebracht in
C. elegans en gingen we na of deze sensor bruikbaar is als indicator van cellulaire energie in intacte C. elegans wormen.
Om de dynamiek van ATP, ADP en AMP in kaart te brengen, was het vooreerst belangrijk grote synchrone populaties van wormen (zowel wildtype als daf-2 mutanten) op te kweken. Zo konden we voor elke leeftijd enkele wormstalen nemen. De wormen werden gehomogeniseerd en de aanwezige adenine nucleotiden gederivatizeerd tot fluorescente etheno(
ε
)-adenine nucleotiden.
1

Summary
Deze adenine nucleotidederivaten werden vervolgens gescheiden door middel van reverse-phase highperformance liquid chromatography (RP-HPLC). Fluorescentiedetectie van de gescheiden
ε
-ATP,
ε
-
ADP en ε -AMP en het gebruik van standaarden lieten toe om exacte oorspronkelijke concentraties van
ATP, ADP en AMP in de wormstalen te berekenen. Uiteindelijk werden nog verschillende indicatoren voor cellulaire energieniveaus (AMP/ATP ratio, ADP/ATP ratio en de ‘energy charge’) berekend.
Uit de resultaten werd geconcludeerd dat, in tegenstelling tot wildtype wormen, langlevende daf-2 mutanten in staat zijn om tijdens het verouderen hogere cellulaire concentraties van zowel ATP als
ADP te behouden. We vermoeden dat dit het gevolg is van verhoging van AAK-2 activiteit door verminderde inhiberende insuline/IGF-1 signaaloverdracht in deze insuline/IGF-1 receptor mutant.
AAK-2 maakt deel uit van een enzymcomplex (AMPK) dat energieniveaus integreert en metabole en transcriptionele veranderingen reguleert om energetische homeostase te bereiken. Sterkere AAK-2 activering leidt tot ATP productie, kan de levensduur verlengen en verhoogt de activiteit van autofagie. Dit laatste zou belangrijk kunnen zijn om hoge energieniveaus te behouden en die kunnen geïnvesteerd worden in onderhoud van de cellulaire functies en in levensduurverlenging.
Omdat cellulaire ATP concentraties geen volledige weergave bieden van de energietoestand van een cel, werd recent een genetisch gecodeerde reporter van ADP/ATP ratios, Perceval, ontwikkeld en getest zowel in vitro als in een celcultuur. Perceval bindt ATP en ADP met een verschillende affiniteit en andere fluorescentierepons tot gevolg. Het in interessant om na te gaan of deze biosensor ook bruikbaar is om energieniveaus in levende Caenorhabditis elegans wormen in ‘real-time’ te meten.
In een eerste stap werd het gen
Perceval
, via Gateway ® klonering, gekoppeld aan verschillende promoters die constitutief tot expressie komen in de meeste weefsels van C. elegans . Vervolgens werden verschillende moleculaire kloneringsstappen uitgevoerd om tot plasmiden (expressievectoren) te komen die het gen Perceval in de juiste conformatie bevatten om expressie in C. elegans te bekomen.
Zeven constructen bestaande uit onder andere
Perceval
in combinatie met een verschillende promoter. werden in de gonaden van verschillende C. elegans hermafrodieten gebracht via microinjectie. Het daaruitvolgende nageslacht bracht slechts één getransformeerde fluorescente worm voort. In de volgende generaties werd mozaïek expressie en een gradueel verlies van Perceval opgemerkt, het gevolg van de aanwezigheid van de plasmiden als grote extrachromosomale ‘arrays’
(aaneengeschakelde plasmiden). Via UV-bestraling werd, tevergeefs, getracht om de constructen in het genoom te integreren. Wormen waarbij Perceval sterk geëxpresseerd wordt in het nageslacht werden geselecteerd en verder opgekweekt.
2

Summary
Stabiele integratie van Perceval in het genoom is belangrijk om ten volle gebruik te kunnen maken van de mogelijkheden die Perceval biedt. Daarom werden wormen ook getransformeerd via biolistische transformatie. Hierbij werden ruim een half miljoen wormen gebombardeerd met goudpartikels. Aan deze goudpartikels werden lineare plasmiden, van het construct dat tot expressie kwam via microinjectie, gebonden die vervolgens konden integreren in the chromosomen. Hoewel transformanten verkregen werden, kwam Perceval hier niet tot expressie. Mogelijks is dit het gevolg van ‘silencing’ door integratie van het construct in heterochromatine waar geen transcriptie plaatsvindt.
Uiteindelijk werd een grote populatie van fluorescente Perceval-transgene wormen opgekweekt vertrekkende van een niet-geïntegreerde lijn. Via fluorometrie werd nagegaan of een verandering in fluorescentierespons van Perceval kan worden waargenomen na inhibitie van mitochondriale respiratie door natrium azide. We stelden vast dat de fluorescentierespons zeer snel en drastisch veranderde. Dit bewijst dat het mogelijk is Perceval te gebruiken om veranderingen in de ADP/ATP ratio in ‘realtime’ en op een niet-invasieve manier te detecteren in levende Caenorhabditis elegans wormen.
Bij een ander experiment werd gebruik gemaakt van confocale laserfluorescentiemicroscopie. Er werden foto’s getrokken van individuele Perceval-transgene wormen, zowel voor als na het toedienen van natriumazide. De foto’s werden bewerkt om een beeld te krijgen van weefselspecifieke expressiepatronen en weefselspecifieke verschillen in fluorescentierespons van Perceval. Hierbij werd enerzijds geobserveerd dat Perceval sterk tot expressie komt in de verwachte weefsels en anderzijds dat er tussen verschillende weefsels duidelijke verschillen zijn in fluorescentierespons van Perceval.
Hieruit kon geconcludeerd worden dat met behulp can confocale laserfluorescentiemicroscopie
Perceval gebruikt kan worden voor het rapporteren van weefselspecifieke veranderingen in de
ADP/ATP ratio, een belangrijke indicator van cellulaire energieniveaus.
3

Summary
Our society is heavily influenced by the human aging process. The past decades many scientists became intrigued by the complexity of this biological phenomenon. An important goal of aging research is decreasing the rate of the aging process. This would allow postponing age-related diseases and discomforts and improving the quality of life for the elderly. From an economical point of view, this might contribute to relieving the societal cost and problems related to aging.
Over the past 20 years, some important breakthroughs in aging biology were achieved by molecular genetics. One of them is the discovery that lifespan can be extended substantially by downregulating the activity of a single gene. It was found that in the model organism Caenorhabditis elegans a reduction-of-function mutation in the daf-
2 gene, encoding the insulin/IGF-1 receptor, doubles lifespan. The impairment of the insulin/IGF-1 signaling pathway results in enhanced longevity through different, not fully characterized mechanisms. Interestingly, long lived mutants experience an extended period of vitality rather than a longer decline. An altered energy metabolism might play a considerable role in this.
This study aims at gaining knowledge about the relation between energy metabolism and aging in
Caenorhabditis elegans
. The research involves two direct goals.
1.
It was tried to obtain a more complete picture of the dynamics of ATP, ADP and AMP in aging wild-type worms as well as in long-lived daf-2
mutants. By determining various cellular energy indicators we were able to propose a possible mechanism that links the energy status to the aging process.
2.
In mammalian cells, the genetically encoded fluorescent sensor ‘Perceval’ allows in vivo and real time detection of intracellular ADP/ATP ratios. In this study, we constructed nematodes expressing Perceval
, and we examined its usefulness as an indicator of cellular energy in intact worms.
To get a better view on the dynamics of ATP, ADP and AMP, we started by culturing large synchronous populations of worms (both wild-type and daf-2 mutants). This allowed us to obtain several worm samples at different ages. The worms were homogenized and the adenine nucleotides derivatized into fluorescent etheno( ε )-adenine nucleotides. Next, they were separated using reversephase high-performance liquid chromatography (RP-HPLC). Fluorescence detection of the separated
ε -ATP, ε -ADP and ε -AMP and the use of external standards allowed us to determine precise initial concentrations of ATP, ADP and AMP in the worm samples. Eventually, several indicators of cellular energy levels (AMP/ATP ratio, ADP/ATP ratio and the ‘energy charge’) were calculated.
4

Summary
Our results confirm that, unlike wild-type worms, daf-2 mutants are able to maintain higher cellular concentrations of ATP as well as ADP during aging. We suspect that this is the result of increased
AAK-2 activity due to a decrease in inhibiting insulin/IGF-1 signaling in this insulin/IGF-1 receptor mutant.
AAK-2 is part of a larger enzyme complex, AMPK, which integrates energy levels and directs metabolic and transcriptional changes in favor of reaching energy homeostasis. Stronger AAK-2 activition leads to ATP production, may extend lifespan and increases autophagic activity. The latter might be important to maintain high energy levels that can be invested in maintenance of cellular functions and in lifespan extension.
Cellular ATP concentrations do not provide a complete pictureof the energy state of a cell. Recently, a genetically encoded reporter of ADP/ATP ratios, Perceval, was developed and tested in vitro as wells as in a cell culture. Perceval binds ATP and ADP with different affinities and a consequential differential fluorescence response. It is interesting to examine whether this biosensor is also useful for real-time measurements of energy levels in living
Caenorhabditis elegans
worms.
In a first step, Gateway ® cloning was used to combine the Perceval gene with various promoters that are constitutively expressed in most tissues of
C. elegans
. Subsequently, several molecular cloning steps were performed to create plasmids (expression vectors) that contain Perceval in a conformation suitable for expression in this nematode.
Seven constructs each comprising Perceval and a different promoter were introduced in the gonads of several
C. elegans
hermaphrodites through microinjection. In the progeny only one transformed fluorescent worm was detected. During the following generations mosaic expression and a gradual loss of Perceval was observed. This may be due to the presence of the constructs as large extrachromosomal arrays. Therefore, we tried to integrate the constructs into the genome by UV irradiation without success. Non-integrated lines of worms with strong Perceval expression were selected and further cultured.
Stable integration of Perceval into the genomes is important to facilitate its use and to ameliorate the reliability of measurements. Therefore, worms were also transformed by biolistic transformation. Over half a million worms were bombarded with gold particles, to which linearized expression vectors were coated. When present in the cell the construct could integrate into chromosomes. Although transformants were obtained, no expression of Perceval was observed. Possibly, this is due to silencing as a consequence of integration into heterochromatin, where no transcription occurs.
5

Summary
Eventually, a large population of transgenic fluorescent Perceval worms was obtained from the nonintegrated line. Through fluorometry, it was examined whether the fluorescence response of Perceval changes upon an increasing ADP/ATP ratio, mediated by treatment with the mitochondrial respiration inhibitor sodium azide. The fluorescence ratio appeared to change quickly and drastically. This proves that Perceval can be used to detect changes in the ADP/ATP ratio in real-time and in a non-disruptive way in living
Caenorhabditis elegans
worms.
In another experiment confocal laser scanning microscopy was used. Pictures of individual Percevaltransgenic worms were made, both before and after administration of sodium azide. Software editing of these pictures allowed us to visualize both expression patterns and tissue-specific differences in fluorescence response of Perceval. It was observed that Perceval was expressed sufficiently in the expected tissues and, that there are explicit differences in fluorescence response of Perceval between tissues. Hence, it was concluded that, by means of confocal laser scanning microscopy, Perceval can be used effectively to report tissue-specific changes in the ADP/ATP ratio, an important indicator of cellular energy levels.
6

Introduction
Aging is the most familiar yet least well-understood aspect of human biology (Kirkwood, 2005). It gets a negative connotation because of its final endpoint: death. Other aging-associations like the many age-related diseases and discomforts further aggravate our feeling about getting older .
Since time immemorial mankind challenged the inevitability of both death and aging to achieve eternal life as a young and vivid human being. Nevertheless, the maximum lifespan potential of humans has remained nearly constant over time (
Figure 1
). Contrary, the average lifespan (or life expectancy ), representing the age at which 50% of a given population survive, showed a dramatic increase during the last centuries. Improved sanitation, diet and health care combined with reduced infant mortality by overcoming infectious diseases, were responsible for this extraordinary life expectancy jump. The last forty years European citizens gained 2.5 extra life years per decade due to progress in combating respiratory diseases and cancer in the 1970s, and cardiovascular diseases in more recent years
(European Commission Demography Report
2008). In some countries the life expectancy even advances by more than five hours per day
(Kirkwood, 2008). Together this has led to a compression of morbidity towards the end of the human lifespan (Troen, 2003). Thus, further average lifespan extension will probably come from tackling old-age-related diseases
like cancer, osteoporosis, neurodegeneration, cardio-vascular diseases, atherosclerosis, sarcopenia and type II diabetes.
Figure 1 | Percent survival curve for humans at different times in history with varying environments, nutrition and health care (Troen, 2003; modified by author).
7

Introduction
Figure 2 | Drugs targeting mechanisms that regulate aging could prevent many age-related diseases
(Curtis, 2005).
As one can imagine, prevention of a disease is inherently more valuable than curing the symptoms.
Having this in mind, Curtis et al. (2005) suggested a complementary approach whereby one develops therapies to treat and prevent diseases by targeting the major risk factor: aging ( Figure 2
). By slowing it down people should remain physically younger for a longer time. As a consequence one should be able, and ethically obliged to society, to work longer. This gives rise to a larger working class and could solve the social-economical issues about retirement payments. The common fear for a catastrophic overpopulation due to life extension is exacerbated by the lack of detailed demographic projections for radical life extension. Recently, however, Gavrilov and Gavrilova (2010) showed that even in case of a dramatic life extension, population growth might not lead to overpopulation. This is due to decreasing fertility rates, which are in the EU-27 (Eurostat) and other industrialized countries
(The World Factbook, CIA) lower than the replacement level, or 2.1 live births per woman.
Pharmaceutical interventions have the objective of decreasing the rate of aging rather than keeping us longer alive as cripple
Table 1 | Common characteristics of aging in mammals
(Troen, 2003).
and needy elderly. Before we can rationally evaluate the potential impact of them, we will need to understand the primary causes of aging
(Vijg & Campisi, 2008). Of key importance one should first know what aging exactly is.
Many definitions circulate in the biogerontology world. A very well worked out definition is proposed by Arking (1998): “
Aging is the time-dependent series of cumulative, progressive, intrinsic, and deleterious functional and structural changes that usually begin to manifest themselves at reproductive maturity and eventually culminate in death
.” Troen (2003) assembled evidence supporting at least five common characteristics of aging in mammals ( Table 1 ). For a long time it was believed that aging is a haphazard process, driven solely by entropy (Kenyon, 2005). This is in high contrast with many other biological processes, such as cell differentiation and development, which increase complexity (Kenyon, 2001).
8

Introduction
Figure 3 | Balancing somatic maintenance with growth and reproduction may determine lifespan. According to the ‘disposable soma theory’, organisms must compromise between energy allocation to growth and reproduction or somatic maintenance and repair (Vijg & Campisi, 2008).
As every biological process, aging is subjected to evolution. Most scientist now accept that aging results from the greater weight placed by natural selection on early survival and reproduction than on vigour at later ages. So, as Medawar stated first in 1952, gene variants promoting early growth and reproduction are favoured by natural selection in harsh environments. If conditions are less hazardous, survival increases and gene variants that promote somatic maintenance can propagate in the population (Vijg & Campisi, 2008). Hence, species specific lifespan is determined by a trade-off between somatic maintenance and early growth or reproduction
(
Figure 3
).
Until two decades ago, extensive human lifespan extension was still a fantasy. Then, with the arrival of molecular genetics, some breakthrough discoveries were achieved. Several single gene mutations, of which the first were age-1 (Friedman & Johnson, 1988) and daf-2 (Kenyon et al., 1993) could extend the three-week lifespan in the nematode Caenorhabditis elegans by 1-2 fold and increase its resistance to many diseases (Curtis et al., 2005). Even a tenfold lifespan extension was described in
PI3K-null mutants of this nematode (Ayyadevara et al., 2007). In humans this is the equivalent of an
800 years old person. Interestingly, these findings revealed that longevity is under genetic control
(Antebi, 2007). At present, hundreds of mutant genes can increase longevity in model organisms, including nematodes, yeast ( Saccharomyces cerevisiae ), fruit flies ( Drosophila melanogaster ) and mice ( Mus musculus ) (Vijg & Campisi, 2008). Most of them act in evolutionary conserved pathways that regulate growth, energy metabolism , nutrient sensing and/or reproduction (Kenyon, 2005).
Lifespan is influenced by genes, environment and stochastic factors (Antebi, 2007). These parameters are responsible for the highly plastic
animal lifespan which is amenable to change in response to environmental signals as nutrients and stress (Kenyon, 2005). The appreciation that lifespan is plastic and under negative influence by genes that favor growth or procreation fuels hope of finding small molecules that target the pathways affected by pro-longevity mutations or dietary restriction 1 (Vijg &
Campisi, 2008).
Many mutations affecting longevity pathways delay age-related disease, and the molecular analysis of these pathways is leading to a mechanistic understanding of how aging and disease susceptibility are linked (Kenyon, 2005). Importantly, long lived mutants experience an extended period of vitality rather than a longer decline (Curtis et al., 2006).
1
Dietary restriction : Underfeeding without malnutrition resulting in extension of lifespan in many species.
9

Introduction
These discoveries resulted in a real paradigm shift : aging was no longer seen as an inexorable process of decay, but rather, like other biological processes, subjected to regulation
by evolutionary conserved pathways (Guarente & Kenyon, 2000). However, the idea of a programmed aging mechanism seems rather odd. It was found that genes affect longevity by adjusting hundreds of mechanisms for maintenance and repair rather than by changing the timing of a mechanism for self-destruction
(Murphy et al., 2003; Lee et al., 2003). In most cases the lifespan extension occurs when activity of a gene product is diminished. Hence such genes appear to shorten lifespan, either by increasing somatic damage, or by decreasing somatic maintenance and repair (Vijg & Campisi, 2008).
Altogether, genetics have revealed us aging pathways and enabled lifespan extension in model organisms . This can inspire scientists to seriously try to slow down human aging (Kenyon, 2005).
However, one has to bear in mind that organismal complexity will possibly limit the extent of the prolonged lifespan by manipulation of metabolic pathways. Also, there could be other layers of control or pathways that are yet to be discovered in complex animals (Vijg & Campisi, 2008). Unfortunately, attempts to understand the causes of aging are limited by the complexity of the problem: aging changes are manifested from the molecular to the organismic level; precisely defined, easily measurable “biomarkers 2 ” are lacking; environmental factors affect experimental observations; and secondary effects complicate elucidation of primary mechanisms (Troen, 2003).
2
Biomarkers : Substance used as indicator of a biological state.
10

Introduction
The long term goal of fundamental research on aging, amongst scientists called “biogerontology”, does not halt at exposing the phenomenon of aging, but rather aims at creating opportunities to interfere with this process. As such one hopes to be able to age at a slower rate, and as a consequence live longer in a healthy way. Because of ethical and practical reasons humans cannot be readily used as research subjects, except for comparative genotyping. The complex physiology of other vertebrates often delivers non-interpretable results due to secondary effects. Smaller and simpler invertebrate model organisms are more comfortable for elucidating primary mechanisms of aging. The wide divergence between humans and these species is a problem that can be partially overcome by the strong evolutionary conservation of the pathways that regulate aging (Kenyon, 2001).
Within the group of invertebrates, nematodes are quite simple multicellular eukaryotic organisms.
Nevertheless, they do show an extended differentiation in organs such as intestine, muscle, gonads and a neuronal system. In the 70’s several nematodes belonging to the family of Rhabditidae - Turbatrix aceti (Kisiel and Zuckerman, 1974), Caenorhabditis briggsae (Zuckerman et al., 1971) and
Caenorhabditis elegans (Klass, 1977) - were used as first organisms to conduct research on aging.
Although Sydney Brenner already proposed
C. elegans
as an excellent model organism in 1963, the real onset of its use merely started in 1974 after Brenner’s milestone paper ‘Genetics of
Caenorhabditis elegans
’ (Brenner, 1974). During the last three decades the vision of this pioneer appeared to be quite right. Due to some great assets, C. elegans has undoubtedly led the way to fundamental insights into the biology of aging.
A first asset is that C. elegans cultures are safe, cheap and easy manageable. It is quite convenient to perform several short aging experiments in a short time on this tiny nematode because of several important characteristics. They show a stereotypical development, have a short lifespan and generation time, and produce a huge number of offspring. Large and synchronized cohorts are easily attained and maintained using standardized methods (Braeckman et al., 2002) (see 4.1.5, Set-up synchronized aging C. elegans cohorts ).
Secondly,
C. elegans
is a nearly ideal organism for genetic analyses since selfing hermaphrodites lead to laboratory clones of a uniform genome. New alleles can be transferred by cross-fertilization by males being homozygous for this gene (Brenner, 1974), or by introducing a recombinant gene in the hermaphrodite through microinjection or gene bombardement (Evans, 2006).
This way, thousands of strains are developed and are easily obtained from the
C. elegans
Genetics
Center ( http://www.cbs.umn.edu/CGC/ ). C. elegans worms can be cryogenically preserved in liquid nitrogen (-195.8°C) indefinitely to shrink the costs and work associated with maintenance of stock cultures (Brenner, 1974).
11

Introduction
Thirdly, C. elegans is transparent in all developmental stages, facilitating research on in vivo localization of proteins and other markers of interest using fluorescence and luminescence techniques.
In addition, a large number of GFP reporter-gene strains have been generated, making it possible to visualize anatomy, developmental processes and many signaling pathways in real-time (Gruber et al.,
2009). This resulted in the manifestation of the largely invariant cellular genealogy.
Fourthly, the
C. elegans
research community has generated an extensive toolkit
for the genetic manipulation and analysis of this organism (http://www.wormbook.org/toc_wormmethods.html).
Double stranded DNA (dsDNA) interference can be accomplished relatively easily, i.e. by feeding worms with bacteria expressing dsRNA for the gene of interest, thereby causing a loss of function phenotype (Timmons & Fire, 1998). In 2006 this discovery was awarded with the Nobel Prize in
Physiology or Medicine.
Fifthly, as a consequence of decades of research on C. elegans an enormous amount of information
is available. For the moment, already more than 22.000 peer-reviewed articles are published. Furthermore, two books summarized a large part of C. elegans research until the nineties
(Wood, 1988; Riddle et al., 1997). In 1998 the entire 97-megabase
C. elegans genome was sequenced by the Caenorhabditis elegans Sequencing Consortium. The current release of the C. elegans genome
(http://www.wormbase.org; release WS225) predicts 20.431 genes and 4599 alternative splice forms.
The 2002 Nobel Prize in Physiology and Medicine winners Brenner (Brenner, 1974), Sulston (Sulston,
1976; Sulston & Horvitz; 1977; Sulston et al., 1983) and Horvitz (Sulston & Horvitz, 1977) analyzed the cell lineage and neuroanatomy in high detail, which lead to the discovery of all 8.400 neuronal connections (Varshney et al., 2011). A significant breakthrough was reached in 2000; an enormous open-sourced web-based database, called Wormbase (http://www.wormbase.org), was launched and includes a gigantic amount of information. Two other databases, Wormbook
(http://www.wormbook.org) and WormAtlas (http://www.wormatlas.org), provide an overview of biology and structural anatomy of C. elegans , respectively. The former also contains a section
WormMethods, which is a collection of protocols for nematode researchers.
The major advantage, however, of using C. elegans as a study object in aging research is the availability of a substantial amount of different long-lived
(and short-lived) mutants
(for an extensive list of knock-out genes and interventions that influence the aging phenotype, see http://uwaging.org/genesdb/index.php). By examinating the biochemistry and physiology of long-lived worms one can discover to what extent changes in these parameters result in lifespan extension.
12

Introduction
2.2.1
Ecology of C. elegans
Émile Maupas was the first one to describe Caenorhabditis elegans in 1900 after isolating it from rich humus in the surroundings of Algiers. It was described since as a terrestrial free-living soil nematode
(roundworm) filter feeding on bacteria through pharyngeal pumping. More than a century later, however, Felix and Braendle (2010) remarked that
C. elegans
is actually a colonizer of various microbe-rich habitats, particularly decaying plant and fruit material, and not a typical soil nematode.
Further on, they noticed that its cosmopolitan presence in temperate environments has been achieved by using, as a dauer larva (see 2.2.3.4, Dauer ), carrier arthropods and gastropods or even frugivore vertebrates for long-distance dispersal. Locally,
C. elegans
dauer larvae disperse actively between food patches. The laboratory habitat of C. elegans comprises Petri dishes with an agar surface seeded with a lawn of
E. coli
cells or liquid cultures containing the same bacteria as food source.
2.2.2
Morphology and Anatomy of C. elegans
Caenorhabditis elegans is a tiny nematode measuring 1.2-1.5 mm in length and being 50 µm wide.
There are two sexes: a self-fertilizing hermaphrodite
(5AA; XX) and a male
(5AA; XO). Males are far less common (ca. 0.1-0.2%; Ward & Carrel, 1979; Hodgkin & Doniach, 1997) and arise by spontaneous meiotic non-disjunction in hermaphrodites. However a higher frequency can easily be reached by mating of a male and hermaphrodite delivering up to 50% males in the next generation.
C. elegans
is a eutelic
organism, meaning that a fixed number of cells is reached and maintained at maturity. Somatic development of C. elegans hermaphrodites generates 1090 cells. An invariant number of 131 specific cells, of which 105 are neurons, undergo apoptosis on fixed times in development, resulting in a body plan composed of 959 somatic nuclei (Sulston & Horvitz 1977;
Kimble and Hirsh, 1979; Sulston et al., 1983). Over its lifetime a wild-type hermaphrodite generates additionally about 2000 germ cells whereby more than half of them die by apoptosis (Gumienny et al.,
1999). Males on the other hand consist of 1031 somatic nuclei .
C. elegans displays a large repertoire of behavior including locomotion, foraging, feeding, defecation, egg laying, dauer larva formation, sensory responses to touch, smell, taste and temperature as well as some complex behaviors like male mating, social behavior and learning and memory (Rankin, 2002; de Bono, 2003). To fulfill these functions its tissues are far more differentiated than what would be expected of the seemingly simple body plan. The body shape is, typically for nematodes, cylindrical, unsegmented and narrowed at the ends, especially at the tail. The general body plan can be viewed as two concentric tubes separated from each other by the pseudocoelomic cavity
(Wood, 1988), which is partially filled with the gonad in adults ( Figure 4 ). In an adult hermaphrodite the pseudocoelom contains three pairs of non-migratory coelomocytes taking position at the somatic musculature of the head, vulva and tail. Owing to their ability of continuously endocytosing and accumulating a diversity
13

Introduction of macromolecules from the body cavity fluid, coelomocytes have been suggested to serve an immune, scavenging and hepatic function (Fares & Grant, 2002; Yanowitz & Fire, 2005).
The outer tube , or body wall, consists of a cuticle and hypodermis, muscles, neurons and excretory system. The latter regulates the internal hydrostatic pressure in the pseudocoelom via the excretory pore at the ventral side of the head. The cuticle, surrounding the whole body surface, is permeable for fluids, provides protection against environmental factors, maintains body shape, and by acting as an external skeleton it permits motility. At each larval transition (there are four), the cuticle is shed and an entirely new one is generated. It is secreted by underlaying specialized epithelial cells. The other category of the epithelial system is the hypodermis, which is, comparable to human skin, crucial for body morphogenesis, muscle development and cuticle structure and function. As in human adipose tissue, the hypodermis acts also in energy storage.
Figure 4 | Nematode body plan with cross section from head to tail
(WormAtlas).
A : Posterior body region. Body wall
(outer tube) is separated from the inner tube (alimentary system, gonad) by a pseudocoelom. Alae are lateral longitudinally oriented ridges, generated by underlaying seam cells, that interrupt circumferential annuli.
B : Section through anterior head. The body wall muscles and nerves are inbedded into a hypodermis, together composing the outer tube.
C : Section through middle of head.
D : Section through posterior head.
E : Section through posterior body.
DNC = dorsal nerve cord, VNC = ventral nerve cord.
F : Section through tail, rectum area.
A total of 95 obliquely striated body wall muscles are arranged in bands of two dorsal and two ventral quadrants along the whole body axis (White, 1988). These dorsal and ventral muscles fire alternating all-or-none action potentials, underlying the sinusoidal locomotion of
C. elegans
(Gao & Zhen, 2011).
A different group, nonstriated muscles, contains 20 pharyngeal muscles, 2 stomato-intestinal muscles,
1 anal sphincter muscle, 1 anal depressor muscle, 8 vulval muscles, 8 uterine muscles and a contractile gonadal sheath.
14

Introduction
The hermaphrodite neural network consists of 302 neurons. The major portion is present in the head around the pharynx. However, a minor share of them is an independent pharyngeal nervous system (20 neurons). The latter is connected, through a pair of interneurons, to the nerve ring 3 of the central nervous system (282 neurons). The cell bodies of most neurons are clustered in ganglia in the head or tail. The few axons are bundled in nerves running longitudinally and circumferential throughout the body. The neurons communicate through approximately 6400 chemical synapses, 900 gap junctions, and 1500 neuromuscular junctions. Among individual animals, the location of chemical synapses is about 75% reproducible (Durbin, 1987). Males possess a nervous system containing 383 neurons of which 89 being sex-specific. The latter are mostly located in the posterior (Sulston & Horvitz, 1977;
Sulston et al., 1980) and many have specific roles in male mating behavior (Emmons & Sternberg,
1997). C. elegans has 24 sensillar organs and various isolated sensory neurons to accomplish perception of environmental cues, like temperature, pH, light, oxygen levels, osmolarity, chemicals and mechanic stimuli. These enable the worm to conduct thermotaxis, aerotaxis and chemotaxis to favorable surrounding, and to escape from harmful and noxious stimuli (Bargmann, 2006; Bergamasco
& Bazzicalupo, 2006). Most sensory neurons have a soma positioned close to the nerve ring. The principal sensory organs, however, are the amphids, which is a pair of lateral sensilla in the head. In the tail similar but smaller sensilla are located, the phasmids.
The inner tube is basically a simple digestive system. Bacterial suspensions are taken up into the mouth by pharyngeal pumping 4 . The ingested food passes through the lumen of the pharynx, which consists of two enlargements, separated by an isthmus or narrowing (peristaltic movement from first to second enlargement). The anterior one is the metacorpus (preceded by a more lean procorpus) and is responsible for the sucking and accumulation part. The posterior expansion is called the bulbus; it presses the accumulated particles through the pharyngeal valve into the intestine (Avery & Horvitz,
1989). After passing the rectal valve and rectum, the unabsorbed content is excreted via the anus, which is located at the ventral side just proximal to the tail whip.
The reproductive system of
C. elegans
(
Figure 5
), located at the pseudocoelom
, comprises three components: the somatic gonad, the germ line and the egg-laying apparatus. In hermaphrodites the gonad consists of two bilaterally symmetric, U-shaped gonad arms. It is built up by five tissues: the distal tip cell (DTC), gonadal sheath, spermatheca, spermatheca-uterine valve, and the uterus. The
DTC
is a large somatic cell located at the tip of both gonad arms and forms a cap over the distal germ stem-cells . Germ cells leave this mitotic zone and migrate proximally while undergoing meiosis.
3
Nerve ring : Circumferential nerve bundle located in the head; together with surrounding ganglia it is also called the worms’ “brain”.
4
Pharyngeal pumping : Uptake of food particles by first sucking up of suspension, then accumulating particles, while fluids are spitted out. Finally, peristaltic movements push the food particles towards the intestine.
15

Introduction
A layer of five pairs of thin gonadal sheath cells
covers the germ-line along each arm and are important, together with the DTC, in germline development (Kimble & White, 1981;
McCarter et al., 1997; Hall et al., 1999).
Hermaphrodites are protandric and syngonic; during the fourth larval stage they produce approximately 150 spermatozoa in both ovotestes (distal gonads) before all of their oocytes are generated (protandric) in the same gonads (syngonic). They are stacked in the spermatheca (receptaculum seminalis) which interconnects the oviduct (proximal gonad) to the uterus. After the nematode transforms into a young adult, oogonia in the ovotestes undergo a first meiotic division, while moving up to the oviduct. Upon arrival in the spermatheca, a spermatozoan invades an oocyte I (in metaphase I) resulting in the continuing of
Figure 5 | Schematic overview of C. elegans’ reproductive system.
meiosis and formation of an oocyte II, and ultimately fusion with the spermatozoan to a zygote. It migrates via the spermatheca-uterus valve to the uterus . This egg chamber links both gonad arms and provides an environment for the embryonic development of the diploid zygotes. When the approximately 30-cell stage is reached, the egg is expelled to the outside through the vulva , which protrudes ventrally at the midbody. A self-fertilizing hermaphrodite generates in ideal circumstances about 300 progeny.
Adult
C. elegans males can easily be distinguished from hermaphrodites by their slim body (despite the higher number of somatic nuclei), a clear ventral gonad and a distinctive tail which bears the copulatory apparatus. The somatic gonad is distally connected to the germ line in the testis
, which generates only sperm. The seminal vesicle is the sperm collector and opens into the vas deferens .
Together these gonad components form a single J-shaped arm that connects proximally via the seminal vesicle and vas deferens to the cloaca . This chamber links the genital (vas deferens) and alimentary tract (proctodeum) to the exterior via the anus. Housed inside the cloaca there’s a pair of copulatory spicules . Upon copulation these cuticular rods probe for the vulval opening of the hermaphrodite and insert fully. As such the cloacal opening is closely opposed to the vulva and ejaculation takes place
(Garcia et al., 2001). A mating male can increase the number of offspring of a hermaphrodite from 300 to 1200-1400, of which up to half is male. Successful mating can be achieved by males for six days after their last larval molt, resulting in approximately 3000 offspring (Hodgkin, 1988).
16

Introduction
2.2.3
Life cycle of C. elegans
2.2.3.1
Introduction
Remarkably, three of the most frequently used lab model organisms, Saccharomyces cerevisiae ,
Drosophila melanogaster
and
Caenorhabditis elegans
, share the same habitat: rotting fruit or plant material. Consequently, they also have a rapid lifecycle in common, which is required to flourish in this ecological context. In
C. elegans
, similar to other nematodes, this lifecycle is comprised of the embryonic stage, four larval stages (L1-L4) and adulthood, all separated by a molt ( Figure 6 ). The main body plan is established at the end of embryogenesis and does not change in later stages.
Figure 6 | Life cycle of C. elegans at 25 °C.
17

Introduction
2.2.3.2
Larval stages
Upon hatching, L1 larvae are triggered by feeding to start the post-embryonic developmental program
(Ambros, 2000). If no food is present, the larvae arrest their further development until food becomes available (Slack & Ruvkun, 1997). In such fasting conditions they are able to survive up to 6-10 days of fasting (Johnson et al., 1984). In food-rich conditions the L1 larvae molt, after half a day (at 22-
25°C), to L2 larvae.
L2 larvae subsequently molt to the L3 stage during which the vulva starts to develop ( Figure 5 ). Male germ line cells undergo meiosis and differentiate into sperm; this continues for their entire life (Klass et al., 1976). In hermaphrodites, meiosis of the germ line occurs at the L3/L4 molt and the cells differentiate into mature sperm. However, the production stops half a day later at the L4/adult molt, while the remaining germ line cells become, upon meiosis, exclusively oocytes. In order to manage the whole process of generating gametes, gonadogenesis proceeds strongly and eventually comes to an end in this last larval stage
, L4
.
2.2.3.3
Aging adult
A newly matured hermaphrodite lays its first eggs approximately at 45-50 hours after hatching at
22°C-25°C. By this it completes its three-day reproductive life cycle (Byerly et al., 1976; Lewis &
Fleming, 1995). Four days later the adult hermaphrodite quits the fertile period by terminating its oocyte production. Nevertheless, the mature adult retires for an additional 10-15 days, hence an adult lifespan of 14-19 days is reached. Males potentially live twice as long as hermaphrodites, however homosexual interaction greatly reduces their lifespan (Gems & Riddle, 2000).
Aging worms show some typically conserved aging phenotypes: decreased fitness, innate immunity and mitochondrial function, genome instability, macromolecular aggregates and sarcopenia 5 (Vijg &
Campisi, 2008). Furthermore, progressive decline of defecation (Bolanowski et al., 1981), pharyngeal pumping and body movement are characteristic for aging C. elegans (Huang et al., 2004). There is also an age-dependent decline in efficiency of autophagic degradation (Salminen & Kaarniranta, 2009) and in protein turnover (Johnson & McCaffrey, 1985; Prasanna & Lane, 1979), resulting in an accumulation of denaturated and carbonylated proteins. In addition, intestinal autofluorescence accumulates with age (Klass, 1977). This is thought to be due to lipofuscin, a pigment that accumulates progressively in aging tissues (in a variety of organisms) as a result of the oxidative degradation and autophagocytosis of cellular components (Garigan et al., 2002).
5
Sarcopenia : Deteriorated muscle integrity because of derangement of muscle fibers and overt changes in nuclear morphology.
18

Introduction
Endomitotic DNA synthesis increases in hermaphrodite germ cells (Golden et al., 2007), as well as the number of necrotic cavities and vitellogenin 6 droplets (Garigan et al., 2002). A decline in rate of mitochondrial oxidative phosphorylation (Lesnefsky & Hoppel, 2006) and abundance and bioenergetic competence of mitochondria (Brys et al., 2010) is also noticed. Of high interest for this master dissertation, AMP/ATP ratios are shown to increase in aging C. elegans (Apfeld et al., 2004).
Remarkably, the
C. elegans
nervous system seems to be resilient to aging; at least structure and reporter expression do not change, although function has yet to be examined (Antebi, 2007).
At the end of their life, worms probably die of pharyngeal and intestinal (anterior and posterior region) bacterial infection, while antibiotics extend life (Garigan et al., 2002). It is plausible that a decline in enteric muscle function leads to impairment of expelling bacteria, thereby resulting in life-threatening infection (Antebi, 2007). The importance of stochastic events in aging is demonstrated by the extensive variability in age-related degeneration and mortality in genetically identical populations of worms (Herndon et al., 2002).
2.2.3.4
Dauer
Interestingly, there is one extra important developmental stage: the dauer larva . This structurally different larva was given its name by Fuchs in 1915. A late L1 or an early L2 larva can transform into a morphologically distinct L2 larva ( L2d) , upon being triggered by the absence of food, a high temperature, drought, or the presence of a dauer pheromone (which indicates a high population density). A high ratio of pheromone compared to food availability promotes dauer entry, the adverse results in dauer exit (Golden & Riddle, 1982). A high temperature further augments the dauer frequency (Golden & Riddle, 1984a). If the environment continues to be adverse, the L2d larva molts into a very thin facultative dauer larva. In this state, the buccal cavity and anus are obstructed such that feeding and defecation are arrested, the cuticle is thickened to withstand stresses, locomotion is notably reduced, and oxidative damage is tolerated (Honda & Honda, 1999). At dauer entry they store mainly fat in the intestine and hypodermis, which they metabolize as dauers (Albert & Riddle, 1988).
It gives them an easy recognizable lean and dark appearance. An apparent metabolic shift towards anaerobic fermentation pathways is observed, while anabolic pathways are down regulated. Energy consumption is rather employed to enhance cellular maintenance and detoxification processes (Burnell et al., 2005). When more favorable conditions are encountered, the worms start feeding and after about ten hours, they molt to the L4 stage. Hence the dauer larva is an alternative L3 larva. Interestingly, unlike the usual two-to-three weeks life cycle, the dauer can persist up to four months in this fasting stage (ultimately dies by fasting), while the dauer duration does not affect the postdauer life span.
Hence the dauer is a non-aging state of Caenorhabditis elegans (Klass & Hirsh, 1976).
6
Vitellogenin : An egg yolk precursor glycolipoprotein, involved in lipid transport, in young nematodes mobilized from intestine to gonad where it is incorporated in embryos.
19

Introduction
In dauers the environmental cues are integrated via more than 30 dauer formation ( daf ) genes (Albert
& Riddle, 1988; Riddle & Albert, 1997). Two categories exist for mutations in these genes. Dauer constitutive mutants (Daf-c) form dauers under normal growth conditions. Several of them develop normally at 15 °C (permissive temperature), while entering the dauer state at 25 °C (restrictive temperature). Dauer defective mutants (Daf-d) cannot produce dauers under starvation (Riddle &
Albert, 1997).
2.2.4
Insulin/IGF-1 pathway in C. elegans
2.2.4.1
Introduction
The daf genes regulate dauer formation and are part of the Insulin/IGF-1, the TGFβ and the cyclic
GMP signal transduction pathways (Burnell et al., 2005). Intriguingly, the insulin/insulin growth factor-like 1 (IGF-1) signaling (IIS) pathway also regulates lifespan (and stress resistance) in C. elegans
.
In the nineties it was discovered that aging is subjected to genetic control. So far, the best characterized pathway is the IIS pathway. Reduction-of-function mutations of age-1
(Klass, 1983;
Friedman & Johnson, 1988) and daf-2 (Kenyon et al., 1993) in C. elegans could extend longevity as much as 100 percent. This lifespan determination seemed to involve neuroendrocine signaling because it was found that age-1 encodes an ortholog of the p110 catalytic subunit of a mammalian phosphatidylinositol-3-OH (PI3) kinase family member (Morris et al., 1996). Another major breakthrough was the discovery that daf-2 is actually an insuline receptor-like gene (Kimura et al.,
1997). Both these genes act in the same pathway (Dorman et al., 1995), with DAF-2 more upstream in the signaling cascade. Epistasis experiments showed that activity of DAF-16, a hepatocyte nuclear factor 3 (HNF-3)/forkhead box O (FOXO) family transcription factor (Lin et al., 1997; Ogg et al.,
1997), is required to achieve life extension in these mutated forms (Kenyon et al., 1993). These findings proved that aging is subject to hormonal regulation (Kenyon, 2005).
20

Introduction
2.2.4.2
Signaling cascade
Upstream of this IIS pathway and at the very beginning of the neuroendocrine system, a high food availability-to-dauer pheromone ratio stimulates a sensory neuron ( Figure 7 ). Binding of acetylcholine to a muscarinic receptor transmits the signal to an insulin-like peptide (ILP) secreting neuron
(Tissenbaum et al., 2000). Impulse conduction and an action potential at the synaps open voltagegated Ca 2+ channels. The consequential rise in intracellular Ca 2+ levels activates UNC-31, a CAPS 7 ortholog. Together with UNC-64, a syntaxin 8 ortholog, they trigger secretion of ILPs (Ailion et al.,
1999).
The
C. elegans
genome encodes 40 putative ILPs ( ins-1-39
and daf-28
) (http://www.wormbase.org; release WS225) (Pierce et al., 2001; Li et al., 2003). They can bind the sole transmembrane insulin/IGF-1-like receptor DAF-2 (Kimura et al., 1997). Some of these peptides antagonize, others activate DAF-2 receptor tyrosin kinase (Pierce et al., 2001). Upon activation, DAF-2 recruits and activates
AAP-1
(Wolkow et al., 2002), a p55-like regulatory subunit of phosphatidyl-inositol-3kinase (PI3K). Consequently, AGE-1 (Morris et al., 1996), a p110 catalytic subunit of PI3K, catalyzes the conversion of PIP
2
(phosphatidyl-inositol-(4,5)-bisphosphate) to PIP
3
(phosphatidyl-inositol-
(3,4,5)-trisphosphate). This reaction is counteracted by the PIP
3
-phosphatase DAF-18 , a human PTEN homolog (Ogg & Ruvkun, 1998). PDK-1 , the Akt/PKB homolog (Paradis et al., 1999), transduces a phosphorylation signal from PIP3 to SGK-1
9 (Hertweck et al., 2004), AKT-1 and AKT-2 (Paradis &
Ruvkun, 1998). The latter are both Akt-PKB serine/threonine homolog’s and form together with SGK-
1 a kinase complex that phosphorylates three conserved regulatory sites and thereby inactivates DAF-
16 , a FOXO transcription factor (Lin et al., 1997; Ogg et al., 1997). Inactivation takes place through
14-3-3 protein-mediated nuclear export and cytoplasmic retention (Brunet et al., 1999; Brunet et al.,
2002). Therefore, the IIS pathway negatively regulates DAF-16 activity by modifying its intracellular localization. Consequently, dampening of the IIS pathway, either by environmental factors or mutation, localizes DAF-16 in the nucleus, where it performs a role as an activator of an enhanced life maintenance program
to increase lifespan (Henderson & Johnson, 2001; Lin et al., 2001).
7
CAPS : Ca
2+
-dependent activator protein for secretion, a mammalian neuronal protein.
8
Syntaxin : Mammalian neuronal protein of the core synaptic vesicle fusion machinery involved in synaptic transmission.
9
SGK-1 : A serum and glucocorticoid inducible kinase homolog.
21

Introduction
Figure 7 | Insulin/IGF-1 signaling cascade in C. elegans (Braeckman et al., 2001).
22

Introduction
2.2.4.3
DAF-16
DAF-16 is a master regulator, meaning it is a key component of an extensive network integrating many upstream signals and controlling a wide variety of downstream genes with diverse functions.
DAF-16 has 103 putative direct targets (promotor regions) (Oh et al., 2006), whereas insulin/IGF-1 signaling was predicted to regulate at least 10 % of all C. elegans genes (McElwee et al., 2004).
Nuclear DAF-16 on its own downregulates lifespan shortening genes (growth and reproduction genes) while upregulating certain antimicrobial, detoxification and metabolism genes (Murphy et al.,
2003). Unexpectedly, its nuclear localization is not sufficient for extending lifespan or dauer formation
(Lin et al., 2001). Oxidative stress, UV light and pathogenic bacteria activate
SMK-1
10 , which colocalizes with and regulates DAF-16 to increase lifespan by inducing gene expression of important antioxidant enzymes, antimicrobial peptides, small heat shock proteins (HSPs) (Wolff et al., 2006), protein and energy metabolism components (Murphy et al., 2003) and the indispensable positive regulators of IIS-mediated longevity OLD-1 (Murakami & Johnson, 2001) and SCL-1 (Ookuma et al.,
2003; Patterson, 2003). Parallel to the IIS pathway a JNK-1/SIR-2.1/14-3-3 pathway is thought to act on lifespan through DAF-16 (Oh. et al., 2005). In response to heat, oxidative and UV stress JKK-1 activates JNK-1 which phosphorylates cytosolic DAF-16 at a non-AKT phosphorylation site, thereby translocating it back to the nucleus. Consequently, SIR-2.1
, a NAD-dependent protein deacetylase involved in transcriptional silencing, acts together with two 14-3-3 proteins , PAR-5 and FTT-2
,
on this extra phosphorylated DAF-16 to promote expression of a subset of DAF-16 genes involved in heat stress response, immunity and detoxification metabolism (Berdichevsky et al., 2006; Wang et al.,
2006). DAF-16 can also be phosphorylated on another different residue by CST-1 , an ortholog of
MST-1 and involved in growth control, resulting in an oxidative stress response and slower aging
(Lehtinen et al., 2006). The same outcome is also induced by BAR-1 , a β -catenin homolog, which acts as a cofactor of DAF-16 (Essers et al., 2005). Recently, a new conserved pathway, the
EAK
11 pathway, was found to act in parallel to the Akt/Pkb pathway. EAK proteins regulate activity of nuclear DAF-16 without influencing its intracellular localization. EAK-2 and EAK-7 influence
C. elegans lifespan and are conserved in mammals (Williams et al., 2010). The master regulator HSF-1
12 mediates heat resistance by inducing various heat shock proteins. These function as chaperones to protect cells and clear misfolded proteins. HSF-1 is, through DAF-16, partially responsible for the longevity phenotype of diminished IIS (Garigan et al., 2002; Hsu et al., 2003; Morley & Morimoto,
2004; Singh & Aballay, 2006). Finally, besides phosphorylation, DAF-16 is also modified and
10
SMK-1 : Supressor of MEK null.
11
EAK : Enhancer-of-
Akt-1.
12
HSF-1 : Heat shock factor 1.
23

Introduction destabilized by ubiquitination by the evolutionarily conserved E3 ubiquitin ligase, RLE-1
13 (Li et al.,
2007).
Recently, however, Kwon et al.
(2010) found in addition to two previously found DAF-16 isoforms,
Daf-16a and Daf-16b (Lin et al.,
1997; Lin et al., 2001; Ogg et al.,
1997), a third DAF-16 isoform
(Daf-16d/f) in C. elegans . Both Daf-
16d/f and Daf-16a are mainly involved in regulating longevity and stress response, while Daf-16b primarily functions in development.
Similar to the four mammalian
FOXO transcription factors, the
DAF-16 isoforms show distinct tissue-specific abundance, different preference for upstream kinases and specific, cooperative and redundant regulation of target genes ( Figure 8 )
Figure 8 | Model depicting a network between DAF-16 isoforms and the upstream kinases modulating the IIS-mediated processes.
The thickness of lines represents the strength of regulation. The dotted line represents minor or no regulation (Kwon et al., 2010).
(Kwon et al., 2010).
2.2.4.4
Spatio-temporal regulation of aging C. elegans
Lifespan in
C. elegans
is mainly modulated by sensory neurons that integrate environmental inputs
(Wolkow et al., 2000). DAF-16 activity in multiple tissues is required to coordinate lifespan (Iser et al., 2007). This master regulator induces production of intercellular insulin-like signals that act on target tissues on a DAF-16 dependent mechanism, so having an autocrine and paracrine effect. This positive feedback mechanism renders it possible for the animals’ system to rapidly and effectively adjust to internal or environmental perturbations (Libina et al., 2003).
C. elegans
aging can also be influenced by gonadal signals. Removal of germline precursor cells extends lifespan with 60 % in a DAF-16 dependent way (Arantes-Oliveira et al., 2002; Hsin and
Kenyon, 1999). Remarkably, further destroyal of the remaining somatic gonadal cells diminishes the lifespan extension. This indicates a contravening effect of a life-extending signal from the somatic gonad towards a life-shortening signal form germ cells.
13
RLE-1 : Regulation of longevity by E3.
24

Introduction
The former signal is a lipophilic hormone synthesized by DAF-9, the cytochrome P-450 homolog. It acts on the intestine where it provokes a response in which KRI-1, an intestinal ankyrin repeat 14 protein, promotes DAF-16 nuclear localization, which in turn regulates lifespan (Berman & Kenyon,
2006).
The IIS pathway is active in both larval and adult worms and is involved in stress resistance, reproduction, longevity and dauer formation. Early in development (L1 and early L2) gonadal signals and environmental factors influence IIS to regulate the decision of dauer formation. The resulting larval DAF-16 activity is irrelevant in case of influencing adult lifespan, which is only increased in case of reduced Insulin/IGF-1 signaling during adulthood (Dillin et al., 2002a).
2.2.4.5
daf-2 mutation
A certain weak reduction-of-function mutation in daf-2(e1370)
15 lowers the level of this insulin-like receptor and extends lifespan twofold
in wild-type
C. elegans
(Kenyon et al., 1993). Apparently low levels of endocrine signaling are sufficient to bypass the dauer checkpoint, whereas high levels are necessary to age normally (Guarente and Kenyon, 2000). daf-2(e1370)
is a temperature sensitive mutant of the Daf-c phenotype category. Hence, they form dauers at 25 °C while developing normally at lower temperatures (Riddle and Albert, 1997). This e1370
mutant allele of daf-
2 belongs to class 1 alleles, which convey normal movement, brood size and reproductive schedules. Such active and fertile animals remain youthful much longer than normal. Thus, lifespan extension is decoupled from certain dauer aspects.
A second class gives a dauer-like, quiescent posture, high fat levels, low fertility and very late reproduction (Gems et al., 1998; Tissenbaum & Ruvkun, 1998). However, daf-2(e1370)
animals and dauers do share a common life maintenance system , which contains small HSPs, anti-ROS 16 defense systems, increased activity of cellular detoxification processes and possibly also increased chromatin stability and decreased protein turnover (Burnell et al., 2005).
Unlike dauers, this daf-2(e1370)
mutant is not hypometabolic, but shows attenuation in age-dependent decline of abundance and bioenergetic competence of mitochondria (Brys et al., 2010). This allele is responsible for greatly increased fat synthesis and storage as triglycerides and phospholipids (Perez and Van Gilst, 2008). It gives these more slender worms a dark intestinal pigmentation.
Downregulation of daf-2(e1370) further delays the onset of protein-aggregation disease (Morley et al.,
2002) and slows down age-dependent decline in tissue integrity and muscle function (Huang et al.,
14
Ankyrin repeat : Very common protein-protein interaction motif.
15 daf-2(e1370) : Substitution C/T (Wild-type/mutant).
16
ROS : Reactive oxygen species, cause cellular damage and lead to oxidative stress, which is thought to be a cause of aging.
25

Introduction
2004). Autophagy and resistance to hypoxia and bacterial pathogens is enhanced (Garsin et al., 2003;
Hansen et al., 2008; Jia et al., 2009; Scott et al., 2002). Consequently, one can say that these long-lived adults seem to experience a high “quality of life” (Gems et al., 1998). This is a perfect example of what gerontologists ultimately try to achieve someday in humans.
2.2.4.6
Conservation of IIS pathway
C. elegans belongs to the phylum Nematoda, which originated and diverged from the human evolutionary branch approximately 1.000 million years ago (Hedges, 2002). So by what means can the knowledge about the extended longevity in
C. elegans daf-2(e1370)
mutants be extrapolated to our highly complex physiology? It seems that lifespan regulation by IIS is remarkably evolutionary conserved. Homologs of this pathway appeared to regulate lifespan, increase ‘healthspan’ and alleviate age-related pathologies, in yeast, Drosophila and mice (Kenyon, 2005; Piper et al., 2008; Tatar et al.,
2003). In humans, mutations in the insulin receptor, one of at least three human homologs of DAF-2, causes diabetes rather than longevity. This might be due to the strength of the mutations, which is too high compared to the weak daf-2(e1370)
mutation (Guarente & Kenyon, 2000). Comparative human genotyping, however, found in centenarians 17 an overrepresentation of heterozygous mutations in the
IGF-IR 18 , which significantly reduced IGF-IR activity (Suh et al., 2008). Some variants of
FOXO1
and
FOXO3 genes, daf-16 homolog’s, seem to be more abundant in people older than 85 (Kuningas et al.,
2007), and women containing gene variants that reduce insulin/IGF-1 signaling are blessed with longer survival (Capri et al., 2006). These promising results, however, do not automatically lead to interventions that make us live longer. This is currently empeded due to our highly complex physiolology and the many remaining gaps in the knowledge about how metabolic pathways operate and interact (Vijg & Campisi, 2008).
2.2.4.7
Evolution of IIS pathway
All metazoans age, except for some species such as Hydra (Martinez, 1998). Lifespan is plastic, as is shown by the highly diverse lifespans in multicellular organisms. In fact, all of them are evolved from a common precursor that probably did not live very long. Hence, mutations in regulatory genes that extend lifespan must have been important in evolution (Guarente & Kenyon, 2000). Others argue that significant longevity was not achieved by single mutations with large effects, since these often increase lifespan at a cost to reproduction or survival under stress. Rather subtle changes in many genes over the course of evolution seem to explain lifespan plasticity (Jenkins et al., 2004; Vijg &
17
Centenarians : People living beyond the age of 100.
18
IGF-IR : Insulin-like growth factor 1 receptor, a homolog of DAF-2 and involved in growth signaling.
26

Introduction
Campisi, 2008). Genes encoding components of the IIS pathway, TOR 19 pathway and mitochondrial electron transport chain constitute pathways that regulate besides growth, energy metabolism, nutrient sensing and reproduction also aging. Actually, the IIS pathway is selected early in evolution not to regulate aging, but rather organismal survival by postponing reproduction in harsh environmental conditions (Antebi, 2007; Kenyon, 2005). This is reflected in C. elegans by the ability to enter the dauer state early in development during bad times. The same genes that protect dauers also protect against endogenous stress that accelerates aging; hence at the same time a means of regulating longevity itself was created (Kenyon, 2005). Thus, hard times are coped with by investing in somatic maintenance by the IIS pathway, whereas in good times growth and reproduction, with negative consequences on adult lifespan, are preferred (Antebi, 2007).
19
TOR : Target of Rapamycin, integrator of nutrient and growth factor signals, coordinator of cell growth and cell cycle progression.
27

Introduction
2.3.1
ADP/ATP and AMP/ATP ratios
Cells are active and organized entities which require a lot of energy input to keep entropy low.
Reduced carbon compounds in food are oxidized (catabolism) which creates free energy that allows mitochondrial ATP synthase to convert ADP and pyrophosphate to the universal energy carrier ATP 20
( Figure 9 , reaction 3). The mitochondrial adenine nucleotide transporter (ANT) exchanges mitochondrial ATP for cytosolic ADP, which increases the cytosolic ATP/ADP ratio. The ATP/ADP ratio is maintained continuously high (± 10/1), about eight orders of magnitude away from their equilibrium ratios (± 10 -7 /1), and functions as an energy store (Hardie et al., 2003). The (Gibbs free) energy is released by hydrolysis of the
γ
- or
β
- and
γ
-acid anhydride bonds in ATP, which also yields
ADP or AMP and pyrophosphate respectively, and is able to perform work in energy-requiring processes in the cell (
Figure 9
, reaction 1 and 2).
In all eukaryotic cells ADP is converted to ATP and AMP by sufficient levels of a very active adenylate kinase to keep the reaction close to equilibrium (
Figure 9
, reaction 4). Consequently, the AMP/ATP ratio varies as the square of the
ADP/ATP ratio (
Figure 9
, lower part). Hence, the more radical changing AMP/ATP ratio is an exquisitely sensitive indicator of cellular energy status (Hardie & Hawley, 2001).
Cellular stress gives rise to an increasing
ADP/ATP ratio when the activity of ATPases exceeds that of ATP synthases. Consequently,
AMP rises, due to the adenylate kinase reaction,
Figure 9 | Reactions interconverting ATP, ADP and
AMP (Box 1, Hardie & Hawley, 2001).
generating a high AMP/ATP ratio, which is a signal that the energy status of the cell is compromised (Hardie, 2003).
20 ATP: When referred to ATP, actually Mg-ATP is meant. Magnesium is required for ATP to be biologically active.
28

Introduction
2.3.2
Control of energy level
The ADP/ATP ratio is usually preserved within very narrow limits. This indicates that the rate of ATP production resembles the rate of ATP consumption (Hardie et al., 2003).
ATP production is strictly regulated by several mechanisms to maintain the concentrations of ATP and ADP in equilibrium.
Control at the level of the electron transport chain involves the activity level of complex IV and V.
ATP adjusts its own production by allosterically inhibiting complex IV (Arnold & Kadenbach, 1999), while the concentration of ADP in the mitoplasm determines, together with the proton flux, activity of complex V. With the discovery that AMP, or the AMP/ATP ratio, regulates the activity of several glycolytic enzymes, it was suggested that many metabolic enzymes involved in anabolic and catabolic pathways are regulated by AMP (Ramaiah et al, 1964). This idea has been elaborated by the discovery that the AMP/ATP ratio is integrated and metabolism adapted by an AMP-activated protein kinase
(AMPK, see 2.3.5.1, AMPK as energy sensor ) (Hardie & Carling, 1997; Kemp et al., 1999).
2.3.3
Energy charge
Besides the AMP/ATP and ADP/ATP ratios, a third index of cellular energy status was proposed: the energy charge (Atkinson, 1968). It indicates to what extent theadenine nucleotides exist as high energy phosphates
.
The numerator represents the number of phosphoric anhydride bonds in the pool; ATP contains two,
ADP one. The denominator adds up the total adenylate pool. The equation is normalized by the factor
½ to keep the energy charge in a range of 0.0 (all AMP) to 1.0 (all ATP) ( Figure 10 ). Usually the energy charge is maintained in a steady state between 0.80 and 0.95
(Atkinson, 1968). Upon higher values anabolism is accelerated and catabolism decreased, while the reverse takes place at a lower energy charge. Hence, in healthy living cells a continuous oscillation of energy charge around the metabolic steady state takes place. The response of ATP-consuming processes on the energy charge exhibits a clear hierarchy (Buttgereit & Brand, 1995). Of all processes investigated, macromolecule biosynthetic pathways are more sensitive to changes in energy supply than sodium and calcium cycling across the plasma membrane. Other ATP consumers and mitochondrial proton leak are the least responsive to changes in energy status.
29

A
Introduction
B
Figure 10 | A : Relative concentrations of ATP, ADP and AMP as a function of energy charge. The adenylate kinase reaction is assumed to be near equilibrium with a standard free energy change of -473
J/mol and K eq
of 1.2. B : Responses of regulatory enzymes to variation in energy charge, which oscillates about a steady-state value (Garrett & Grisham, 2010).
2.3.4
Aging and adenine nucleotides
Homeostasis of cellular energy supplies in response to changes in nutrient availability or stress stimuli is essential to all life. Dysregulation of this balance is associated with aging and many diseases.
In contrast, lifespan can be extended by limited caloric intake (decreasing energy availability) and alteration of certain single gene activity within defined pathways related to the control of metabolism
(Curtis et al., 2005; Katic & Kahn, 2005). These genes affect, amongst others, the insulin pathway
(Kenyon, 2005; Braeckman & Vanfleteren, 2007; Kaletsky & Murphy, 2010), the Sir2 pathways that sense the cellular redox state via NAD + -dependent deacetylation (Nakagawa & Guarente, 2011), and mitochondrial pathways resulting in lower energy levels and lifespan extension (Dillin et al., 2002b;
Hekimi & Guarente, 2003; Rea, 2005). Hence, lifespan regulating pathways also integrate distinct energy supplies to modulate the level of cellular maintenance, and as such, play central roles in energy homeostasis (Curtis, 2005).
In aging C. elegans
worms
ATP
and
ADP
levels gradually decrease
. The long-lived daf-2 (e1370) mutant, however, maintains higher concentrations of ATP and ADP (Houthoofd et al., 2005; Brys et al., 2007; Brys et al., 2010). A potential mechanism involved is inhibition of the intrinsic F1-ATPase activity by enhanced levels of the mitochondrial F
1
-ATPase inhibitor protein (IF
1
) in daf-2 mutants
(Lebowitz & Pedersen, 1996; McElwee et al., 2006). A recent proteomics study revealed that the
C. elegans IF-1 encoding gene mai-2 was found at higher concentration in a daf-2 mutant background
(personal communication, G. Depuydt). However, no higher levels were found in daf2 mutants in a recent qPCR study (Brys et al., 2010). Hence, the role of F1-ATPase in maintaining higher ATP levels in daf-2 mutants remains uncertain.
30

Introduction
Remarkably, higher ATP levels are no prerequisite for the observed lifespan extension. The ucp-4(0) mutant is not long-lived despite its elevated ATP levels (Iser et al., 2005). Contrary, downregulation of several mitochondrial genes by RNAi extends lifespan while ATP is lowered significantly (Dillin et al., 2002b).
As described above, ADP/ATP and AMP/ATP ratios are reliable indicators of the cellular energy state and could have a profound influence on lifespan. The adult life of
C. elegans
is characterized by a remarkable exponential rise in
AMP/ATP (
Figure 11
) (Apfeld et al., 2004).
Interestingly, the most active worms of the same chronological age display a lower ratio and lived longer. It was observed that differences in the
AMP/ATP ratio reflected the effect of stochastic events on lifespan. Day 1 adults of daf-2 mutants
Figure 11 | Aging C. elegans worms display an increasing AMP/ATP ratio (Apfeld et al., 2004).
display the same AMP/ATP and ADP/ATP ratio as day 1 wild-type
worms. This indicates that the mechanism by which daf-2
mutants extend lifespan must be independent of the AMP/ATP ratio (Apfeld et al., 2004). Besides C. elegans , also replicative senescent human fibroblasts contain a higher AMP/ATP ratio compared to young fibroblasts (Wang et al., 2003).
31

Introduction
2.3.5
AMPK as energy sensor
2.3.5.1
Introduction
The cellular ADP/ATP ratio or the energy charge are adequately maintained within very narrow limits regardless of cellular activity, nutrient availability or hypoxia. It allows cells to respond to ATPlowering stresses by inducing ATP-generating pathways and inhibiting non-essential ATP-utilizing functions. The key player is 5' adenosine monophosphate-activated protein kinase ( AMPK ), an evolutionary conserved, ubiquitously expressed, multi-substrate serine/threonine protein kinase (Shin et al., 2011). AMPK is an intracellular feedback regulator that is activated by a dual mechanism involving phosphorylation upon allosteric activation by high AMP/ATP ratios (Hardie et al., 2003).
Higher AMP and reduced ATP levels are the consequence of various environmental or metabolic stresses that interfere with ATP production or increase ATP consumption. Activated AMPK functions as a ‘ metabolic switch ’ by upregulating ATP-producing processes and downregulating ATPconsuming pathways (Hardie, 2007) ( Figure 12 ). To achieve this, it phosphorylates metabolic enzymes to provoke instant effects, and transcription factors and coactivators to induce longer-term effects. AMPK was also shown to integrate the cellular redox potential. NAD + activates AMPK is a dose-dependent manner, whereas NADH inhibits its activity (Rafaeloff-Phail et al., 2004).
Figure 12 | Regulation of energy homeostasis by AMPK (Hardie, 2007).
32

Introduction
2.3.5.2
Regulation of AMPK activity
Mammalian AMPK is a heterotrimeric
complex that comprises one catalytic subunit ( α ) and two regulatory subunits (
β
and
γ
); for each there are several isoforms with tissue dependent expression. AMPK senses the low energy signal by antagonistic binding of AMP or ATP to the regulatory γ subunit. This subunit has the configuration of a line of four cystathionine β -synthase
(CBS1-4) domains; two of these bind AMP or ATP in a highly cooperative manner (Scott et al., 2004). Under normal physiological conditions two (Mg-) ATP molecules are bound rendering AMPK in its inactive form. A third CBS domain always binds AMP irreversibly, while the fourth remains unoccupied (Xiao
Figure 13 | Activation mechanism of
AMPK as result of allosteric AMP binding
(Nagata & Hirata, 2010). AID is autoinhibitory domain, K is kinase domain.
et al., 2007). When the AMP/ATP ratio increases, the higher concentration of AMP outcompetes the
ATP molecules at the regulatory sites. The binding of AMP causes a conformational change which has two effects: (1) allosteric activation
of AMPK by 10-fold, and (2) inhibition of dephosphorylation of threonine 172 (in mammalian AMPK) in the T-loop of the catalytic α subunit.
Thr-172 is probably continuously phosphorylated by LKB1 (or Thr-243 by
PAR-4
in
C. elegans
; Lee et al., 2008), which has a high basal activity, but is immediately dephosphorylated by a phosphatase in the absence of AMP (Davies et al., 1995; Sanders et al., 2007). Hence, the conformational change induced by AMP binding prevents action of phosphatases and results in a phosphorylated kinase. This releases the inhibition by the autoinhibitory domain and leads to a 100-fold activation of AMPK
(Nagata & Hirata, 2010) ( Figure 13 ). Both effects combined yield a 1000-fold activation of the kinase
( Figure 14 ) and as such AMPK acts as an ultrasensitive sensor for a drop in energy charge .
Next to LKB1, the calmodulin-dependent kinase kinase b (CaMKKb) was found to phosphorylate and activate AMPK in response to a rise in cytosolic calcium concentrations (Hawley et al., 2005; Woods et al., 2005; Hurley et al., 2005). It might anticipate a demand for ATP since rising calcium levels often trigger ATP-consuming processes (Hardie, 2011).
33

Introduction
Figure 14 | Regulation of kinase activity of AMPK (Nagata &
Hirata, 2010).
The β subunit of AMPK comprises a domain which acts as a platform for both other subunits, and a glycogen-binding domain . The latter permits AMPK to bind to glycogen particles (Hudson et al.,
2003; Bendayan et al., 2009), of which it can sense the structural state (McBride et al., 2009).
Possibly, AMPK monitors the status of glycogen stores and precludes glycogen depletion. Altogether,
AMPK is a guardian of cellular energy as it senses both immediate energy availability and mediumterm glycogen reserves (McBride & Hardy, 2009).
2.3.5.3
Function of AMPK
2.3.5.3.1
AAK-2 gene dosage determines lifespan
Gene orthologs for mammalian AMPK are found in other eukaryotic kingdoms, ranging from protists
(with the very primitive mitochondria-lacking Giardia lamblia ), plants, fungi and metazoans (Hardie et al., 2003; Hardie, 2011). The ancestral role of AMPK was probably a sensor responsive to starvation for a carbon source. C. elegans contains two homologs of the catalytic α subunit, aak-1 and aak-2 , of which the latter regulates lifespan is a dose-dependent manner (Apfeld et al., 2004).
Upon starvation and heat stress, AAK-2 is catalytically activated by phosphorylation at Thr-181, upon which it phosphorylates CRTC-1 (CREB 21 -regulated transcription coactivator 1) at conserved 14-3-3 binding sites. Consequently, CRTC-1 is cytosolically sequestered and inactivated, which renders
CRH-1 (CREB homolog-1) transcription factor inactive. This pathway is responsible for lifespan extension obtained by overexpression of AAK-2 (Mair et al., 2011). Further, AAK-2 is required for the longevity phenotype of daf-2 mutants. Hence, AAK-2 is regarded as a sensor that couples lifespan to information about energy levels
and insulin-like signals
(Apfeld et al., 2004).
21
CREB : cAMP response element binding
34

Introduction
2.3.5.3.2
AAK-2 is involved in regulation of reproduction
Sublethal doses of ATP-lowering stresses in early adulthood (e.g. starvation, treatment with glycolytic and mitochondrial inhibitors, heat shock) increase lifespan and decrease fertility by activating AAK-
2 (Apfeld et al., 2004; Schulz et al., 2007). The drop in fertility is due to germ line stem cell (GSC) quiescence which requires activation of AAK-2 by the combination of PAR-4 and STRD-1 (Narbonne et al., 2010). During reduced insulin signaling in dauer formation and in the L1 stage, AAK-2 is activated by PAR-4 and cooperates, in parallel to the TGFβ pathway, with DAF-18 to inhibit germ line proliferation and alter morphogenesis (Narbonne & Roy, 2006; Baugh & Sternberg, 2006). In
C. elegans gonads AAK-2 is expressed in distal tip cells, spermatheca, and gonadal sheath cells (Lee et al., 2008). It might have an essential role in regulation of genes involved in sex differentiation, vulval development, gamete generation, regulation of meiosis, cell division, fertilization, and oviposition, as these genes are upregulated in unstressed aak-2(gt33)
mutants (Shin et al., 2011). Thus, AAK-2 acts as a sensor in a mechanism that executes the trade-off between fertility and lifespan under energylimiting conditions (Apfeld et al., 2004). It delays aging in response to stresses during early life to postpone reproduction to times with improved conditions. This is compatible with the ‘disposable soma’ theory of aging (Kirkwood & Austad, 2000; Hardie et al., 2007).
2.3.5.3.3
AAK-2 mediates long-term survival of dauer larvae
Mutant aak-2
dauers show reduced stress resistance and longevity upon starvation or crowding
(Narbonne & Roy, 2006; Narbonne & Roy, 2009). Wild-type dauers survive long periods of low caloric intake by slowly releasing their stored energy. Triglycerides stored in the hypodermis are progressively hydrolyzed through inhibitory ATGL-1 (adipose triglyceride lipase 1) phosphorylation by highly active AAK-2. The primary cause of aak-2
mutant lethality is failure of osmoregulation as a consequence of energy depletion. Hence, AAK-2 activity is critical for long-term osmoregulation during energetic stress by rationing triglycerides (Narbonne & Roy, 2009).
2.3.5.3.4
AAK-2 induces oxidative stress resistance through DAF-16
Active AAK-2 enhances DAF-16-dependent transcription by
phosphorylating DAF-16 in vitro
at non-Akt sites, independent of its subcellular localization. As such, AAK-2 might increase oxidative stress resistance and prolong lifespan (Greer et al., 2007).
2.3.5.3.5
AAK-2 regulates lipid metabolism
In response to oxidative stress, the energy-consuming lipid synthesis
is downregulated
through negative regulation of ∆ 9 fatty acid desaturases by active AAK-2 (Shin et al., 2011). These enzymes are involved in production of monosaturated fatty acids which are components of triacylglycerides and phospholipids (Brock et al., 2007). Also vitellogenins, important in intercellular fat transport and storage, are downregulated by active AAK-2 (Shin et al., 2011).
35

Introduction
2.3.5.3.6
AAK-2 shifts glycolytic to mitochondrial metabolism
Glycolysis in impaired by glucose shortage or 2-deoxy-D-glucose treatment which results in a mild decrease of ATP levels the next two days (Sols & Crane, 1954, Schulz et al., 2007). In response to the lower ATP production, AAK-2 initiates a shift towards mitochondrial respiration to enhance ATP supplies. Consequently, more reactive oxygen species are produced, catalase activity induced, and oxidative stress and survival increased. This is consistent with the ‘ mitohormesis’ hypothesis of lifespan extension (Schulz et al., 2007, Ristow & Zarse, 2010).
2.3.5.3.7
AAK-2 induces autophagy
Both low energy levels and reduced insulin signaling activate AAK-2, which consecutively induces autophagy through phosphorylation of UNC-51/ULK1 (Egan et al., 2011). Autophagy is a survival mechanism that ensures availability of critical metabolic intermediates and eliminates damaged organelles under nutrient-poor and low-energy conditions (He & Klionsky, 2009).
Besides direct ULK1 phosphorylation, mammalian AMPK also regulates autophagy through inactivation of mTORC1 (mammalian target of rapamycin complex 1). To this end, activated AMPK phosphorylates raptor (regulatory associated protein of mTOR) at two 14-3-3 sites, causing it to be sequestered cytosolically (Gwinn et al., 2008). Consequently, raptor can no longer assist mTOR in formation of mTORC1, which results in absence of phosphorylation of the site on ULK1 that disrupts the activating AMPK phosphorylation of ULK1 (Kim et al., 2011).
2.3.5.3.8
Mammalian AMPK regulates TOR mTORC1 regulates homeostasis and growth by coordinating catabolism and anabolism with growth factor signaling, energy, nutrient, and oxygen levels (Sengupta et al., 2010). It is especially an important enhancer of protein synthesis
by stimulating translation. As mentioned above (see
3.1.3,
Energy charge ) protein synthesis is one of the cellular processes that consume the most energy.
Hence, in unfavorable conditions a decline of protein synthesis saves a lot of energy that can be diverted to somatic maintenance and repair, contributing to longevity (Artal-Sanz & Tavernarakis,
2008). In
C. elegans
, TOR and raptor are encoded by let-363
and daf-15
respectively. When nutrients are scarce, LET-363 activity is reduced by decreased insulin signaling and DAF-15 transcription is repressed by DAF-16 which leads to dauer morphogenesis, fat accumulation and lifespan extension
(Vellai et al., 2003; Jia et al., 2004). A similar AMPK-mediated inhibition of TOR as in mammals has not yet been examinated, but an AAK-2-mediated decline in translation via the TOR pathway has been suggested (Greer et al., 2007).
36

Introduction
2.3.5.3.9
AAK-2 influences behavior
Although C. elegans AMPK performs a similar metabolic role as mammalian AMPK, its cell type- and tissue-specific localization as well as its exclusive cytoplasmic restriction is different from the ubiquitous expression and partial nuclear presence of mammalian AMPK. AAK-2 was found in the pharynx, body wall muscle, ventral cord, some neurons, excretory cell, somatic gonad, and vulva, suggesting a more important role in the neural and muscular systems than in the hypodermis. Its expression in neurons allows AAK-2 to modulate body bending during locomotion and foraging behavior (Lee et al., 2008).
2.3.5.4
AAK-2 is a key node in the lifespan regulating network
Tuning of cellular metabolism towards maximal survival is the molecular basis of longevity (Artal-
Sanz & Tavernarakis, 2010). AAK-2 represents an important component of the mechanisms involved because it monitors energy levels, redox potential and insulin/IGF-1 signals, continually from L1 to old aged adults , to adjust metabolism and control the lifespan (Curtis et al., 2006). Multiple pathways involved in aging intersect at AAK-2 ( Figure 15) . Environmental stresses or mitochondrial dysfunction extend lifespan partly by activation of AAK-2 due to high AMP/ATP ratios (Apfeld et al., 2004; Curtis et al., 2006). Although the complete mechanism by which long-lived daf-2
mutants extend lifespan is largely enigmatic (Brys et al., 2010), AAK-2 and DAF-16 both act in parallel to mediate the daf-
2 mutant longevity. During reduced insulin/IGF-1 signaling
AAK-2 mediates lifespan extension independent of the AMP/ATP ratio , as the latter is reportedly identical to that of wild-type animals (Apfeld et al., 2004). DAF-16 was found to upregulate transcription of AMPK subunits (Schuster et al., 2010).
Dietary restriction (DR) has the ability to extend lifespan and improve healthspan in virtually all organisms ranging from yeast to mammals (Masoro, 2005). Many different DR regimens have been described and they act through different pathways resulting in additive lifespan prolonging effects
(Greer & Brunet, 2009). The AMP/ATP ratio enhancing effect of solid DR 22 ( sDR ) induces stress resistance and longevity through the AAK-2 – DAF-16 pathway (Greer et al., 2007).
Other DR regimens extend lifespan through SIR-2.1 and the PHA-4, SKN-1 23 and HSF-1 transcription factors (Greer & Brunet, 2009; Mair et al., 2009). The red wine component resveratrol slows down C. elegans
aging through AAK-2 independently of DAF-16 (Greer & Brunet, 2009). Germline stem cell signals reduce lifespan through inhibition of DAF-16 activity. AAK-2 does not determine longevity caused by loss of germline cells (Curtis et al., 2006).
22
Solid DR : Bacteria are diluted on solid plates.
23
SKN-1 : Only the SKN-1 isoform expressed in the ASI neuron pair is involved in lifespan extension through
DR.
37

Introduction
Altogether, the diverse response to a wide range of environmental and physiological conditions renders the
C. elegans
lifespan remarkably adjustable. AAK-2 represents a key node in this lifespan regulating circuit in C. elegans .
Figure 15 | Model of inputs and pathways regulating longevity. Models of Curtis et al. (2006), Greer et al.
(2007), and Greer & Brunet (2009) combined and updated with new insights.
38

Introduction
2.4.1
ATP detection
As one can imagine ATP production and consumption is highly tissue- and cell-specific. Intracellular organization entails further compartmentalization of ATP pools (Hoffman, 1997; Hardie, 2011). The effect of spatial ATP restriction on cellular physiology can be investigated using ATP reporters.
Usually, whole organism
ATP levels
are measured
on homogenized worms using an
HPLC
(High
Performance Liquid Chromatography) or by an in vitro luciferase based luminescence method (Brys et al., 2010). An in vivo
method based on bioluminescence through expression of transgene luciferase
(fireflie) has also been described in C. elegans (Lagido et al., 2001). This enzyme uses ATP to convert administered luciferine together with oxygen into CO
2
, AMP, and excited oxyluciferine. Generally, one reaction generates one photon that is captured by a photon-counting charge coupled device (CCD) camera (Wilson & Hastings, 1998). The luminescence is proportional to the consumed ATP, although, it delivers rather low signal levels and bad temporal and spatial detection. Other drawbacks are the need for exogenous delivery of luciferin and presence of molecular oxygen, and perturbation of the energy balance (Berg et al., 2009). Finally, this bioluminescence mechanism reports the ATP level; however, ADP/ATP and AMP/ATP ratios are much more reliable indicators of cellular metabolism
(see
2.3.1, ADP/ATP and AMP/ATP ratios
). Especially detection of the
ADP/ATP
ratio is valuable since it represents the actual free energy available through ATP hydrolysis (Hardie & Hawley, 2001).
2.4.2
Perceval
2.4.2.1
Structure
Recently, Berg et al. (2009) constructed a fluorescent sensor for ADP/ATP ratios, named Perceval.
The sensor function is executed by the backbone of Perceval which consists of a tandem trimer of
GlnK1 protomers, each connected through a ‘13-amino acid’ linker. The trimeric GlnK1 is a Mg-
ATP-binding, and ammonia transport regulating, bacterial protein of the PII family. Binding of Mg-
ATP alters its conformation drastically; the very loose disordered ‘ T-loop ’ becomes compact and ordered (
Figure 16
) (Durand & Merrick, 2006; Yildiz et al., 2007). Changes in fluorescent characteristics are mediated by using circularly permuted 24 monomeric Venus ( cpmVenus ). This yellow circularly permuted fluorescent protein (cpFP) is integrated into the T-loop of the first GlnK1 protomer. The second and third protomer contain no cpmVenus insertion and have the T-loop deleted to ameliorate the compactness and eliminate the possible negative cooperation reported in some PII proteins (
Figure 167
) (Ninfa & Jiang, 2005; Berg et al., 2009).
24
Circular permutation : Connection between original N and C termini via a peptide linker and creation of new
N and C termini near the sensor protein.
39

Introduction
Figure 16 | Ribbon representation of one subunit of the GlnK1 protein without a ligand (gray/blue) or with Mg 2+ -
ATP (gray/green/yellow, with ligand in ball-and-stick form). The T-loop (colored) becomes compact and ordered in the presence of Mg-ATP. Blue is one of many alternative and disordered structures observed for the unliganded
T loop. Yellow region indicates insertion point for cpFP (Berg et al., 2009).
Figure 17 | Construction of tandem trimer gene Perceval. The gene encodes three GlnK1 protomers. The first protomer (A) is full length, with cpmVenus inserted between position 51 and 52. The second (B) and third (C) protomers have a deletion of the T-loop region ( ∆ T). The protomers are connected through linker sequences
(black residues). Numbered subscripts indicate position in monomeric wildtype GlnK1 (Berg et al., 2009).
40

Introduction
2.4.2.2
Excitation spectrum
The basal excitation spectrum of Perceval is similar to that of YFP (yellow fluorescent protein) and displays a prominent emission peak at 530 nm when excited at 490 nm. An additional smaller peak was observed by excitation at 405 nm. Binding of Mg-ATP closes the T-loop and results in a substantial change in the excitation spectrum ( Figure 18 ). At a saturated concentration of ATP the ratio of fluorescence emitted by excitation at 490 nm to that by excitation at 405 nm (F
490
/F
405
) increases threefold. GlnK1 can also bind ADP, and although this yields no secure T-loop closure
(Yildiz et al., 2007), the same ratio of fluorescence enhances 1.4-fold. This ratiometric change is an ideal property for Perceval as it allows normalization of the signal independent of its concentration
(Berg et al., 2009).
Figure 18 | At the right, excitation spectra of purified Perceval construct under control conditions (gray) and after addition of 50 µM Mg-ATP (black) or 10 µM ADP (red) (emission at 530 nm). ATP addition leads to an increase in the 490 nm peak and a decrease in the 405 nm peak. Upon ADP addition, these effects are only minimal (Berg et al., 2009).
2.4.2.3
Fluorescence response to ADP/ATP changes
In cells, when both adenine nucleotides are present, ATP and ADP compete for the binding site. Tloop closure and a maximal change in fluorescence take place if only ATP is bound; competition by
ADP lowers the affinity for ATP with a concomitant decrease in the ratio of fluorescence (F
490
/F
405
)
(
Figure 19
). The sensor’s affinity for Mg-ATP is, compared to the normal intracellular millimolar concentrations, very high (< 100 nM) and doubles that for ADP.
41

Introduction
This means that if the ADP/ATP ratio is 2/1, the fluorescence response of Perceval is half-maximal
(K
R
25 is 0.5). Since
C. elegans
worms have ADP/ATP ratios of approximately 1/14 (Apfeld et al.,
2004; Curtis et al., 2006), interesting rises in this ratio (e.g. upon metabolic inhibition) should be measurable with very fast kinetics (detectable changes in fluorescence within seconds). However, the use of Perceval as a sensor in C. elegans has not been reported yet. If ATP and ADP are present,
Perceval is not influenced by other purine nucleotides, such as AMP, GTP, and NAD + .
In summary, Perceval displays differential fluorescenct properties depending on the ADP/ATP ratio.
Therefore, it can be used to monitor variation in (sub) cellular energy levels.
Figure 19 | Perceval, with a K
R of ~0.5, responds to a wide variety of ATP/ADP ratios. Fluorescence response (490 nm / 405 nm) increases by higher [ATP] / [ADP], and is normalized to a value of 1 in the presence of saturating ADP (Berg et al., 2009).
2.4.2.4
Advantages of Perceval
To measure energy levels using HPLC, worms have to be homogenized. The use of Perceval transgenic worms, on the other hand, would allow non-disruptive in vivo
measurements of cellular energy levels. Compared to the bioluminescence method, which uses an ATP hydrolysis reaction, energy levels are not perturbed since Perceval binds ATP and ADP competitively. The fluorescence response of Perceval shows very fast kinetics and responds to a wide variety of ADP/ATP ratios, which make it an ideal sensor to monitor real-time changes in energy levels (Berg et al., 2009).
In conclusion, Perceval allows non-disruptive, in vivo, real-time measurements of cellular energy levels on both total worms (fluorimetry) and tissue-specific (Confocal Laser Scanning Microscopy or tissue-specific promoters).
25
K
R
: Ratio at which the Perceval fluorescence response is half maximal; analogous to the dissociation constant
(K d
) of a receptor.
42

Goals and experimental outline
The goal of this master dissertation is to contribute to the knowledge about the role of energy levels in aging Caenorhabditis elegans worms. The research involves two main subjects.
1) A more complete picture of the dynamics of adenine nucleotides (ATP, ADP and AMP) in aging wild-type as well as long-lived daf-2(e1370) mutants is aspired. Although ATP and
ADP dynamics are already known, AMP dynamics in aging C. elegans have never been reported. Exact concentrations of all three adenine nucleotides at each day of adulthood of both strains will be obtained by homogenizing several thousands of worms, derivatizing the adenine nucleotides and measuring the derivatized adenine nucleotides using reverse-phase high performance liquid chromatography. Therefore, large synchronized cultures will be established. Since the absolute amount of ATP is a less reliable parameter for cellular energy levels than the AMP/ATP, ADP/ATP ratios and the energy charge, the latter will also be compared between both aging strains. Finally, their contribution to the longevity phenotype of daf-2(e1370) mutant worms will be discussed.
2) The recently developed genetically encoded sensor ‘Perceval’ allows detection of ADP/ATP ratios in vivo
in real-time
.
In this study, we try to validate the usefulness of this fluorescent sensor in Caenorhabditis elegans . Therefore, we will first perform several molecular cloning steps to obtain plasmids (‘expression clones’) that contain the Perceval gene in a conformation suitable for expression in C. elegans . Subsequently, nematodes will be transformed either by
‘microinjection’ or ‘biolistic transformation’. To assess the functionality of Perceval in transgenic fluorescent worms, they will be treated with sodium azide, which inhibits ATP production and should result in higher ADP/ATP ratios. The effect of this treatment on the fluorescent properties of Perceval, yielding an estimate for ADP/ATP ratios, will be analyzed both by fluorescence spectroscopy and confocal laser scanning microscopy.
43

Materials and methods
All experiments were performed at the lab of the Research Group for Aging Physiology and Molecular
Evolution in the Faculty of Sciences at Ghent University.
4.1.1
Used strains
Three C. elegans strains were used to perform the experiments for this master dissertation. The strain used as reference wild-type was the N2 Bristol CGC 26 male stock. In the early 1950s a
C. elegans strain was isolated from compost in a mushroom farm in Bristol, UK; as such it was called the Bristol strain (Nicholas et al., 1959; Fatt & Dougherty, 1963). From this stock, two lines were established out of a hermaphrodite; a male-rich one and a line of hermaphrodites propagating by self-fertilization.
These are the founder and so called N2 male /female stock of which thousands of mutants are derived and characterized. In 1969 John Sulston preserved the N2 strains by freezing (Brenner, 1974). Because of the many years before freezing, the N2 isolate was adapted to artificial lab environments and has undergone numerous genetic bottlenecks. This resulted into phenotypes and underlying alleles different to wild C. elegans . Therefore one has to be careful with extrapolations of biological observations of N2, which captures a very limited range of genetic variation, to other
C.
elegans genotypes (Felix & Braendle, 2010).
The daf-2(e1370)
mutant (strain CB1370) carries a point-mutation in the insulin/IGF-1 receptor and lives twice as long compared to N2 (see 2.2.4.5, daf-2 mutation ). The strain was used together with the
N2 strain in the HPLC assay (see
4.2, HPLC-fluorometric assay
). The HT1593 strain has a mutation in unc-119(ed3) , which encodes a highly conserved protein amongst metazoans that is required for development of the nervous system. UNC-119 may be part of a signal transduction pathway that mediates axonal patterning in response to external developmental cues, as human UNC-119 functions as a receptor-associated activator of signal transduction and rescues C. elegans unc-119 mutants
(http://www.wormbase.org; release WS225). Microinjection and biolistic transformation with Perceval
(see 4.3, Perceval: proof of concept ) were performed using this strain.
26
CGC
: Caenorhabditis Genetic Center; it collects, maintains and distributes stocks of
C. elegans
( http://www.cbs.umn.edu/CGC/).
44

Materials and methods
4.1.2
Freezing and thawing strains
C. elegans strains are kept frozen in liquid nitrogen (-195.8°C) for two reasons; first, it permits conservation of the genomic uniformity over time, and secondly, it strongly reduces the work and costs associated with the maintenance of stock cultures (Brenner, 1974).
To preserve five aliquots of worms, two or three NA plates (see
4.1.3.1, Nutrient agar plates
) that contain many (starved) juveniles were rinsed of with S-buffer (43.55 mM KH
2
PO
4
+ 6.45 mM
K
2
HPO
4
+ 100 mM NaCl in distilled water, pH 6.0) and poured into a 15 ml tube. The worms were spun down (2 min., 3000 rpm) and supernatant was removed. S-buffer was added to the pellet up to the three ml mark. Three extra ml S-buffer containing glycerol (30%) was added. Glycerol is a freezing point lowering cryoprotectant which decreases the intracellular osmotic pressure by passing the cell membrane and acting as a salt buffer. As a result water leaves the cell and as such, crystallization in the cell is precluded.
The worms were resuspended and 1 ml aliquots were transferred to six cryoval tubes, which were labeled with strain name and current date. The tubes were placed in a container, called Mr. Frosty
(C#1562 -Nalgene, Sigma-Aldrich ® , Saint Louis, Missouri, USA), which contains isopropanol (200 ml) at room temperature. Subsequently, the container with tubes was set in a -80 °C freezer until next day. Isopropanol functions to decrease the temperature of the tubes gradually, at about 1°C per minute, allowing the water to move out of the worm cells before it freezes.
Next day, one tube was thawed and worms were placed on an NGM plate (see 4.1.3.2, Nutrient growth medium plates ) to check if the worms survive the freezing/thawing process. The thawed worms usually start moving within the time window of minutes to half an hour. Upon such a positive viability test, the data of these strains are entered into a database and the remaining vials are stored in liquid nitrogen. To start-up a culture from frozen
C. elegans
strains, these were thawed and placed on an
NGM plate on which glycerol is rapidly diluted. This plate contains no Escherichia coli ( C. elegans food) because in presence of glycerol the bacteria grow rapidly with detrimental effects on the worms.
The next day, the culture was rinsed of, brought onto NA plates (see 4.1.3.1, Nutrient agar plates ) that were seeded with
E. coli
and placed at 17 °C. From this point, the nematodes were cultured according to standardized procedures (see 4.1.5, Set-up synchronized aging C. elegans cohorts )
45

Materials and methods
4.1.3
Agar plates
4.1.3.1
Nutrient agar (NA) plates
Nutrient Agar is a solid, rich medium used to culture C. elegans by covering it with a lawn of E. coli cells.
A 500 ml duran bottle was filled with Nutrient Agar (11 g - Life Technologies, Paisley, Scotland), distilled water (400 ml) and cholesterol 27 (0.2 ml, 1%, 10 mg/ml in EtOH). The NA was sterilized by autoclaving and cooled to 65 °C. To control pH, sterile KH
2
PO
4
(10 ml, 1 M, pH 6.0) was added to the solution. With one such a bottle petriplates (25 of 10 cm diameter) were filled with Nutrient Agar solution (15 ml). After solidification of the agar plates on a flat surface, they were stored in a sealed bag at 4 °C.
4.1.3.2
Nutrient growth medium (NGM) plates
NGM is a solid medium used to culture transfected
C. elegans
. The plates are seeded with OP50, a uracil-requiring mutant of E. coli . NGM contains a limited amount of uracil to prevent the bacteria to overgrow the plate into a thick layer and as such blur the worms (Brenner, 1974).
A 500 ml duran bottle was filled with NaCl (1.2 g), SoyPepton (1 g,), agar N° 1 (6.87 g, Life
Technologies, Paisley, Scotland), distilled water (390 ml) and cholesterol (0.2 ml, 1%, 10 mg/ml in
EtOH). The NGM was sterilized by autoclaving and cooled to 65 °C. To control pH, sterile KH
2
PO
4
(25 mM, pH 6.0) was added to the solution. In addition, both sterile CaCl
2
(1 mM CaCl
2
.H
2
O) and
MgSO
4
(1 mM MgSO
4
) were added to supply the medium with necessary salts.
27
Cholesterol : Required for proper development in all stages of C. elegans (Shim et al., 2002).
46

Materials and methods
4.1.4
Escherichia coli
4.1.4.1
Lysogeny Broth (LB) medium
LB medium is a liquid, nutritionally rich medium used to grow bacteria rapidly. Sterile practice is required to avoid contamination with other competitive microorganisms in the rich and non-selective
LB medium.
A 500 ml duran bottle was filled with LB (6.4 g, Life Technologies, Paisley, Scotland) and distilled water (400 ml). The LB medium was sterilized by autoclaving. 250 ml LB medium was inoculated with a fresh culture of
E. coli and incubated overnight at 37 °C on a rotary shaker (120 rpm). When the bacteria reached the stationary phase, the culture was preserved at 4 °C.
4.1.4.2
LB agar
LB agar is a solid, nutritionally rich medium used to grow selective E. coli colonies.
A 500 ml duran bottle was filled with LB (12.8 g, Life Technologies, Paisley, Scotland) and distilled water (400 ml). The LB was sterilized by autoclaving and cooled to 65 °C. To obtain selective medium for cloning purposes (see
4.3.1.2, Transformation E. coli
) 0.1 mg/ml filter-sterilized ampicillin was added.
4.1.4.3
K12 on NA plates
The NA plates were seeded with a lawn of E. coli K12 cells by adding and spreading 0.5 ml E. coli
K12 suspended in LB medium. The plates were incubated (37 °C) overnight and stored at 4 °C.
4.1.4.4
OP50 on NGM plates
The NGM plates were seeded with a thin lawn of E. coli OP50 cells by transferring E. coli OP50 suspended in LB medium (0.1 ml).
47

Materials and methods
4.1.5
Setting up synchronized aging C. elegans cohorts
Our experimental setup required hundreds of thousands equally-aged worms. Therefore we started synchronous worm cohorts by performing 2 steps. First, gravid 28 adults were bleached (or ‘chloroxed’) to yield eggs in S-buffer; next, the eggs were allowed to develop into L1s overnight in S-buffer, resulting in a further synchronization by L1 arrest (Sulston & Hodgkin, 1988; Vanfleteren & De
Vreese, 1996).
4.1.5.1
Monoxenic plate cultures
NA plates were used for upscaling as they support fast and nearly synchronous development and high fecundity (Braeckman et al., 2002). Plates with enough gravid adults were rinsed of with S-buffer and transferred into a falcon tube (15/50 ml, depending on the amount of plates). The excess of E. coli was washed out three times with S-buffer: while adult worms settled to the bottom, supernatant with
E. coli was removed and S-buffer was added to resuspend the worms and bacteria. The chlorox mixture 29 (8 ml double distilled water (bidi), 2 ml NaOCl (bleach, 12 ° Chl) and 0.5 ml NaOH (10 M) in a 15 ml tube or proportionally increased for a use in a 50 ml tube) was added to the worm pellet and shaken strongly (4 min) 30 . The solution was centrifuged (3 min, 3000 rpm) and supernatant was removed. The pellet of eggs was washed 3 times with S-buffer to get rid of the bleach mixture: S-buffer was added, solution was shaken and centrifuged (2 min, 3000 rpm) and supernatant was removed. Finally, the eggs were suspended in a few ml of S-buffer and transferred into a 100 ml jar. The suspension should not be too dense and sufficient air access should be permitted, to oxygenate embryonating eggs and
L1s. The solution was incubated (17 °C) and shaken (120 rpm) in a shaking incubator (New
Brunswick Scientific, NJ, USA). Although synchronized arrested L1 larvae can survive up to 10 days in S-buffer, worms were transferred onto bacteria containing plates the next day (see
4.1.5.2,
Monoxenic plate cultures ).
28
Gravid : Containing eggs.
29
Mechanism of NaOCl and NaOH :
NaOCl
NaOH
Na + + OCl
Na + + OH -
HOCl H + + OCl - (pK a
= 7.5)
Because of the low K a
(-7.5) especially HOCl is formed, which has a stronger reactivity than OCl . Its strong oxidizing capacities damage biomolecules. Subsequently, toxic Cl
2
is formed.
HOCl + H + + Cl - Cl
2
+ H
2
O
30 The cells of both C. elegans and E. coli are destroyed by the oxidizing HOCl . The eggs are protected by 3 layers: an external vitelline layer, a middle chitinous layer and an internal lipid layer (Bird & Bird, 1991).
Alltogether the process of shaking and centrifuging should not take longer than 10 minutes to prevent breakdown of eggs.
48

Materials and methods
For synchronous culture, each 15 ml plate is seeded with an estimated 10000 juveniles. The time of development is temperature dependent; worms reach adulthood after approximately 2.5 days at 24 °C and 5 days at 17 °C. The next 3 days they are gravid and ready to be bleached to further upscale the nematodes. After every bleaching procedure, the amount of worms is scaled up by about four fold.
4.1.5.2
Monoxenic liquid cultures
Monoxenic liquid cultures were used to grow several synchronized C. elegans cohorts of which worm samples were taken daily.
Synchronous cohorts were obtained by culturing them as described above (see
4.1.5.1, Monoxenic plate cultures ) at 17 °C. This temperature suppresses dauer formation of the daf-2(e1370) mutant and allows the worms to further reach adulthood. At the late L4 stage the worms were rinsed of the plates with S-buffer and transferred in 50 ml falcon tubes. The worms were washed 3 times in S-buffer.
Next, the L4s were transferred to a Fernbach flask containing 10 µg/ml cholesterol in S-buffer.
To prevent reproduction, the DNA synthesis inhibitor 5’fluorodeoxyuridine (FUdR 31 ) was added
(Hosono, 1978), at 200 µM final concentration. As a food source, the worms were fed frozen
E. coli cells 32 (23.08 g/l). These worm suspensions were incubated (24 °C) and shaken (120 rpm). The bacterial density was checked on a daily basis by measuring turbidity with the Varian spectrophotometer (absorption at 550 nm) and proper amounts of bacteria were added to maintain an
OD
550 nm
of 1.8. The day after transferring the L4 worms to Fernbach flasks, they were considered as day 0 adults. Harvesting started at day 1.
31
FUdR FUdMP
Deoxyuridine dUMP dTMP dTTP: DNA building block, required for DNA replication. Lack of dTTP leads to inhibition of mitosis. Hence, germ cells do not develop oocytes.
32
Frozen E. coli cells : Prepared by dripping a solution of washed E. coli cells in equal S-buffer volumes into liquid nitrogen. These frozen beads were stored at -75 °C until further use.
49

Materials and methods
4.1.6
Sampling
Two C. elegans strains, N2 and daf-2(e1370) , were upscaled to 100.000 - 400.000 worms per replicate and transferred to a liquid culture using the standardized methods described above (see
4.1.5, Settingup synchronized aging C. elegans cohorts ). Starting from day 1 adults, each morning several replicates of 4000 worms were sampled. Worms were washed in 50 ml tubes with S-buffer to remove bacteria and waste products. Dead worms were cleared by performing a Percoll 33 washing step. Percoll (9 ml,
23% w/w in water) was diluted in S-buffer (16 ml) and with a syringe 20 ml of this solution was brought beneath a 20 ml suspension of nematodes. While living worms settle to the bottom, the less dense dead worms float on the Percoll and can be removed. After another washing step, the worms are transferred to a 15 ml tube in 4 ml of S-buffer. Next, a sucrose wash was carried out to remove remaining bacteria. 8 ml sucrose (60 %) was added to the nematode suspension, which was shaken and centrifuged (1 min, 3000 rpm). Since bacteria absorb the sucrose and become denser than the nematodes they are spun down by this procedure. The nematodes float on the sucrose medium and are transferred to another 15 ml tube, followed by two S-buffer washes. The clean worm suspensions were divided into 100 µl aliquots in several 2 ml sampling tubes. One of them was immediately used to derivatize the adenine nucleotides (see
4.1.2, Formation of fluorescent derivatives
), the others were stored at -75 °C, until further use for the BCA assay (see 4.2.3, BCA assay for protein quantitfication ).
To detect ATP/ADP/AMP differences between liquid cultures and plate cultures, a plate (NA, K12) with N2 worms was grown. Two aliquots of day 1 adults were washed, either with or without Percoll, to test the effect of Percoll. Further handling was the same as that of liquid cultures.
33
Percoll : A sterile colloidal suspension comprised of polyvinylpyrrolidone coated silica particles of 15-30 nm in diameter. It is a non-toxic density gradient medium.
50

Materials and methods
4.2.1
Formation of fluorescent derivatives
For each replicate of each strain (and both plate samples), an aliquot of 100 µl live worm suspension in a 2ml sampling tube was used to extract the adenine nucleotides. In order to break open the worms and precipitate the proteins, glass beads (0.2 g) and HClO
4
(400 µl, 8% v/v) were added respectively and the mixture was homogenized using a Mini-Beadbeater (1 min, 5000 rpm, Biospec Products, OK,
USA). KOH (520 µl, 1.33 M) was added to neutralize the solution. The mixture was vortexed and centrifuged (8 min, 4 °C, 14000 rpm) to sediment the salts and to remove worm debris and glass beads. The neutralized supernatant (720 µl), containing the adenine nucleotides, was brought into a new 1.5 ml tube. The acidity was brought to pH 4.2 by addition of 80 µl NaAc/HAc buffer (5.3 ml 1
M NaAc + 14.7 ml 1 M HAc). Finally, to derivatize the adenine nucleotides to fluorescently detectable etheno-adenine nucleotides, chloroacetaldehyde 34 (30 µl, Sigma-Aldrich ® , Saint Louis, Missouri,
USA) was added and the solution was incubated (40 min, 80 °C) in a thermomixer (Eppendorf) under a fume hood. After derivatization ( Figure 20 ) samples were centrifuged (8 min, 4 °C, 14000 rpm) and supernatant was transferred to HPLC tubes. These were stored at -75 °C, until use.
Chloroacetaldehyde
AMP etheno-AMP
Chloroacetaldehyde
ADP etheno-ADP
ATP
Chloroacetaldehyde etheno-ATP
Figure 20 | Derivatization of AMP/ADP/ATP to etheno-AMP/ADP/ATP by chloroacetaldehyde.
34
Chloroacetaldehyde : Highly electrophilic and alkylating agent; useful intermediate in synthesis of many compounds. Molecular formula: C
2
H
3
ClO.
51

Materials and methods
4.2.2
RP-HPLC analysis
4.2.2.1
Hard- and software
The liquid chromatograph consisted of a
Series 200 Micro Pump (Perkin Elmer) and a Series 200 HPLC Autosampler
(Perkin Elmer). Separation was achieved on a reverse-phase column
(Hypersil Gold, ThermoScientific, C18,
4.6 X 250 mm, 5 µm particle size). The fluorescent etheno( ε )-adenine nucleotides were detected at an excitation wavelength of 340 nm and an emission wavelength of 420 nm using a
Figure 21 | Flow scheme HPLC.
Series 200 Fluorescence Detector
(Perkin Elmer). Programs were edited and data was acquired, processed and reported by TotalChrom ®
Chromatography Data Systems (CDS) software (Perkin Elmer) ( Figure 21 ).
4.2.2.2
Solvents
Two buffers were used to separate the e-adenine nucleotides. Buffer A was an aqueous solvent: phosphate buffer (0.1M, pH 6.0). Buffer B consisted of an organic solvent: 50% buffer A/50% methanol (v/v). Buffer A was prepared out of a stock solution (1 M, pH 6.0) of K
2
HPO
4
(0.6 M) and
KH
2
PO
4
(0.4 M).
After the last run the column was flushed with HPLC grade water to get rid of salts. These could precipitate into a non-salt containing solvent and cause corrosion or plugging of the column. The columns were stored on 50/50 methanol/water. Before the first run the column was flushed with water to bring it back to the equilibrating condition. The first run was always performed on a blank sample 35 .
35
Blank sample : Bidi on which the derivatization procedure was performed.
52

Materials and methods
4.2.2.3
Separation
Etheno-AMP,-ADP and -ATP (2 -5 µl depending on the concentration, calculated from sample protein content (see 4.2.3, BCA assay for quantitative comparisons ) were automatically injected by the autosampler injection needle into the mobile phase of the column (
Figure 21
). A polar solvent, phosphate buffer (A), was used as mobile phase during the injection. The column (Hypersil Gold,
Thermo Scientific) has an internal diameter of 4.6 mm and a length of 250 mm. The internal stationary phase consists of tiny silica particles (5 µm) attached to non-polar hydrophobic C18 alkyl chains (-
CH
2
-(CH
2
)
16
-CH
3
) that interact with the analyte. At a flow rate of 300 µl/min., different gradients of buffer A and B were, under a high pressure (around 3000 psi), passed through the column (
Table 2
).
Both buffers have a high but different polarity. Because ε -adenine nucleotides are polar, they move with the buffers rapidly through the column. Due to its high solubility in aqeous buffers
ε
-ATP migrates faster in the phosphate buffer than ε -ADP. ε -AMP is the least soluble in this buffer and hence, interacts more with the stationary phase by weak Van der Waals dispersion forces 36 . The less soluble in the mobile phase, the longer it takes to find the way through the column. In an increasing methanol gradient, the weak intermolecular Van der Waals forces are dominated by properties of the polar hydroxyl group of methanol and, consequently, the less aqeous-soluble ε -AMP dissolves in the highly polar methanol. Due to their different solubilities ε -ATP elutes first and is followed by ε -ADP and finally, ε -AMP. The combination of the component and column properties, along with the experimental setup results in characteristic, reproducible retention times (time between injection of the sample and elution from the column for the components of interest).
Table 2 | Schedule of solvent gradient flow.
Step
4
5
6
2
3
0
1
Time (min)
0.5
15.0
10.0
5.0
5.0
10.0
10.0
Flow (µl/min)
0.3
0.4
0.3
0.3
0.3
0.3
0.3
A (%)
100
100
0
0
100
100
100
B (%)
0
0
0
0
0
100
100
Curve
1
0
0
1
0
0
0
36
Van der Waals dispersion forces : Sum of attractive or repulsive forces between dipoles. Compared to chemical bonds they are relatively weak. These forces define the solubility of organic substances in polar and non-polar media.
53

Materials and methods
4.2.2.4
Chromatogram
The fluorescence detector excites the eluting fluorescent ε -adenine nucleotides at a wavelength of 340 nm. Emission signals, at a wavelength of 420 nm, are captured by the fluorescence detector and plotted in function of time to obtain a chromatogram in TotalChrom ® software (
Figure 22
). This software was also used to quantify peak retention times and peak surface areas. Component identification is based on retention time, while peak area under curve at a peak (baseline was manually extended) is proportional to the concentration of the component. Serial dilutions of known amounts of
ATP/ADP/AMP (1 µM, 2 µM, 5 µM, 10 µM and 20 µM) were derivatized as described above ( 4.2.1,
Formation of fluorescent derivatives
) and HPLC analyzed to establish specific retention times and to correlate the peak area. Peak retention times of these standard curves were used to identify components in the injected samples, while standard curve peak surface areas were used to correlate the peak areas from injected worm samples to absolute amounts of ATP, ADP and AMP.
ATP
ADP
Baseline
AMP
Baseline
Figure 22 | Example of chromatogram. Adenine nucleotides and baseline are indicated with arrows.
Excitation at 340 nm, emission at 430 nm.
54

Materials and methods
4.2.3
BCA assay for protein quantification
Both adenine nucleotide and body mass generally change with age and among strains. For quantitative comparisons between age groups and strains all experimental data should be normalized correctly. To achieve this, the results for ATP, ADP and AMP levels were normalized to total protein content of the worm samples, as determined with a bicinchoninic acid (BCA) assay (Smith et al., 1985).
The samples (100 µl) stored at -75 °C were dried in a Savant Speed Vac Concentrator overnight. The dry pellets were immersed in 180 µl 1 N NaOH and heated (70 °C for 25 min) with intermittent vortexing. NaOH was further diluted to 0.1 N by adding 1620 µl bidi. The tubes were gently mixed and centrifuged (10 min, 14000 rpm). On 96 wells transparent microtiter plates 4 replicates of 10 µl portions of the supernatant of each sample were pipetted into the wells. As an internal standard, 2 replicates of 10 µl portions of a dilution series of bovine serum albumin (BSA, 1mg/ml) in 0.1 N
NaOH were made on each plate. Next, 200 µl of the BCA reagent mix (Pierce, Rockford, IL) was added to each well. The microtiter plates were covered with sealing tape and incubated at 37 °C for 1 h. Finally the sealing tape was removed and absorbance was measured at 560 nm in the Victor
Multilabel Counter.
55

Materials and methods
4.3.1
Molecular cloning techniques
4.3.1.1
Plasmid preparation by LR reaction
•
Principle
Eight different constructs (
Table 3
) containing Perceval, preceeded by a constitutive promoter (
Table
4 ), were developed by performing the Multisite Gateway ® Pro LR Recombination Reaction
(Invitrogen). The Gateway ® Technology is a universal cloning method based on the bacteriophage lambda site-specific recombination system. It provides a rapid and highly efficient way to transfer heterologous DNA sequences into multiple vectors systems. In phage lambda, recombination occurs through specific recombination sequences denoted as att ( att for attachment) sites. During LR recombination DNA segments that are flanked by att sites are exchanged upon incubation with the LR
Clonase™ II Plus enzyme mix. Using this technology, multiple DNA segments can be inserted into a vector in a single reaction. In the experiments described below, we recombined a promoter sequence with the Perceval gene into a ‘destination vector’, resulting in the desired ‘expression clone’ (
Figure
23 ). The att sites become hybrid sequences comprised of sequence donated by each parental vector
( att
L + att
R att
B (expression clone) + att
P (by-product, not shown in
Figure 23
)).
Construct N° Promoter Gene
1 Prps-0 Perceval
2 Prpl-17 Perceval
3
4
5
6
7
8
Prpl-24.1 Perceval
Prpl-32 Perceval
Prps-0
Prpl-17 gas-1tp::Perceval gas-1tp::Perceval
Prpl-24.1 gas-1tp::Perceval
Prpl-32 gas-1tp::Perceval
Table 3 | Eight plasmids consisting of a C. elegans ribosomal promoter and attached gene, either Perceval or gas-1tp::Perceval.
P : promoter; e.g. Prps-0 is the promoter region of the rps-0 gene. gas-1tp : gas-1 transit peptide is an N-terminal mitochondrial targeting sequence consisting of 40 amino acids (Kayser et al., 2001), separated of Perceval by a GA polylinker.
56

Materials and methods
Table 4 | Genes, of which the promoter region was used, their products and expression pattern
(http://www.wormbase.org; release WS225).
Gene rps-0 rpl-17 rpl-24.1 rpl-32
Type of protein small ribosomal subunit SA protein large ribosomal subunit L17 protein large ribosomal subunit L24 protein large ribosomal subunit L32 protein
Expression pattern (adult)
Pharynx, intestine, stomato-intestinal muscle, anal depressor muscle, body wall muscle, hypodermis, nervous system, tail neurons
Pharynx, intestine, anal sphincter, reproductive system, vulval muscle, body wall muscle, nervous system, nerve ring, head neurons, neurons along body, tail neurons
Pharynx, intestine, anal depressor muscle, rectal epithelium, reproductive system, vulval muscle, spermatheca, body wall muscle, hypodermis, excretory cell, nervous system, head neurons
Pharynx, intestine, reproductive system, distal tip cell, body wall muscle, seam cells, excretory cell, nervous system, nerve ring, ventral nerve cord, head neurons, neurons along body, tail neurons
Figure 23 | LR reaction. An entry clone, bearing the gene, and a destination vector, bearing the ampicillin resistance gene, were recombined by LR Clonase™ II, resulting in a donor vector and an expression clone, bearing both the gene and ampicillin resistance gene.
57

Materials and methods
•
Protocol
For each of 8 different constructs, the entry clone bearing the promoter (5 fmol) was mixed with the entry clone bearing Perceval or gas-1tp-Perceval (5 fmol) and the destination vector pDESTMB14-
NOGFP-UNC54UTR (10 fmol) into a 1.5 ml microcentrifuge tube at room temperature. 1X TE Buffer
(pH 8.0) was added to reach a volume of 4 µl. The LR Clonase ™ II Plus enzyme mix was removed from -80 °C and thawed on ice (2 min) and restored directly after use. LR Clonase ™ II Plus enzyme mix was briefly vortexed twice (2 sec each time) and subsequently 1 µl of this mix was added to all tubes. This mix was briefly vortexed twice (2 sec each time) and incubated overnight (25 °C, 16 h).
The next day, proteinase K solution (0.5 µl) was added to each reaction, followed by incubation (37
°C, 10 min). The mix was chilled on ice.
4.3.1.2
Transformation E. coli
For each of eight constructs, one vial of DH5
α chemically competent E. coli cells was thawed on ice and MultiSite Gateway ® Pro LR recombination reaction (3 µl) was added, followed by a gentle mixing and incubation on ice (30 min). The cells were heat-shocked (30 sec) in a warm water-bath (37 °C) without shaking and directly replaced on ice. The rich SOC medium (250 µl, room temperature) was added to improve plasmid uptake and stabilize the bacterial competent cells after transformation. The vials were incubated and shaken gently at 37 °C for 1 h. For each transformation 20 µl and 200 µl of these recovered bacteria were streaked on selective LB agar plates (100 µg/ml ampicillin). The plates were inverted and incubated (37 °C, overnight).
The next day, three distinct colonies of each construct were inoculated from the 20 µl or 200 µl plate
(dependent on the density of colonies) and grown into 3 different tubes containing 5 ml LB medium and 0.1 mg/ml ampicillin (37 °C, overnight, shaken at 120 rpm).
Upon a first bacterial transformation, no colonies for construct ‘4’ (Prpl32::Perceval) were present.
Therefore, the recombination reaction and bacterial transformation were repeated. To catch up with the other constructs, four colonies were selected to perform an immediate ‘colony PCR’ (see 4.3.1.5.2,
PCR )
58

Materials and methods
4.3.1.3
Plasmid purification
•
Principle
Expression plasmids were isolated using the GenElute™ Plasmid Miniprep Kit (Sigma-Aldrich ® , Saint
Louis, Missouri, USA). For all 3 37 replicates of each construct the overnight recombinant E. coli culture was harvested with centrifugation and subjected to a modified alkaline-SDS lysis procedure.
Plasmid DNA (pDNA) was adsorbed onto silica of a binding column in the presence of high salt conditions. A spin-wash step was performed to remove contaminants and, finally, the bound pDNA was eluted in water. It was predominantly present in the supercoiled form and ready for immediate use in restriction digestion, PCR and sequencing.
•
Protocol
Before starting and prior to initial use, 13 µl (for the 10 prep-package) of the RnaseA Solution was added to the Resuspension Solution and the Wash Solution Concentrate was diluted with 10 ml of
100% ethanol. The Resuspension Solution was stored at 4 °C.
3 ml of the overnight recombinant E. coli culture was pelleted by centrifugation (14000 rpm, 1min) and supernatant was discarded. The bacterial pellet was resuspended with Resuspension Solution (200
µl) and homogenized by vortexing. Cells were lysed with Lysis Solution (200 µl). To avoid shearing of genomic DNA, resulting in chromosomal DNA contamination in the final recovered pDNA, the contents were mixed by gentle inversion (6-8 times). Incubation in lysis solution was no longer than 5 min, because prolonged alkaline lysis might permanently denature supercoiled pDNA, rendering it unsuitable for downstream applications. The cell debris was precipitated by adding
Neutralization/Binding Solution (350 µl) and the tube was gently inverted (4-6 times). The cell debris, proteins, lipids, SDS and chromosomal DNA were pelleted by centrifugation (14000 rpm, 10 min).
Meanwhile a GenElute™ Miniprep Binding Column was inserted into a microcentrifuge tube. Column
Preparation Solution (500 µl) was added to the miniprep column to maximize the binding of pDNA to the membrame resulting in more consistent yields. The column was centrifuged (14000 rpm, 1 min) and the flow-through liquid was discarded. After the 10 min centrifugation of the lysate, the neutralized supernatant was transferred to the prepared miniprep column and centrifuged (14000 rpm,
1 min). The flow-through was discarded and diluted Wash Solution (750 µl) was added to the column.
The column was centrifuged (14000 rpm, 1 min) twice to remove residual salts, other contaminants and excess ethanol, and the flow-through liquid was discarded. Finally, the column was transferred to a fresh collection tube and sterile bidi (100 µl) was added to elute the pDNA.
37
3 Replicates : 5 replicates were made for construct ‘4’: 3 in first LR reaction, 2 replicates in a second one.
59

Materials and methods
4.3.1.4
pDNA quantitation
•
Principle
The recovery and purity of the plasmid DNA was determined by spectrophotometric analysis.
Absorbance of the pDNA at A
260
and of proteins at A
280
was measured. The ratio of absorbance at
A
260
/A
280
should be between 1.8 and 2.0 to have pure plasmids. Absorbance at A
260
was converted to pDNA concentration.
•
Protocol
In preparation for pDNA quantitation, pDNA samples were centrifuged (14000 rpm, 10 min). The pDNA concentration was determined using the NanoDrop ® ND-1000 spectrophotometer (Life
Science, De Meern, the Netherlands). First, 1 µl bidi was pipetted on the Micro-Volume pedestal and a calibration check was executed. Then, for each pDNA sample 1 µl was pipetted on the Micro-Volume pedestal. The Micro-Volume UV spectrophotometer measures the absorbance of the pDNA at A
260
and proteins at A
280
. By a pre-configured method 38 , absorbance at A
260
was converted to pDNA concentration.
4.3.1.5
Plasmid control
To check for the presence of the promoter and Perceval sequence in the purified plasmids, both a restriction digest and a PCR reaction were performed, followed by gel electrphoresis. For one construct a colony PCR was performed. After this preliminary check, good candidate plasmids were also sequenced to ensure the absence of frameshifts or other mutations in the expression vectors.
4.3.1.5.1
Restriction Digest and Gel Electrophoresis
•
Principle
The bioinformatics tool Vector NTI Advance™ 11.0 (Invitrogen) was used to choose restriction enzymes for each construct that cut at 3 restriction sites in the pDNA. This resulted in 3 clearly separated bands on an agarose gel that could be scored in length (bp). As a negative control undigested pDNA of each construct was loaded on the gel.
38
Pre-configured method : Used Beer-Lambert law and extinction coefficient for dsDNA of
0.020 (µg/ml) -1 cm -1 .
60

Materials and methods
•
Protocol
The purified pDNA, restriction enzymes (Fermentas and New England Biolabs) and associated buffers were thawed on ice. In a first restriction digest pDNA (5 µl) of all 3 replicates of 7 39 constructs was added to 0.5 ml microcentrifuge tubes. The chosen restiction enzyme (1 µl) was added together with its restriction enzyme buffer (1 µl). For some constructs, a double digest (using 2 restriction enzymes) was performed to get 3 restriction sites according to the manufacturer’s instructions
(www.fermentas.com). Sterile bidi was added to reach a total volume of 10 µl. After the mix was incubated at 37 °C for 2 hours, 5 µl of the mixture was transferred to a new 0.5 ml microcentrifuge tube, CoralLoad 40 Concentrate (10% w/w = 0.5 µl) was added and sterile bidi (4 µl) completed the mix, followed by a shortspin. Also full length pDNA (5 µl) of each construct and replicate was mixed with CoralLoad Concentrate (10% w/w = 0.5 µl) and bidi (4 µl), followed by a shortspin.
Restriction fragments were separated by gel electrophoresis. A 2% agarose gel was made by first adding agarose 41 (2g) to TAE buffer 42 (200 ml, 1x). The agarose was dissolved by heating in a microwave until a homogenous solution was attained. For a big gel (containing 17 slots), a 50 ml falcon tube was filled with agarose solution (45 ml) and ethidium bromide 43 (1.8 µl stock solution).
The solution was thorougly mixed by inversion and poured in a gel tray. The comb was inserted and gel was solidified for 40 min. The comb was removed and the gel was transferred to the electrophoresis chamber and submersed in TAE buffer (1x). The gel slots were loaded with the pDNA-CoralLoad mixes of 15 different samples and a DNA ladder (GeneRuler 1kb plus, Fermentas), to compare loaded pDNA fragment size.
39
7 constructs : The restriction digest for construct ‘4’, pRPL-32::Perceval, was postponed since no bacterial colonies were observed upon transformation. For this construct the LR-reaction and bacterial transformation were repeated, followed by plasmid purification.
40
CoralLoad : A loading dye made with a high concentration of sugar which is heavier than the buffer solution.
It causes the dye, and the pDNA within, to sink to the bottom of the slots in the gel during the gel loading. It functions also to visualize how far the DNA has migrated in the gel during the separation.
41
Agarose : Composed of long unbranched chains of uncharged carbohydrate without cross links resulting in a gel with large pores.
42
TAE buffer : Tris/Acetic acid/EDTA. Tris Acetic acid solution buffers in slightly basic conditions (pH 8.0), which keep DNA deprotonated and soluble in water. EDTA protects DNA against enzymatic degradation by chelating divalent ions, which are necessary co-factors for possible contaminant nucleases.
43
Ethidium bromide : DNA intercalating agent which fluoresces orange (20-fold more intense after binding to
DNA) upon UV excitation.
61

Materials and methods
For small gels (8 slots) the same protocol was used, except a 50 ml falcon tube was filled with 25 ml agarose solution and 0.9 µl ethidium bromide. The electrophoresis chamber was closed and a current
(200 V/cm) was applied for 23 min. The DNA migrated to the positive electrode. Shortly after cessation of electrophoresis, the gel was placed in an ultraviolet transilluminator (BioDoc-it, UVP) and a photographic image was captured for analysis.
Two restriction digests and gel electrophoresis runs were carried out to include the 8 th construct (N° 4:
Prpl-32::Perceval), the full Perceval and Gas-1tp::Perceval pDNA and to try new restriction enzymes for 2 other constructs (Prps-0::Perceval and Prps-0::gas-1tp::Perceval), which gave inadequate results upon first restriction digest and gel electrophoresis.
•
Analysis
The positions on the gel of the bands, which represent the fragments (or full pDNA), were compared with the DNA ladder of which the bands have known lengths. The fragment (or full pDNA) length should be equal to the theoretical restriction fragment (or full pDNA) length on Vector NTI
Advance™ 11.0 (Invitrogen TM , Carlsbad, California, USA).
4.3.1.5.2
PCR
•
Principle
The presence of the transgene (
Perceval
gene and promoter) on the pDNA of 7 constructs was also checked by performing a PCR (Polymerase Chain Reaction). The target DNA sequence (3’-end of the promoter, a 5’-part of Perceval and the transition in between) was exponentially amplified and separated by gel electrophoresis. Fragment length was determined by comparing with a DNA ladder.
For construct ‘4’, Prpl-32::Perceval, a colony-PCR was executed to control the presence of the transgene. The procedure was faster because 4 colonies were directly picked from the LB plates after bacterial transformation. The plasmids had not to be upscaled and purified, but underwent the PCR together with the purified plasmids of the 7 other constructs.
•
Protocol
A forward primer in each promoter and a common reverse primer in Perceval were selected using the
‘Primer Design’ tool of Vector NTI Advance™ 11.0 (Invitrogen TM , Carlsbad, California, USA). The selection was based on several parameters; primers consisted of at least 18bp, G-C content was between 35-60 %, melting temperature (Tm) was higher than 45 °C, nucleotide repeats, dimers, palindromes and hairpin loops were avoided. Importantly, for PCR the forward and reverse primers were selected to be 1000-3000 bp apart.
62

Materials and methods
The primers that were ordered at Invitrogen™ (Carlsbad, California, USA) and used for the PCR reaction were: Fwrps0 (5’- GGC AAC GAG AAA TAG GAA A -3’), Fwrpl17 (5’- CGC CTT TAA
ATA CAT TTC G -3’), Fwrpl24.1 (5’- CGC ACA GCC GAA ACG AGT GTT A -3’), Fwrpl32 (5’-
TTG GTT CAG ACG GTT CAG TGG TTT -3’) and RePCV (5’- ATC TAC TAT GGT GCC GTT -
3’). For all primers a stock solution (100 µM) was made by adding Tris (10 mM, pH 8.0). The primers were diluted (25 µM) in bidi before use. For each sample a 200 µl PCR tube was filled with template
(0.125 µl), forward primer (0.25 µl, 25 µM), reverse primer (0.25 µl, 25 µM), Taq DNA polymerase
(QIAGEN ® , Hilden, Germany) (0.125 µl), dNTPs 44 (0.5 µl), MgCl
2
45 Solution (2 µl), 10X TopTaq
PCR buffer (2.5 µl), 10X CoralLoad Concentrate (2.5 µl). Sterile bidi (16.75 µl) was added to reach a volume of 25 µl. The reaction mixture was shortly vortexed, shortspinned and incubated in the
Eppendorf thermal cycler for PCR. The target sequence was amplified using the following conditions: initialization step (95 °C, 10 min), 30 cycles of denaturation (95 °C, 45 sec), annealing of forward and reverse oligonucleotide primers (± 60 °C, 30 sec) and DNA polymerization (72 °C, 2 min) by Taq
DNA polymerase 46 (QIAGEN ® , Hilden, Germany) final elongation (72 °C, 5 min) and final hold (12
°C,
∞
) . After PCR, gel electrophoresis (see
4.3.1.5.1, Restriction Digest and gel electrophoresis
) was used to estimate the length of the PCR product. Two agarose gels were loaded to include all samples.
In the second gel, a non template control was included.
The colony PCR was executed by touching a single colony with a pick and rubbing it in sterile bidi
(30 µl) of a 0.5 ml microcentrifuge tube. 1.25 µl of this mix was used as template for PCR as described above.
• Analysis
The length of the PCR product was compared with the DNA ladder and should be the theoretical distance (Vector NTI) in bp between the 5’-ends of both primers.
44 dNTP : Deoxynucleotide triphosphates: dATP, dGTP, dCTP, dTTP.
45
MgCl
2
: Cofactor of Taq DNA polymerase; it increased the productivity and fidelity of the enzyme.
46
Taq DNA polymerase : A thermostable DNA polymerase isolated from the thermophilic bacterium Thermus aquaticus which lives in hot springs and hydrothermal vents.
63

Materials and methods
4.3.1.5.3
Cycle Sequencing
Finally, to ensure the absence of frameshifts and other mutations in the constructs, the expression plasmids were also sequenced.
Cycle sequencing was performed (by Andy Vierstraete) using an Applied Biosystems ABI 3130XL
Genetic Analyzer. The resulting sequences were compared with the theoretical sequence (from Vector
NTI) with the aid of an online pairwise sequence alignment tool (EMBOSS-needle, pairwise sequence alignment, http://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html).
Primers used for sequencing were: FwExp (5’- CGG GTT TCG CCA CCT CTG ACT -3’), rps0 (5’-
GGC AAC GAG AAA TAG GAA A -3’), rpl17 (5’- CGC CTT TAA ATA CAT TTC G -3’), rpl24.1
(5’- CGC ACA GCC GAA ACG AGT GTT A -3’), rpl32 (5’- TTG GTT CAG ACG GTT CAG TGG
TTT -3’), RePCV (5’- ATC TAC TAT GGT GCC GTT -3’), ReExp (5’- GCC CAG ACG TGC GAA
GAA ATA AA -3’). The primers were selected as in
4.3.1.5.2, PCR
.
4.3.2
Transformation of C . elegans by microinjection
4.3.2.1
Microinjection
•
Principle
C. elegans unc-119(ed3)
mutants were transformed to introduce the fluorescent ADP:ATP sensor protein Perceval. The plasmids containing the correct sequence are injected into the distal arm of the gonad because it contains a syncytium that is shared by many germ cell nuclei (Mello et al., 1991;
Mello & Fire, 1995). As such, a lot of transgene progeny can be acquired. Microinjection is a common transformation technique which usually generates large extrachromosomal DNA arrays with many copies of the transgene (Mello & Fire, 1995). In addition to Perceval, the injected plasmids contained a selectable marker gene, unc-119(wt) . Transgene expression of unc 119(wt) rescues the loss-offunction mutant gene unc-119(ed3) and induces a dominant wild-type phenotype, which can be easily distinguished from the ‘uncoordinated’ phenotype.
•
Protocol
A DNA injection mix (10 µl) was made by adding DNA ladder (0.5 µl) to pDNA (70 ng/µl final concentration). Sterile bidi was added to complete the mix when the measured pDNA concentration was equal or higher as 73.70 ng/µl. Just before microinjection, the DNA injection mix was centrifuged
(14000 rpm, 10 min). Per construct 6 well-fed young adult hermaphrodite unc-119 mutant worms were injected (by Marjolein Couvreur), each with 1 µl DNA injection mix. They recovered in S-buffer for
30 min and were transferred onto NGM plates at 20 °C.
64

Materials and methods
During a first microinjection the four samples 1-3 (Prps-0::Perceval), 2-1 (Prpl-17::Perceval), 3-2
(Prpl-24.1::Perceval) and 6-1 (Prpl-17::Gas-1tp::Perceval) were injected. During a second one, the three samples 4-2 (Prpl-32::Perceval), 7-3 (Prpl-24.1::Gas-1tp::Perceval) and 8-2 (Prpl-32::Gas-
1tp::Perceval) were injected.
•
Analysis
Several days after microinjection, F1 progeny were screened for transgenes. A transgenic C. elegans worm is wild-type and could easily be distinguished from the non-transgene unc-119
mutants by their rescued mobility. Wild-type worms show typical sinusoidal movement while unc-119 mutants move rather uncoordinated. In cases of doubt, the worms are touched in the head region with a needle.
Consequently, a wild-type worm shows backward locomotion, while in unc-119(ed3) mutants no alteration in direction of displacement is observed. Additionally, a fluorescence dissecting microscope
(Olympus FZX12) was used to screen for worms showing green fluorescence (excitation 460-
490nm/emission 510-550). Transgenic worms were picked with a needle and transferred to a new
NGM plate. Transgenic lines were established and upscaled by selecting those animals that segregate the arrays in their progeny.
4.3.2.2
UV irradiation of transgenic C. elegans
•
Principle
The microinjected plasmids are present in transgene C. elegans cells as extrachromosomal arrays, which segregate randomly and can be lost. To obtain stable transgenic lines, these arrays can be randomly integrated into a chromosome upon ultraviolet irradiation. UV irradiation presumably induces chromosomal breaks and ligation of arrays to chromosomes during DNA repair.
Consequently, mutations can arise in treated worms, so recovered integrated strains should be outcrossed by mating with wild-type males.
•
Protocol
To each of two NGM plates (without bacteria) 30 L4 unc-119 mutant worms were picked. The plates were placed in a Stratagene UV crosslinker, with the lids removed. Power was set to ~4000
µwatts/cm2 and the energy of the UV bulb decreased with time until exhausted after half a minute.
The UV irradiated worm were transferred to OP50 seeded NGM plates.
65

Materials and methods
•
Analysis
After two weeks, the plates were screened for fluorescent worms. 80 highly fluorescent individual worms were transferred to separate plates and checked for high transmission of the transgene. Of those plates that contained ‘high transmission’ (more than 90%) clones, 30 worms were separated to fresh plates. Also 20 worms of ‘intermediate transmitters’ were cloned to new plates. Other animals were discarded. Of the following generations only ‘high transmitters’ were selected, separated and cultured.
4.3.3
Biolistic transformation of C. elegans
Transgenic
C. elegans
worms can also be obtained by biolistic transformation. With this technique unc-119(ed3) mutant worms are bombarded with gold particles to which the transgenic DNA is coated. A considerable share of transformants generated this way has integrated the DNA (usually containing a low copy number of the transgene). Besides integrants, gene bombardment also generates extrachromosomal arrays. However, the unc-119(wt) gene is used as a selectable marker which helps to enrich for integrants (Praitis et al., 2001). unc-119(ed3) mutants move uncoordinated and cannot survive dauer formation (Maduro & Pilgrim, 1995). Hence, unc-119 rescued worms survive starvation and probably carry transgenes integrated at various non-homologous sites in the genome (Evans,
2006).
Since construct ‘1’ (replicate 3) was the only one to give good expression upon microinjection, we decided to only use this construct for biolistic transformation.
4.3.3.1
Plasmid purification
•
Principle
Biolistic transformation of 1,200,000 worms on 6 plates required 30 µg pDNA, therefore the amount of pDNA had to be upscaled. To this end, a bacterial culture of
E. coli
containing the plasmid with
Prps-0::Perceval (construct ‘1.3’), was grown in 150 ml LB with ampicillin, and plasmid DNA was isolated using the GenElute™ Plasmid Maxiprep Kit (Sigma-Aldrich ® , Saint Louis, Missouri, USA).
The principle is the same as of a ‘miniprep’ (see 4.3.1.3, Plasmid purification ), but higher amounts of plasmid can be recovered.
•
Protocol
Before starting and prior to initial use, RnaseA Solution was briefly spinned and 750 µl (10 prep kit) was added to the Resuspension Solution, which was stored at 4 °C. Wash Solution 2 was diluted with
120 ml of 95-100% ethanol.
66

Materials and methods
Isolation of plasmids with a maxiprep starts by harvesting the overnight E. coli culture (150 ml). The cells were centrifuged (5000 rpm, 10 min) and supernatant discarded. Resuspension/RNaseA Solution
(12 ml) was added, followed by vortexing. Lysis Solution (12 ml) was added and the mixture was inverted (6-8 times). After 3-5 minutes the lysed cells were neutralized by adding chilled
Neutralization Solution and gently inverting (4-6 times). Binding Solution (9 ml) was added, followed by inverting (1-2 times). The cell lysate was poured immediately into the barrel of a filter syringe of which the plunger was removed. After 5 minutes of incubation a white aggregate (cell debris, proteins, lipids, SDS, and chromosomal DNA) should float to the top. Meanwhile a GenElute HP Maxiprep
Binding Column was placed into a 50 ml collection tube, Column Preparation Solution (12 ml) was added to the column, followed by centrifugation (3000 rpm, 2 min) and eluate was discarded. The filter syringe barrel was held above the Binding Column and, gently, pressure was applied to the plunger to expel half of the cleared lysate into the column. The collection tube with the column was centrifuged (3000 rpm, 2 min), eluate was discarded and rest of the cleared lysate was added to the column and the centrifugation was repeated. After discarding the eluate, Wash Solution 1 (12 ml) was added to the column, which was subsequently centrifuged (3000 rpm, 2 min). Eluate was discarded and Wash Solution 2 (12 ml) was added, followed by centrifugation (3000 rpm, 5 min). The binding column was transferred to a clean 50 ml collection tube and sterile bidi (3 ml) was administered. A final centrifugation step (3000 rpm, 5 min) was taken to recover as much plasmids as possible. pDNA concentration was measured with the Nanodrop spectrophotometer as described previously.
4.3.3.2
pDNA precipitation
•
Principle
Biolistic transformation requires a pDNA concentration of around 1000 ng/µl. To achieve this concentration, plasmid DNA from the maxiprep preparation was concentrated using a method based on alcohol precipitation.
•
Protocol
The recovered pDNA was transferred to a clean 1.5 ml microcentrifuge tube. Sodium Acetate Buffer
Solution (0.1 volumes, 3.0 M, pH 5.2) and isopropanol (0.7 volumes) were added, followed by gentle inversion and centrifugation (15000 rpm, 4 °C, 30 min). Supernatant was discarded carefully and the undisturbed pellet was rinsed with ethanol (1.5 ml, 70%). After centrifugation (15000 rpm, 4 °C, 10 min) supernatant was discarded and pellet was air-dryed until residual ethanol was evaporated. The pellet was resuspended in 40 µl sterile bidi and pDNA concentration was measured (see 4.3.1.4, pDNA quantitation
).
67

Materials and methods
4.3.3.3
Restriction Digest and Gel Electrophoresis
•
Principle
Biolistic transformation requires linearized plasmids in order to avoid that crucial pDNA regions are disrupted upon integration of the plasmid into chromosomal DNA. Linearization was achieved using the restriction enzyme NotI, which cuts at 1 site prior to the promoter region. The absence of unwanted additional NotI restriction sites was checked in VectorNTI software.
•
Protocol
80 µl pDNA (300 ng/µl), 8 µl buffer O (Fermentas) and 8 µl NotI (Fermentas) were added to a 0.5 ml microcentrifuge tube, gently mixed and shortspinned. The mixture was incubated overnight at 37 °C and efficiency of the restriction digest was confirmed by agarose gel electrophoresis.
4.3.3.4
Preparation of golden beads
A 1.5 ml silicon eppendorf tube was filled with golden beads (18 mg, 1 µm; Bio-Rad) and ethanol (1 ml, 70%). The mix was vortexed (5 min), the beads were settled (15 min), a shortspin was executed and supernatant was removed. Three cycles of adding sterile bidi (1 ml), vortexing (1 min), settling (1 min), shortspinning and removing of supernatant were performed. Glycerol (300 µl, 50%) was added and the mix was vortexed until completely resuspended. This gold beads-glycerol mixture was stored at 4 °C.
Just before the biolistic bombardment, the beads were further prepared. The mixture was vortexed (5 min). 300 µl of this mix was transferred to a new 1.5 ml silicon tube, followed by vortexing (1 min).
Linearized pDNA (80 µl), CaCl
2
(300 µl, 2.5M) and spermidine (120 µl, 0.1 M freshly prepared;
Sigma-Aldrich®, Saint Louis, Missouri, USA) were added while in between each addition the mix was vortexed (1 min). The final mixture was vortexed (at least 3 min), beads were settled, mix was shortspinned and supernatant removed. The pellet was resuspended in 70% ethanol (900 µl), shortspinned and supernatant removed. A second and third resuspension step used 100% ethanol (900
µl and 300 µl). The final mix was vortexed (at least 3 min).
It was always checked that beads were not aggregating. If so, they were additionally vortexed or sonicated.
68

Materials and methods
4.3.3.5
Culture of unc-119 mutant worms
For biolistic transformation, around 1,200,000 worms on 6 agar plates were required (for 6 bombardments). unc-119(ed3) mutant worms were cultured on NA plates (see 4.1.5.1, Monoxenic plate cultures
) until 100,000 worms were obtained and chloroxed. Around 300,000 chloroxed L1 unc-
119 mutants were divided over 3 Fernbach flasks, supplied with 250 ml S-buffer, 23.08 g/l frozen E. coli cells and 10 µg/ml of cholesterol. These worm suspension cultures were incubated (24 °C) and shaken (120 rpm). On a daily basis the worms were feeded with sufficient amounts of bacteria (around
10 g/l). When the worms reached young adulthood, supplementation of food was halted to obtain an
L1-arrested population in the next generation. When approximately 1,200,000 L1’s were present, adults were settled to the bottom and L1 were transferred to 3 new Fernbachs (with same conditions).
Several days later the young adults were settled, washed with S-buffer and maximum 1 ml of worm pellet was spread on each of 6 plates (NA, K12). The plate lids were removed and plates were dried in the laminar flow bench. Afterwards, the plates were placed at 4 °C to avoid movement of the worms during the bombardment.
4.3.3.6
‘Biolistic transformation instrument’ or ‘Particle delivery system’
•
Principle
The gene gun used was the biolistic PDS-1000/He Hepta instrument ( Figure 24 ; Bio-rad, #165-2258), which accelerates subcellular-sized gold microparticles coated with DNA at a very high velocity to transfect cells. The acceleration is possible by a combination of vacuum circuits and building up of helium pressure. When the fire button is pressed, a rupture disk bursts under the high pressure. This generates a helium wave shock through the hepta adapter towards the bombardment chamber where the plate with worms is located. The shock hits the macrocarriers (plastic films carrying the DNAcoated microparticles) towards the target worms. Between the worms and the macrocarriers a stopping screen was placed, which retains the plastic disks while the coated gold microparticles are allowed to pass through and penetrate the worms.
69

Materials and methods
Rupture disk
Macrocarriers
Helium
Stopping screen
Plate (worms)
Vaccuum chamber
Figure 24 | Biolistic PDS-1000/He Hepta instrument. Right: Main chamber containing the macrocarrier launch assembly and the bombardment helium pressure gauge. Enlarged: Hepta adapter. Left: The gauge monitors the vacuum within the chamber, both lower knobs adjust the vacuum flow and vent rates. The three upper switches are the on/off, fire, and vaccuum switch. Helium entry, vaccuum chamber, position of the plate with worms, stopping screen, rupture disk and one of 7 macrocarrier positions are indicated by arrows.
•
Protocol
Before the first biolistic transformation, a test bombardment was executed to test all components of the
PDS-1000/He Hepta instrument and to check if all channels were filled with Helium. During the test no macrocarriers and worms were used. The system was set up, a rupture disk (1350 psi) was submersed in isopropanol (70%) and placed wet in the retaining cap. The retaining cap en Hepta adapter were screwed with a torque wrench. Next, the instrument and the vaccuum pump were switched on, the Helium tap turned open (1500 psi) and subsequently vacuum switched on. When pressure reached 27 inch Hg, the ‘vacuum’ button was switched to ‘hold’, ‘fire’ button pressed until rupture disk ruptured. Next, the ‘fire’ button was released and ‘vacuum’ button switched to ‘vent’.
When pressure reached normal levels, the chamber was opened and the instrument shut down.
The actual bombardment of the worms started by first cleaning the Particle Delivery System (PDS) with ethanol (70%). A ‘dessicator chamber’ was set up on a cushion to prevent vibrations and to allow uniform evaporation of ethanol. The chamber consists of a big petridish filled with CaCl
2
47 beads and a round filter paper.The ‘macrocarrier holder’ was placed upon the dessication chamber and the macrocarriers were brought into the holder by means of a ‘seating tool’.
47
Calciumdichloride : A hygroscopic salt which is used to absorb air moisture.
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Materials and methods
DNA-coated gold particles (6 µl) were pipetted out of a tube, which was vortexed at the moment of pipetting. The beads were spread out on a macrocarrier. The same amount was spread on the 6 other macrocarriers. Ethanol was damped out, while the hepta stopping screen was sterilized by submersing it in ethanol (70%) and air-dried. The stopping screen was placed on the macrocarrier holder and, subsequently, the rupture disk was submersed in isopropanol (70%) and placed wet in the retaining cap. The retaining cap and Hepta adapter were transferred to the top of the PDS and screwed with the
‘torque wrench’. Underneath, the macrocarrier holder containing the macrocarriers was placed in alignment with the hepta adapter, with the particles aiming downwards to the stopping screen. A cooled, uncovered plate with worms was placed on the ‘target shelf’ at a distance of 6 cm to the macrocarrier holder. The worms were bombarded as described above.
Between different bombardments, all components were cleaned with ethanol (70%). Worms were spread to 12 plates (NA, K12) after at least one hour of recovering from the bombardment.
•
Analysis
All 96 plates were screened for wild-types, since transformation with the co-transformation marker gene unc-119(wt) dominantly rescues the severe uncoordinated and dauer defective phenotypes of unc-119(ed3)
mutants. During one to two weeks after bombardment, the plates were starved. A fresh plate (NGM, OP50) was cut up into little chunks (1 cm 2 ), which were each placed in the middle of a starved plate, with the side containing bacteria upright (O’Connell, 2010). The next days the bacterial lawn on top of the transferred chunks was screened for rescued worms. These show normal wild-type movement and have a significant advantage, compared to non-rescued uncoordinated worms, in climbing to the top of the chunk to reach the food. In addition, the rest of the plates could also be screened for dauers, since unc-119(ed3)
mutants cannot survive starvation by developing as a dauer.
Phenotypic wild-type worms were transferred to new plates (NGM, OP50) and screened for fluorescence with a fluorescence stereo microscope (Olympus FZX12).
4.3.3.7
DNA extraction
•
Principle
Several worms displayed a wild-type, rescued phenotype, but lacked fluorescence as analyzed by fluorescence microscopy (excitation 460-490nm/emission 510-550). To check if these worms were transformed with the Perceval gene, several individuals were picked and genomic DNA (gDNA) was extracted.
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Materials and methods
•
Protocol
Of those rescued worms that lacked fluorescence, 1 was picked for each bombarded plate and transferred to 0.5 ml microcentrifuge tube. Of the first 3 bombarded plates, 10 rescued worms were pooled. The tube contained 20 µl Worm Lysis Buffer (50 mM KCl, 10 mM Tris pH 8.3, 2.5 mM
MgCl
2
, 0.45% NP 40 (Tergitol ® , Sigma-Aldrich ® , Saint Louis, Missouri, USA) and 0.45% Tween 20).
Next, the tubes were frozen (-80 °C, 10 min) and 1 µl proteinase K was added. The mix was incubated
(65 °C, 1 h and 95 °C, 10 min) and centrifuged (14000 rpm, 1 min).
4.3.3.8
PCR
•
Principle
A PCR was performed on the extracted gDNA to detect the presence of Perceval.
•
Protocol
Of each tube with extracted gDNA, 10 µl was used as template in the PCR. As a forward primer rps0
(5’- GGC AAC GAG AAA TAG GAA A -3’) was used, as reverse primer RePCV (5’- ATC TAC
TAT GGT GCC GTT -3’). Negative controls, gDNA of wild-type N2 and GA804, and a positive control, pDNA of construct 1-3, were included in the PCR. For protocol, see 4.3.1.5.2, PCR .
•
Analysis
The length of the PCR product was compared with the DNA ladder and should be the theoretical distance (Vector NTI) in bp between the 5’-ends of both primers.
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Materials and methods
4.3.4
Measuring ATP/ADP ratios in vivo with Perceval upon sodium azide treatment
4.3.4.1
Fluorescence spectroscopy measurement
•
Principle
Fluorometry, or fluorescence spectroscopy, was used to excite Perceval at 490 nm and 405 nm, and detect the resulting emission. Sodium azide was added, in different concentrations, to Perceval transgenic worms to detect differences in the ratio of emission upon excitation at both wavelengths.
•
Protocol
Several plates containing a mix of fluorescent, mosaic fluorescent and non-fluorescent worms
(obtained from microinjected and UV irradiated culture, see 4.3.2.2, UV irradiation of transgene C. elegans
) were washed 3 times with S-buffer to get rid of the bacteria. Approximately equal amounts of
N2 worms were washed. The worms were allowed to settle to the bottom and supernatant was removed. 100 µl of a dense pellet (1000-4000 worms) was loaded in a well of a black 96 wells microtiter plate. In 4 columns 3 or 4 replicates of N2 worms were loaded, in 4 other columns 4 replicates of Perceval-transgene worms.
Fluorescence was measured for 15 repeats at 25 °C with a Victor 2 1420 Multilabel counter (Perkin
Elmer, Boston, MA) with 490-nm (ATP-bound Perceval) and 405 nm (ADP-bound Perceval) excitation filters and a 535-nm emission filter. In between the readings, the microtiter plate was mixed during 0.5 seconds.
To verify the reactivity of Perceval to ATP changes, sodium azide with final concentrations of 0 mM,
5 mM, 10 mM and 15 mM in sterile bidi was added to 4 columns of both strains. The plate was transferred back to the Victor as quickly as possible. Fluorescence was measured for 100 repeats.
After measurement all worms of each well were resuspended in sterile bidi and transferred to 2 ml sampling tubes. Protein content was measured using the BCA assay (see
4.2.3, BCA assay for quantitative comparisons ).
•
Analysis
Raw data were processed with Excel 2007. The ratio of fluorescence upon excitation at 490 nm and
405 nm was calculated and plotted over time.
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Materials and methods
4.3.4.2
Confocal laser scanning microscopy measurement
•
Principle
By means of a confocal laser scanning microscope (CLSM) Nikon Eclipse C1si optical in-focus images were taken (from a selected depth) of Perceval -transgenic C. elegans worms (obtained from microinjection), both with and without administration of sodium azide. Perceval was excited with a
488-nm Argon diode laser and a 405-nm Multi-line Argon laser. Emission was detected through a
525/50 nm narrow bandpass filter. Several images were taken at different enlargements.
•
Protocol
Several highly fluorescent worms were transferred to a 50 µl drop of S-buffer on an NGM plate without bacteria. After a while a few of them were picked and put on a glass substrate in the centre of a square drawn with a hydrophobic Dako Pen. The worms were paralysed by adding levamisole (10 mM). The square was gently covered with a cover glass and sealed with nail polish. The glass slide was transferred to the CLSM and an overview shot was captured using a 10x objective. Several headto-tail images (with the same zoom) were taken with 40x and 60x scopes. The 60x objective was always used with immersion oil. A second glass slide was prepared in the same way, except sodium azide (5 mM) instead of levamisole was added. Besides its paralysing effect, sodium azide also shuts down the electron transport chain, resulting in a drop of ATP. During 10 minutes several pictures
(with the same zoom) were taken of the head region of 1 worm.
•
Analysis
Saved images were first processed with both Nikon EZ-C1 3.40 software, analysed and visualised by
Fiji, an Java based open source packaged version of ImageJ (W. Rasband, National Institues of Health,
Bethesda, Maryland, USA) (http://pacific.mpi-cbg.de/wiki/index.php/Downloads). Head-to-tail images were stitched automatically by running the macro ‘StitchMacro2D b.ijm’ in Fiji. Fiji was also used, with the macro ‘Create INR img Macro d.ijm’, to create an intensity normalized ratio (INR) image, which displays the Perceval ratio of different areas in a worm by integrating the fluorescence intensity levels. Two images were used, one of the channel to detect the fluorescence of ATP-bound
Perceval and a second of the channel to detect fluorescence of ADP-bound Perceval. A mere ratio image, of which values of one image were divided by the other image, also displays the ratio of areas where no fluorescence is present, thus complicating data interpretation. An INR image overcomes this problem, because the ratio of both fluorescence channels is represented by a shade, while the saturation of this shade depicts the average intensity of both channels. Next to the INR image, also a
‘value’ image was obtained in the same way. It represents the mean values of fluorescence upon excitation at both 490 nm and 405 nm.
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5.1.1
Chromatograms
The fluorescence fetector of the HPLC excites eluting fluorescent etheno-adenine nucleotides at a wavelength of 340 nm. The amount of emission, captured at a wavelength of 420 nm, was plotted in a chromatogram over the retention time (minutes), which represents the timespan between injection and elution. A chromatogram example of a typical run (55 minutes) is shown beneath (
Figure 25
).
Signal intensity (mV)
Baseline
?
Baseline
Retention time (min)
ATP ADP AMP Adenine
Adenosine
Signal noise
Figure 25 | Chromatogram representing the signal intensity (mV) as a function of retention time (min). Injection volume of 5 µl. Retention times of ATP, ADP and AMP are indicated by arrows. Question mark indicates a relatively large unidentified peak. The increase in baseline signal between 25 and 45 minutes is probably an artifact due to changing mobile phase. The retention time for adenine and adenosine falls within this timespan.
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By using standards the retention times of ATP, ADP and AMP were determined. Usually, they have signal peak at retention times of 11, 13 and 21.5 minutes respectively. Although retention times of
ATP and ADP are close to each other, the separation is good enough to virtually reach the baseline in between both peaks. A single relatively large peak (indicated by the question mark) represents the signal from an unidentified compound. Adenine and adenosine peaks are not fully detectable because their retention times fall within the signal noise.
Because of technical issues not all of the samples yielded a chromatogram as represented in Figure 25 .
A great deal of HPLC measurements resulted in a chromatogram as shown below (
Figure 26
).
Signal intensity (mV)
NADP +
NADPH
ATP ADP AMP
NAD +
?
Adenine
Adenosine
Signal noise
Retention time (min)
Figure 26 | Chromatogram with incomplete separation of AMP. Possible identities of smaller peaks that were formerly unknown are indicated. Injection volume of 5 µl. Arrows indicate retention times of several adenine nucleotides. Question mark indicates the possible presence of a yet unidentified adenine nucleotide.
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Instead of an AMP peak preceeded by an unknown peak ( Figure 25 ), in the chromatogram represented by
Figure 26
only one peak is present, with a retention time of 19 minutes. Retention times of ATP and ADP are slightly increased (1 minute) in the latter chromatogram.
Since ATP, ADP and AMP are not the only adenine nucleotides that can be derivatized potentially,
NADP + 48 , NADPH 49 and NAD + 50 were derivatized and injected to identify the relatively large unidentified peak with retention time of 17 minutes in the chromatogram shown in
Figure 25
. Both
NADPH and NADP + elute unseparated with a retention time of 6 minutes and show a low signal peak, while NAD + has the same retention time as AMP (
Figure 26
). Neither NADH 51 nor cAMP 52 could be injected due to limitation in time.
For some samples 2 µl was injected instead of 5 µl. In these cases, high levels of adenine nucleotides were anticipated because of high protein concentrations of the samples. Therefore, lower amounts of derivatized sample were injected to avoid signal saturation. Contrary to our expectation, these 2 µl injections resulted in very low signals intensities for the ATP, ADP and AMP-containing peaks
( Figure 27 ). The signal intensity of these peaks is several fold lower than usual, while the noise signal remains largely the same. Retention times of all three peaks were shifted to 13, 15 and 21 minutes respectively.
48
NADP
+ : Oxidized form of nicotinamide adenine dinucleotide phosphate.
49
NADPH : Reduced form of nicotinamide adenine dinucleotide phosphate.
50
NAD
+ : Oxidized form of nicotinamide adenine dinucleotide.
51
NADH : Reduced form of nicotinamide adenine dinucleotide.
52 cAMP : cyclic adenosine monophospate.
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Signal intensity (mV)
ATP ADP AMP
NAD +
?
Adenine
Adenosine
Signal noise
Figure 27 | Chromatogram with very low signal detection. Injection volume of 2 µl.
Retention time (min)
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ResultsResults
5.1.2
Absolute concentrations of adenine nucleotides
5.1.2.1
Introduction
Four replicates of two C. elegans strains, N2 (wild type) and the daf-2(e1370) longevity mutant, were age-synchronised, cultured in liquid medium and sampled each day, for up to 16 days maximum.
Because of technical problems during nematode culture such as unsufficient yield of nematode population or asynchronous culture, not all aging series could be sampled completely.
Quantities of ε -ATP, ε -ADP and ε -AMP were measured which resulted in a chromatogram for each sample. The original amount of ATP, ADP and AMP in the worms sampled was calculated by correlating the obtained peak area with standards and correcting for protein content and injection volume.
Due to technical issues only few reliable data were obtained (separation as in Figure 25 ). In the following figures these data are plotted as bar charts and are called ‘final’ data. Other data were less reliable because of two reasons: (1) either the separation was incomplete as shown in Figure 26
(which was the case for both some N2 and daf-2 datapoints), (2) or the separation was incomplete and the signal intensity was too low as in Figure 27 (which was the case for only a few N2 datapoints).
The latter error leads to lower levels measured and larger standard deviations when combined with
‘final’ data. The former error might yield an overestimation of the absolute level of AMP, since the peak that we annotated as AMP (19 min, Figure 26) possibly combines the two peaks, AMP (21 min) and the unidentified peak (17 min), depicted in
Figure 25
. ATP and ADP peaks were separated well and are reliable.
The less reliable data were supplemented with the ‘final’ data and are plotted as scatter charts with the term ‘preliminary’.
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5.1.2.2
ATP
‘Final’ and ‘preliminary’ ATP levels of N2 and
daf-2(e1370)
worms in function of age are depicted in
Figure 28 . The number of biological replicates are indicated by the table below each chart. Error bars represent standard deviations.
ATP levels of N2 worms progressively decline with age. Day 1 adults of N2 contain about 40 nmole/mg protein and this is halved within 10 days. Young daf-2(e1370) adults contain ATP of about
70 to 90 nmole/mg protein, while daf-2(e1370) mutants of 10 to 16 days old adults have ATP levels of about 35 to 80 nmole/mg protein. Consequently, daf-2(e1370) worms maintain higher ATP levels than
N2 worms, and attenuate the age-dependent depletion of ATP.
At days 6, 7 and 9, “N2 | preliminary” displays lower standing levels and larger error bars compared to the surrounding data points. This is due to incorporation of data with low signal intensities. The very low ATP levels (about 3.0 to 1.7 nmole/mg protein) at days 11 to 15 in this curve are entirely accountable by two biological replicates with low signal intensities. Therefore these data are unreliable and consequently ATP levels of very old N2 worms cannot be compared with those of equally aged daf-2(e1370)
worms.
Thus, in N2 worms the standing ATP levels decline progressively with age. In daf-2(e1370) worms the standing levels of ATP are higher and decline less than those of N2 and are maintained at a higher level.
Figure 28 | Level of ATP (nmole/mg protein) in function of age (days). N2 versus daf-2(e1370) mutant; ‘final’ versus ‘preliminary’. Error bars indicate standard deviation (n = different for each data point and is displayed in the table below).
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5.1.2.3
ADP
In the chart below (
Figure 29
) standing levels of ADP are plotted over age.
ADP levels gradually decline in aging N2 worms. Young adults contain about 8 to 10 nmole/mg protein while day 10 adults possess 3 to 6 nmole/mg protein. Hence, in N2 worms ADP declines approximately at the same rate as ATP. Young daf-2(e1370) adults contain ADP of about 16 to 22 nmole/mg protein, while daf-2(e1370) mutants of 10 to 16 days old adults have ADP levels of about 6 to 11 nmole/mg protein.
Overall, ADP levels are higher in daf-2(e1370) mutants and the decline over age is attenuated as compared to N2.
Figure 29 | Level of ADP (nmole/mg protein) in function of age (days). N2 versus daf-2(e1370) mutant; ‘final’ versus ‘preliminary’. Error bars indicate standard deviation (n = different for each data point and is displayed in the table below).
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5.1.2.4
AMP
In the figure below (
Figure 30
) standing levels of AMP are plotted over age. AMP levels of aging N2 worms remain largely the same at concentrations between 3 and 6 nmole/mg protein. ‘Preliminary’
AMP in N2 adults is at several points lower than ‘final’ AMP, because of artificially low signal intensities in the former measurements. As for ATP and ADP, standing levels of AMP cannot be compared between old aged N2 and daf-2(e1370) worms because of the artificially low signal for N2 worms.
Day 1 daf-2(e1370) adults contain ‘final’ AMP concentrations of 14 nmole/mg protein, although both biological replicates show highly divergent concentrations (3.5 and 25 nmole/mg protein, data not shown) rendering it hard to interpret. From day 6 on the concentration of ‘final’AMP decreases nearly
4-fold from 7.5 to 2 nmole/mg protein at day 16. ‘Preliminary’ AMP levels, are generally higher in daf-2(e1370) compared to N2 and “ daf-2 | final”. From day 4 on the AMP level declines slowly from
10 nmole/mg protein to 8 nmole/mg protein at day 10 and to 2 nmole/mg protein at day 16.
In conclusion, the changes in AMP levels over age in N2 versus daf-2(e1370) are hard to interpret due to lack of robustness of the experimental data. However, the general trend seems to be that in aging N2 worms standing levels of AMP remain largely the same, while in aging daf-2(e1370) worms AMP levels decline.
Figure 31 | Level of AMP (nmole/mg protein) in function of age (days). N2 versus daf-2(e1370) mutant;
‘correct’ versus ‘preliminary’. Error bars indicate standard deviation (n = different for each data point and is displayed in the table below).
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5.1.2.5
Adenine nucleotide pool
To compare the progression of the total adenine nucleotide pool in N2 with that in daf-2(e1370)
, ATP,
ADP and AMP were added up for each strain. The sum was plotted over age in the chart below
(
Figure 32
). Generally, the total adenine nucleotide pool of daf-2 appears to be higher than that of N2.
In both strains the pool declines with age, but seemingly to a lesser extent in daf-2(e1370) compared to
N2. Estimation of adenine nucleotide pool size of N2 at ages beyond 10 days is unreliable due to low signal intensity.
Thus, the adenine nucleotide pool decreases gradually with age in both strains, although in older daf-
2(1370)
worms this decline is attenuated.
Figure 32 | Adenine nucleotide pool (ATP + ADP + AMP) in function of age (days). N2 versus daf-2(e1370) mutant; ‘correct’ versus ‘preliminary’. Error bars indicate standard deviation (n = different for each data point and is displayed in the table below), calculated as square root of sum of squares.
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5.1.3
Indexes of cellular energy
Cellular energy levels are examinated by using sensitive indexes as the AMP/ATP ratio, ADP/ATP ratio, and the energy charge.
5.1.3.1
AMP/ATP ratio
The AMP/ATP ratios of N2 and daf-2(e1370) are plotted over age in the chart below ( Figure 33 ). In
N2 worms the ratio increases steadily from 0.1 at day 1 to 0.23 at day 11. At older ages the AMP/ATP ratio increases to nearly 0.6 at day 15.
For daf-2(e1370)
a different trend is found. For both ‘final’ and ‘preliminary’ values the AMP/ATP ratio gradually declines with age. The ‘final’ data for this mutant show an unchanging AMP/ATP ratio of approximately 0.12 at days 1, 6 and 9. From day 12 to 16 the ratio rapidly decreases from 0.08 to
0.02. The ‘preliminary’ data show a partly similar trend, but the AMP/ATP ratio is higher, most probably due to the additive effect of other components to the AMP signal.
So, while the AMP/ATP ratio of aging N2 worms increases with age, this ratio decreases in aging daf-
2(1370) adults.
Figure 33 | Ratio of AMP over ATP in function of age (days). N2 versus daf-2(e1370) mutant; ‘correct’ versus
‘preliminary’. Error bars indicate standard deviation (n = different for each data point and is displayed in the table below).
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5.1.3.2
ADP/ATP ratio
On the chart below (Figure 34) the ADP/ATP ratios of N2 and daf-2(e1370)
are plotted over age.The
ADP/ATP ratio appears to be slightly higher in N2 compared to daf-2(e1370). While the ADP/ATP ratio in aging N2 worms remains largely unaltered, in aging daf-2(e1370)
worms this ratio declines with 20 to 40% in old adults.
Figure 34 | Ratio of ADP over ATP in function of age (days). N2 versus daf-2(e1370) mutant; ‘correct’ versus
‘preliminary’. Error bars indicate standard deviation (n = different for each data point and is displayed in the table below).
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5.1.3.3
Energy charge
The energy charge is measured as (ATP + ADP/2) / (ATP + ADP + AMP) and indicates to what extent the adenine nucleotides exist as high energy phosphates.
In the chart below, the energy charge of N2 and daf-2(e1370)
is plotted over age (
Figure 35
). In N2 worms the energy charge slowly decreases over the first 11 days from 0.85 to about 0.76. The next 4 days the energy charge drops at a faster rate from about 0.76 to 0.62 (preliminary data). In daf-
2(e1370) worms the energy charge remains largely unaltered around 0.9 for all 16 days.
While in N2 worms the energy charge seems to decreases at old age, this is not the case for daf-
2(e1370)
mutant worms.
Figure 35 | Energy charge ((ATP + ADP/2) / (ATP + ADP + AMP)) in function of age (days). N2 versus daf-
2(e1370) mutant; ‘correct’ versus ‘preliminary’. Error bars indicate standard deviation (n = different for each data point and is displayed in the table below).
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5.1.4
Effect of culture and sampling method on energy indexes
To check the effect of the sampling method on all three energy indexes, two plates (NA, K12) with N2 worms were sampled at day 1. For one plate worms were sampled the usual way; for the other plate no percoll was used. As such the effect of percoll-induced stress (or extended sampling time) can be derived. By comparing these indexes with those of day 1 N2 worms that were sampled from a liquid culture, the effect of the culture and sampling method on the energy indexes can be deduced.
The cellular energy indexes, AMP/ATP, ADP/ATP, and energy charge, are plotted in function of culture and sampling method in the bar chart below (
Figure 36
). When worms are cultured on plates the AMP/ATP ratio almost triples, and the ADP/ATP ratio doubles those of worm cultured in liquid.The energy charge for both plate cultures (0.71 – 0.72) is about 15% lower compared to liquid culture (0.85). Percoll wash does not seem to have an explicit effect on any of the cellular indexes.
Figure 36 | Three cellular energy indexes of day 1 N2 worms in function of three different culture and sampling methods used. Error bars indicate standard deviation (n = 3).
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5.2.1
Obtaining Perceval transgenic C. elegans
We had to perform several steps to produce Perceval transgenic worms. Expression clones with different ubiquitous promoters and the
Perceval
gene, were constructed by Gateway ® cloning.
Figure
37 shows an overview of a representative expression vector that was used. The most important features are indicated.
Figuur 37 | Circular presentation of the expression clone bearing the ‘Prps-0::Perceval’ construct.
The expression vectors were all checked for the possession of the promoter and Perceval sequence.
Therefore a restriction digest and subsequent gel electrophoresis were executed on all replicates of the eight expression clones.
Figure 38
depicts the gel resulting from the restriction digest of the expression clone bearing construct 1 (Prps-0::Perceval). In this case, the restriction enzyme HincII was used. The restriction sites are indicated on
Figure 37
and the theoretical resulting fragments are 8208,
2833 and 306 base pairs in length.
Figure 38 | Gel loaded with a DNA ladder and restriction fragments of 3 replicates of construct 1
(Prps-0::Perceval). Number at top: constructreplicate. Left: Indication of the length in basepairs corresponding to several relevant bands in ladder.
Right: Loading position (slot) and theoretical length of 3 restriction fragments.
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Figure 39 | Gel loaded with a DNA ladder and full expression vectors of 3 replicates of construct 1 (Prps-
0::Perceval). Number at top: construct-replicate. Left:
Indication of the length in basepairs corresponding to 2 relevant bands in ladder. Right: Loading position (slot) and theoretical length of full expression vector.
Indeed, the gel on
Figure 38
indicates three fragments of about the expected fragment lengths. As a negative control undigested expression clones of each construct were loaded on the gel. Figure 39 depicts such a gel of construct 1. We notice, as expected, only 1 band, which is more or less the theoretical size.
As a second control for the insertion of both Perceval and the promoter, a PCR reaction was performed. Subsequently the PCR products were loaded on two gels, which are visualized, together with their expected size, on Figure 40 and Figure 41 .
Figure 40 | Gel 1 of PCR products. Left: Indication of the length in basepairs corresponding to three relevant bands in DNA ladder. From left to right: DNA ladder; three replicates of constructs 1, 2, 3, 5 and 6; DNA ladder.
Upper row: construct-replicate. Lower row: expected PCR product length.
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Figure 41 | Gel 2 of PCR products. Left: Indication of the length in basepairs corresponding to three relevant bands in DNA ladder. From left to right: DNA ladder; three replicates for construct 7, 8, and 4; one replicate for construct 4 (4-4b) obtained from a colony PCR; one non template control; DNA ladder. Upper row: construct-replicate. Lower row: theoretical PCR product length.
Clearly, both gels show shorter PCR products than expected from their theoretical lengths. A possible explanation for this systematic discrepancy might be an error in the selection or the production of the common reverse primer ‘RePCV’. Since the Perceval DNA contains some repeated sequences, it is possible that the primer has bound the DNA sequence of Perceval much more upstream. However, the relative differences in PCR product length between all constructs reflect the relative differences of expected PCR product lengths. In the lane of non template control there is no band present. This suggests that the smear detected above and beneath the most obvious band in each lane is the result of non-specific binding of the primers to the pDNA. For construct 4, no band appears. This should mean that Perceval and the promoter (Prpl-32) were not detected by the primers and hence, possibly are not present. A second PCR reaction on construct 4, however, indicated that both DNA sequences are in fact present in plasmids (gel not shown).
In conclusion, all eight expression clones appeared to be in the right conformation.
The final checkpoint comprises sequencing of both Perceval and the promoter to ensure the absence of frameshifts or other mutations in the expression vectors. It appears that in all constructs, except construct 4, there is at least one replicate of which it is certain that no mutations are present (data not shown).
The seven correct expression vectors were used for microinjection. This yielded one highly fluorescent worm (construct 1, ‘Prps-0::Perceval’) that we used to establish several lines of Perceval transgenic worms. Since this worm carried the Perceval gene without a mitochondrial targeting sequence, we only performed experiments on worms with cytosolically located Perceval. Figure 42 represents a picture of a descendent of the first fluorescent worm under a fluorescence dissecting microscope.
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Figure 42 | Image of a Perceval transgenic worm through a fluorescence dissecting microscope (Olympus
FZX12). Excitation at 460-490 nm, emission at 510-550 nm.
We observed mosaic expression and gradual loss of
Perceval
between successive generations. Thus, we tried to alleviate this by attempting to integrate the DNA construct into a chromosome, thereby using UV irradiation to induce non-homologous, double-strand DNA break repair (Mello & Fire,
1995). However, no stable integrants could be obtained. To acquire sufficient numbers of fluorescent worms, for further experiments, we selected and cultured highly transmitting worm lines.
Eventually, we also tried to obtain stable and homozygous Perceval integrants by doing a biolistic transformation on unc-119(ed3)
mutant worms. We used the ‘Prps-0::Perceval’ construct (1-3) since microinjection with this expression vector yielded a fluorescent transformant. Biolistic transformation produces low-copy chromosomal insertions which reduce the variations in expression level and pattern, often exhibited by microinjected worms that contain extrachromosomal arrays (Praitis et al.,
2001). Because of cotransformation with the unc-119(wt)
rescue allele (a selection marker) transformed worms can be easily distinguished by their wild-type phenotype. Although many transformants were identified this way, none of them showed Perceval mediated fluorescence. We checked for the presence of the
Perceval
transgenic construct by doing a PCR using the Fwrps0 and
RePCV primers (see 4.3.1.5.2, PCR ). For each bombarded plate one phenotypically ‘wild-type’ worm was isolated and lysed. For the first three plates an additional ten worms were lysed. These samples, two negative controls (genomic DNA from a N2 and the GA 804 strain) and a positive control (pDNA from expression vector 1-3) went through the PCR reaction. The subsequent gel electrophoresis reveals the presence of Perceval in lane 1, 6, ‘10*1’ and of course in lane ‘1-3’. The band of approximately 500 base pairs in lane 1 and ‘10*2’ might represent the same product that was found on the gel of Figure 40 .
Thus, the Perceval gene appeared to be present in at least several transformed but non-fluorescent worms. We cultured the transformed worms for several weeks, but no fluorescence was detected.
Hence, for some reason expression of Perceval was silenced.
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Figure 43 | Gel of PCR products of Perceval and promoter three relevant bands in

ResultsResults
5.2.2
Sodium azide treatment of Perceval transgenic worms
Sodium azide (NaN
3
) is a potent inhibitor of mitochondrial respiration. In particular, it inhibits both cytochrome c oxidase (Duncan & Mackler, 1966) and ATP synthase (Herweijer et al, 1985). Upon sodium azide treatment, ATP production is largely abolished while ATP consumption still goes on, resulting in an increase of ADP/ATP ratio. Since Perceval mediated fluorescence response depends on the ADP/ATP ratio (see 2.4.2, Perceval ), we expect a decrease in the ratio of fluorescence emission
(535 nm) when excited at 490 nm over 405 nm.
5.2.2.1
Fluorescence spectroscopy measurement
Perceval transgenic worms, derived from transformation by microinjection with expression plasmid
Prps-0::Perceval (see
4.3.1.1, Plasmid preparation by LR
) were treated with sodium azide in order to check whether this fluorescent reporter can adequately reflect ADP/ATP ratios in living worms. Since no stable transformants could be obtained, some worms of this population did not express the Perceval construct, or were only partly fluorescent (mosaic animals). A suspension of these worms was loaded in 4 wells of a microtiterplate. As a control, comparable amounts of N2 worms were distributed in the same way. The fluorescence (emission at 535 nm) upon excitation at both 490 nm and 405 nm
(F
490
/F
405
) was measured with a fluorimeter and the ratio of both was calculated. After 18 minutes (15 measurements), different concentrations of sodium azide were added (0 mM, 5 mM, 10 mM and 15 mM, respectively) to both N2 and Perceval transgenic worms. Subsequently, fluorescence was measured for an additional 2 hours (100 measurements). The result is depicted in the scatter plot below ( Figure 44 ).
Before addition of sodium azide, the ratio of fluorescence (F
490
/F
405
) in Perceval transgenic worms is about 2.5 as compared to about 0.75 in wild-type. Upon administration of NaN
3
to Perceval transgenic worms, the F
490
/F
405
drops immediately and drastically within several minutes to just above 1.0. Since the lower concentrations of NaN
3
were administered first, the fluorescent reponse also starts earlier.
However, at higher concentrations of NaN
3 the drop in F
490
/F
405
itself is more rapid. After the ratio
(F
490
/F
405
) reaches its minimum (just above 1.0), it remains constant for at least 2 hours. For Perceval transgenic worms to which no NaN
3
was added, the F
490
/F
405
declines gradually at a low rate to reaches a minimal F
490
/F
405 of between 1.5 and 1.75 after more than 1 hour.
In wild-type worms (N2) the F
490
/F
405 decreases only few upon NaN
3 treatment. The absolute fluorescence emission (535 nm) upon excitation at 490 nm is about 10-fold lower in N2 than in
Perceval transgenic worms before NaN
3
treatment. When excited at 405 nm, the emission is about 4fold lower. Hence, it can be expected that the fluorescent reponse of Perceval transgenic worms is hardly affected by changes in autofluorescence in this experiment.
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ResultsResults
Generally, standard deviations are relatively small when worms were treated with NaN
3
and a bit larger when untreated. Average standard deviations represent +/- 5% of the ratios for Perceval transgenic worms, and +/- 12 % for wild type worms. Standard deviations are not shown in Figure 37 to improve clarity of the graph.
Figure 44 | Ratio of fluorescence emission at 535 nm upon excitation at 490 nm over 405 nm (F
490
/F
405
) in function of time (hour:min). N2 versus Perceval transgenes (PCV); no NaN
3
addition versus different NaN
3 concentrations (5 mM, 10 mM, 15 mM). Arrow and vertical, dotted line indicates time of sodium azide treatment. Data points represent the mean of 3 to 4 biological replicates.
In conclusion, NaN
3
treatment lowers the ratio of fluorescence (F
490
/F
405
) significantly in Perceval transgenic worms. The emission at 530 nm upon excitation at 405 nm almost reaches the same emission values as when excited at 490 nm. The ratio shifts rapidly to a baseline level (just above 1) which is maintained for at least 2 hours.
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ResultsResults
5.2.2.2
Confocal laser scanning microscopy measurement
Several highly fluorescent Perceval transgenic worms, derived from transformation by microinjection with expression plasmid ‘Prps-0::Perceval’ (see 4.3.1.1, Plasmide preparation by LR reaction ), were selected to analyze Perceval fluorescence using confocal laser scanning microscope (cLSM). For one worm, images from head to tail were shot (zoom was 10x60x) and stitched to obtain a detailed overview of the fluorescent worm. A ‘value’ and an ‘Intensity Normalized Ratio’ (INR) image were created.
The ‘value’ image displays the mean of fluorescence intensities upon both excitation at 490 nm and
405 nm. Hence, it is a measure of the expression level of Perceval. On the value image depicted below
( Figure 45 ), the highest values are represented by yellow, while orange, red, purple, and blue represent lower values in a decreasing order. At regions where no color is present, there was no detectable emission upon excitation at 490 nm and 405 nm, which means that Perceval is not expressed in these cells. Highest expression is mostly found in the intestine and the pharynx. In the gonads, only low expression levels are found, while the eggs lack any expression of Perceval.
An INR image displays the Perceval ratio at each pixel by integrating the fluorescence intensity levels.
However, this ratio is not the same as measured with fluorimetry as it ranges from 0 to 2. Although no precise ratio of F
490
/F
405
can be derived, an INR image especially allows comparing between different cells, tissues, and time intervals. An INR image of an adult Perceval transgenic worm is depicted below ( Figure 46 ). The red color represents the highest and purple the lowest ratio of F
490
/F
405
. The intestine and epidermis are colored red, which indicates that these tissues should have the lowest
ADP/ATP levels (or the highest energetic capacity).
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ResultsResults
2
0
Figure 45 | ‘Value’ image derived from confocal projections of a Perceval transgenic adult worm.
Colors indicate mean values of fluorescence intensity upon excitation at 490 nm and 405 nm. Yellow indicates the highest value, blue the lowest.
Figure 46 | ‘INR’ image derived from confocal projections of a Perceval transgenic adult worm.
Colors indicate ratios of fluorescence upon excitation at 490 nm and 405 nm. Red indicates the highest ratio,
2. Blue indicates the lowest, 0.
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ResultsResults
A single worm was treated with sodium azide in order to check whether this fluorescent reporter can adequately reflect ADP/ATP ratios in living worms. INR images of the head were made 5, 8 and 9 minutes after NaN
3
treatment. These are depicted below ( Figure 47 ), together with a transparent image of the head in an aligned position to get an idea of the location of specific tissues in the INR image.
After 5 minutes the pharynx shows red, yellow and green signals. This indicates that the ratio of fluorescence upon excitation at 488 and 405 nm remains quite high. Several minutes later, the red and yellow signals seem to fade away while more blue and purple tints are present. This indicates that the ratio has dropped upon NaN
3
addition. Hence, it seems that the changing fluorescence of Perceval upon increasing ADP/ATP ratios can be detected by using cLSM.
Figure 47 | Confocal projections of the head of a Perceval transgenic worm upon sodium azide treatment. A:
Transparent image 5 minutes after NaN
3
addition. Pharynx outlined and indicated by arrow. B, C, and D:
Sequential INR images, 5, 8 and 9 minutes after NaN
3
addition. Red indicates the highest ratio of F
490
/F
405
, purple the lowest.
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DiscussionResultsResults
5 min A
2
5 min B
6.1.1
Evaluation of the assay
6.1.1.1
Reliability of data
The HPLC-fluorometric assay allows accurate measurements of ATP, ADP and AMP. Etheno-adenine amount of fluorescence at each timepoint is represented in a chromatogram. Since a specific type of compound has a certain affinity for the mobile and the liquid phase, different compounds will display different retention times. Hence, chromatograms depict separate fluorescence peaks, each
8 min C 9 min D
2 and AMP retention times and signal intensities are obtained for each molecule. This way, ATP, ADP and AMP peaks are identified ( Figure 25 ) and original concentrations of worm samples determined.
Although the HPLC assay is both accurate and sensitive, its robustness appeared to be problematic during this master dissertation. In a first set of measurements, chromatograms as depicted in Figure 25 were obtained. In addition to distinct ATP, ADP and AMP peaks, a relatively large, unidentified peak
0
0 could be distinguished. A second group of measurements produced chromatograms as in Figure 26 , with a peak for ATP, ADP and another peak that probably contained both AMP and the unidentified component.
We annotated this peak as AMP, thus it should be noted that in this set of results
(designated as 'preliminary), AMP concentrations are most probably overestimated.
Since only derivatized adenine nucleotides are expected to deliver high emission signals upon excitation at 340 nm, the unidentified component probably is an adenine nucleotide. Therefore, it would be interesting to identify this component, as it would allow further clarification of the complete adenine pool in aging
C. elegans
. It can be excluded that the unidentified component is either adenine or adenosine as both were previously found to have a much larger retention time (F. Matthijssens, personal communication). The most likely candidates are cAMP, NADH, NAD + , NADPH and
NADP + . Since cAMP and NADH were not in stock at time of measurement, only the latter three compounds were tested. NADP + and NADPH appeared to have a short retention time of ± 5.25 minutes and constitute a small peak together (
Figure 26
). It is likely that primarily NADH and NAD + constitute the unknown peak because of two reasons. Firstly, during the second set of measurements, the retention times of NAD + and AMP were found to be identical; thus at least NAD + and AMP constitute the merged peak.. Secondly, the unknown peak is too large to be designated to cAMP, since its concentration is usually three orders of magnitude lower than AMP (Charest et al., 1985).
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DiscussionResultsResults
Anyway, it would be interesting to further identify the composition of the peaks by performing extra runs on the HPLC with cAMP, AMP, NAD + and NADH standards.
A third and smaller set of measurements resulted in chromatograms as in Figure 27 . In addition to lack of separation of the unknown and AMP peak, unexpectedly low signal intensities were recorded for the ATP, ADP and AMP-peaks. These results are unreliable for interpretation of quantitative amounts of the adenine nucleotides, but relative ratios are assumed to be reliable.
In conclusion, not all data obtained from the HPLC-fluorometric assay are entirely reliable. Results depicted as ‘final’ can be interpreted as trustworthy.
6.1.1.2
Comparison with other assays
When comparing our results with values found in other studies, some differences can be found.
Unlike the strong decrease in ATP content of N2 worms from ± 40 nmol/mg protein to ± 5 nmol/mg protein after 10 days as reported by Brys et al. (2010), we only observed a limited decrease to ± 20 nmol/mg protein (
Figure 28
). The initial value at day 1 is more or less identical. An important difference between both assays is that we did our perchloric acid extraction and derivatization on living worms, while Brys and coworkers performed their perchloric acid extraction on frozen worm tissue. It was found previously that freezing the worm samples prior to derivatization decreases the levels of ATP and increases those of AMP and ADP, most probably by hydrolysis of ATP during this process (personal communication, F. Matthijssens). Therefore, our method may represent the original cellular levels of ATP, ADP and AMP more accurately. It is conceivable that the progressing frailty of aging worms can aggravate the loss of ATP by hydrolysis upon freezing. This could be a possible explanation for the steeper decrease in ATP levels observed by Brys et al. (2010).
Brys and coworkers (2010) also found in wild-type and daf-2(e1370) worms identical values for ATP and ADP concentration at day 1 adults. Over age the levels decrease slowly and gradually, thereby following a linear trend. However, we observed higher ATP and ADP levels in daf-2
mutants compared with wild-type animals and no obvious trend due to a rather high variation in our results
(
Figure 28
and
29
). Technical inaccuracies during our sampling could explain this discrepancy.
Finally, for all our values the AMP/ATP and ADP/ATP ratios are higher and the energy charge is lower compared to those reported by Apfeld et al. (2004) and Curtis et al. (2006). This might be due to differences in detection and sampling methods. While Apfeld et al. detected the nucleotides by measuring UV absorption at 260 nm, we used a detection system based on derivatization of the nucleotides and subsequent fluorescence detection, which would be expected to be more sensitive and specific.
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DiscussionResultsResults
Another difference is the way the nematodes were cultured. Apfeld et al. (2004) sampled small amounts of hand-picked worms, while we sampled thousands of worms simultaneously from a liquid culture. Liquid cultures might be more stressful for the nematodes due to limited oxygen availability.
This should result in lower energy levels and possibly less age-synchronized worm populations. In contrast, we find ADP/ATP and AMP/ATP ratios for 1 day old wild-type worms that are lower when liquid cultured are used compared to plate cultures (
Figure 36
). The energy charge (see
2.3.3, Energy charge ), which represents the extent to which adenine nucleotides exist as high energy phosphates, is higher for the liquid cultures and confirms the higher energy state of the nematodes’ cells reflected by the ADP/ATP and AMP/ATP ratio. Longer sampling time by performing a percoll step seemed to have no explicit effect on energy levels (
Figure 36
). Further experimental comparisons between the method of Apfeld and the one described in this thesis could clarify the discrepancy between our results and those by Apfeld's group.
Altogether, ATP, ADP and AMP in worm samples can be accurately measured using the HPLCfluorometric assay. However, extra work is needed to find out the optimal sampling method and to improve the robustness of the assay in order to obtain reliable and accurate measurements.
6.1.2
Aging daf2 mutants maintain high energy levels
Deregulation of homeostasis of cellular energy supplies is characteristic for normal aging organisms.
Hence, knowledge about the dynamics of the universal energy carrier in living cells, ATP, is of profound importance to unravel the mutual effects of aging and energy levels. Although not all the results of the HPLC-fluorogenic assay are entirely reliable, we confirm the progressively declining trend of ATP levels in aging wild-type worms (
Figure 28
), as previously reported several times
(Braeckman et al., 1999; Braeckman et al., 2002; Dillin et al., 2002; Houthoofd et al., 2005; Brys et al., 2007; Brys et al., 2010). Impairment of insulin/IGF-1 signaling (IIS) by the weak reduction-offunction allele e1370 of daf-2 causes nematodes to contain higher standing levels of ATP, while over age the decline in ATP levels is strongly attenuated (
Figure 28
). This also confirms previous findings
(Dillin et al., 2002; Houthoofd et al., 2005; Brys et al., 2007; Brys et al., 2010).
ATP production is under control of several mechanisms (see 2.3.2, Control of energy level ). A main player is ADP; therefore also ADP concentrations were measured over age in both strains. ADP levels showed a similar trend as described for ATP in both aging wild-type and daf-2(e1370) mutant worms.
The ADP concentration in daf-2
worms tend to decline a little steeper than the ATP concentration, however. In older (> 10 days) daf-2 animals the decline in ADP content is completely attenuated, similar to ATP. The steeper decrease in ATP concentration in N2 animals compared to daf-2 worms was also reported by Brys et al. (2010).
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DiscussionResultsResults
The instantly utilizable energy source ATP is not a reliable parameter for the cellular energy level.
ADP levels strongly influence the availability of the free energy provided by ATP. ATP-dependent activity of many cellular processes relies on the competition between both high energy adenosine phosphates. Therefore we determined the ADP/ATP ratio, which is kept low to provide free energy and indicates the energy status of a cell more reliably. Wild-type aging worms show, if any, a slightly increasing trend for the ADP/ATP ratio (
Figure 34
). In daf-2
worms, on the contrary, this ratio gradually declines from approximately 0.25 to nearly 0.20 at day 10 and around 0.15 at day 16. Hence, it seems that the available free energy increases with age in these long-lived mutants.
A second and even more reliable parameter of the cellular energy status is the AMP/ATP ratio. Our results show an exponential increase for aging N2 animals, with a ratio of approximately 0.1 at day 1 and nearly 0.6 at day 15 ( Figure 33 ). Although the absolute concentrations of the nucleotides are not reliable due to abnormally low signal intensities, the relative ratios are probably not affected. This assumption is supported by the nearly identical exponential rise of the AMP/ATP ratio found by
Apfeld et al. (2004) ( Figure 11 ). Interestingly, the AMP/ATP phenotype of daf-2(e1370) animals is quite different (
Figure 33
). A more or less stable ratio during the first nine days is followed by a progressive decline at older ages. Hence, over age the AMP/ATP ratio alters in opposite directions in both strains. The difference between both strains can be partly attributed to the attenuated decline of
ATP levels over age in daf-2 mutants compared to wild-type worms ( Figure 28 ). On the other hand absolute AMP concentrations could also have an important impact on the AMP/ATP ratio in function of the age. However, absolute AMP concentrations were never reported before for both strains. Here, we discovered that AMP standing levels hardly change over age in N2 (lower AMP values of “N2 | preliminary” are due to low signal intensities and are not taken into account), while daf-2 animals seem to contain lower AMP concentrations upon getting older ( Figure 31 ). In summary, the diminishing AMP/ATP ratio in aging daf-2 animals can be assigned to high, sustained ATP levels and declining AMP levels. In wild-type worms ATP levels decline rapidly while AMP levels remain more or less unaltered.
We also found a larger adenine nucleotide pool (ATP, ADP and AMP) in daf-2 animals ( Figure 32 ).
In both strains ATP is the predominant adenine nucleotide; therefore similar trends are observed as for the standing ATP levels ( Figure 28 ). In both wild-type and daf-2 worms the total pool declines with age, although this trend is attenuated in old daf-2
worms. The smaller pool size might implicate that aging worms are less able to cope with strong energy demands. In addition, the energy-containing adenine nucleotides seem to be better preserved in daf-2
worms upon aging. Importantly, ATP becomes relatively more abundant in the pool since both the AMP/ATP ( Figure 33 ) and ADP/ATP ratio (
Figure 34
) decline over age in daf-2
animals. In wild-type worms the opposite is true. Hence, daf-2 worms have a lot of free energy to their disposal.
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DiscussionResultsResults
Assessment of cellular energy levels is based on the ADP/ATP and AMP/ATP ratios. The former has a more instant effect by controlling ATP synthesis; the latter influences a wider array of metabolic and cellular maintenance processes. AMPK is a cellular energy sensor that acts as a metabolic switch by integrating the AMP/ATP ratio (see
2.3.5, AMPK as energy sensor
). Importantly, the low AMP/ATP ratio we found might be the result of enhanced AMPK activity in daf-2 mutants.
The ‘energy charge’ is a good indicator to assess if AMPK is able to maintain the energy level in a steady state between 0.80 and 0.95 (Atkinson et al., 1968) ( Figure 10 ). We discovered that wild-type worms are able to do so for more or less the first nine days (with values of approximately 0.80-0.85).
At older ages a steep decline in the energy charge to nearly 0.62 is noted. On the contrary, daf-
2(e1370)
mutants maintain high energy charges (around 0.90) over the entire period measured (
Figure
35 ). Hence, unlike old daf2 worms, old wild-type worms lose their ability to enhance ATP production and demote ATP consumption to reach the ‘point of metabolic steady state’ ( Figure 10 ). Since this regulation is mainly under control of AMPK, we assume impairment of AMPK abundance, activity
(determined as target phosphorylation) or responsiveness. Qiang et al. (2007) reported that the expression of the catalytic subunit of rat AMPK, i.e. AMPK
α
(of which AAK-2 is a homolog), did not alter in 24-month old rats compared with 4-month old rats. However, they did find significantly decreased AMPK
α
activity in the older rats. Hence, declined AMPK responsiveness to increasing
AMP/ATP ratios seems to be responsible for impaired AMPK activity in this case.
In
C. elegans
the LKB1 homolog PAR-4 activates AAK-2 under reduced insulin/IGF-1 signaling in dauer formation (Narbonne and Roy, 2006). Thus, reduced IIS may have a direct, positive impact on
AAK-2 activity, which on its turns directs metabolism to maintain high cellular energy levels.
Furthermore, local and temporal rises in the AMP/ATP ratio, which cannot be detected with this
HPLC assay, could activate AMPK. Altogether, it will be interesting to determine in future research
(1) the AMPK activity in response to the different dynamics in AMP/ATP ratio between aging wildtype and daf-2 worms, and (2) the effect of locally and temporally increasing AMP/ATP ratios on
AMPK activity.
Apfeld and coworkers (2004) proposed that daf-2 mutants extend lifespan using a mechanism independent of the AMP/ATP ratio, because the ratio and energy charge they found was identical to that of wild-type. However, they only sampled day-1 adults, while we measured the adenine nucleotides over lifetime and found obvious differences over age. Interestingly, the AMP/ATP ratio was lower and lifespan longer for more active worms of identical chronological age (Apfeld et al.,
2004). This and our observation suggest that the AMP/ATP ratio and a well functioning AMPK/AAK-
2 might play a role in longevity and vitality, also in daf-2 mutants. AAK-2 was found to be important for the longevity phenotype of mitochondrial mutants ( isp-1(qm150)
and clk-1(qm30)
) (Curtis et al.,
2006). These long lived nematodes have higher AMP/ATP ratios at day 1 compared to wild-type.
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DiscussionResultsResults
In summary, unlike wild-type worms, longlived daf-2(e1370) animals are able to attenuate the agerelated decline in standing levels of both ATP and ADP. A possible mechanism underlying the preservation of this condition is elevated AAK-2 activation as a consequence of reduced insulin/IGF-1 signaling in this insulin/IGF-1 receptor mutant. Possibly, AAK-2 alters metabolic enzyme activity and gene transcription in favor of ATP production and extends the lifespan through a ‘CRTC-1 – CRH-1’ pathway as described by Mair and colleagues (Mair et al, 2011). Furthermore, increased autophagy and mitochondrial turnover may be important to sustain the high energy levels that are required for somatic maintenance and lifespan extension (Ryazanov and Nefsky, 2002; He & Klionsky, 2009).
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6.2.1
Obtaining Perceval transgenic C. elegans
Standing ATP levels do not give a complete picture of the cellular energy state, the ADP/ATP ratio is a more reliable indicator (as discussed above). Besides measuring this ratio in total worms by using the HPLC, it might be interesting to investigate tissue-specific differences in the ADP/ATP ratio in more detail. Here, we tried to find out if the genetically encoded fluorescent sensor ‘Perceval’ is useful in whole animal and tissue-specific detection of ADP/ATP ratio in vivo in real-time in Caenorhabditis elegans
.
Microinjection with seven constructs each comprising Perceval with a different constitutive promoter yielded only one worm with sufficient expression of Perceval. This worm was transformed with the
‘Prps-0::Perceval’ construct. From this worm several highly transmitting lines were established and further cultured.
To obtain stable integrants worms were biolistically transformated with the ‘Prps-0::Perceval’ construct. Several phenotypic ‘wild-type’ worms were detected, hence these were transformed.
Although the Perceval gene was present ( Figure 43 ), these worms did not express Perceval. Probably this is the result of silencing.
One possible explanation for the silencing might be that the transgene was integrated in constitutive or facultative heterochromatin. These silenced chromosomal regions could then be responsible for silencing of the transgene. Therefore it might be interesting to use the Mos1 (transposase) mediated
Single Copy Insertion (MosSCI) method developed by Frøkjær-Jensen et al. (2008). This allows insertion of single copies of a transgene into specific, defined sites in the genome of
C. elegans
and should overcome silencing of the integrated transgene. A huge amount of potential integration sites is provided by the NEMAgenetag consortium (Bazopoulou and Tavernarakis, 2009).
Another possibility is that the transgene is not integrated but assembles into extrachromosomal arrays.
Silencing is suggested to be a possible consequence of recognition of such arrays as repetitive or heterochromatin-like (Kelly et al., 1997). If this is the case, the MosSCI method might also counter this problem.
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DiscussionResultsResults
6.2.2
Sodium azide treatment of Perceval transgenic C. elegans
Berg et al. (2009) used the glycolytic inhibitor 2-deoxyglucose to reduce ATP production and proved that Perceval reports the ADP/ATP ratio in mammalian cells. Here we used sodium azide (NaN
3
), a potent inhibitor of mitochondrial respiration which was shown to increase, at 1mM concentrations, the
AMP/ATP ratio (and therefore also the ADP/ATP ratio) drastically in
C. elegans worms (Apfeld et al.,
2004). Hence, we expected altered fluorescence responses upon sodium azide treatment of the
Perceval transgenic worms.
6.2.2.1
Fluorescence spectroscopy measurement
In a first experiment we used fluorescence spectroscopy on a mix of fluorescent, mosaic and nonfluorescent worms (since no stable integrants were obtained) to measure the fluorescence response upon addition of different concentrations (5, 10 and 15 mM) of sodium azide. We observed a drastic decline in the ratio of fluorescence (F
490
/F
405
) within minutes after sodium azide was administered
( Figure 44 ). At higher concentrations the drop in F
490
/F
405
was steeper. Yet, an identical baseline ratio
(of approximately 1.1) was reached in rapid succession for all three concentrations of sodium azide and maintained for two hours until the end of the measurements. In Perceval transgenic worms that were treated with a blank, only a very limited and gradual decline was observed. A possible reason for the decline is the restricted oxygen availability which lowers mitochondrial respiration and consecutively ATP production.
In conclusion, a changing ADP/ATP ratio caused by sodium azide treatment clearly alters the fluorescent properties of Perceval with fast kinetics. This delivers proof that Perceval can be used for detection of alterations in the ADP/ATP ratio in real-time in living Caenorhabditis elegans .
6.2.2.2
Confocal laser scanning microscopy
In a second experiment we used confocal laser scanning microscopy (cLSM) on individual Perceval transgenic worms to analyze, on a tissue level, the fluorescence response upon sodium azide treatment.
In a first step, we obtained an image of a complete worm. This picture was edited to create two images. On one hand a ‘value’ image which depicts fluorescence intensity levels. These indicate the expression levels of Perceval in different areas of the worm. On the other hand an ‘INR’ (intensity normalized ratio) image which displays the Perceval ratio at these areas.
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DiscussionResultsResults
Usually, the rps-0 gene is constitutively expressed in the pharynx, intestine and hypodermis, while its expression is repressed in the reproductive system (contrary to our other constructs) (
Table 4
). We can confirm this expression pattern with our observation of the expression of Perceval on the ‘value’ image (
Figure 45
). The highest expression of Perceval is observed in the pharynx and intestine, while the hypodermis shows rather low to intermediate Perceval expression. Perceval is not detected in the gonads.
We wondered if we could detect differences in cellular energy levels between tissues in a living worm.
Perceval expression on its own gives us no information on energy levels. Therefore we created an INR image ( Figure 46 ). The colors represent the ratio of fluorescence (F
490
/F
405
) of Perceval, independent of its expression level. We note the lowest ADP/ATP ratio, or highest energy levels, in the intestine and hypodermis. In the pharynx higher ADP/ATP ratios can be found. In the latter and other organs or tissues, we notice a lot of variation in the Perceval ratio. Hence, it appears that Perceval may allow detection of tissue-specific differences in energy levels.
To check if Perceval could be used to visualize tissue-specific changes in energy levels a single
Perceval transgenic worm was treated with sodium azide. During the subsequent minutes three images from the head region were shot ( Figure 47 ). After five minutes still a relatively large Perceval ratio is remarked, however, three and four minutes later this ratio declines to a very level. Therefore we can conclude that, by means of confocal laser scanning microscopy, Perceval can be used for reporting tissue-specific alterations in ADP/ATP ratios.
In summary, Perceval is an interesting tool to examine cellular energy levels in Caenorhabditis elegans
. It allows non-disruptive in vivo and in real-time measurements of the ADP/ATP ratio, both on whole worm level using fluorescence spectroscopy, and tissue-specifically by means of confocal laser scanning microscopy. However, more research is necessary to optimize the use of Perceval as an accurate and reliable sensor of cellular energetics. Most importantly, a stable line of Perceval transgenic worms should be obtained for further research. One major limitation is the sensitivity of its fluorescence response to intracellular pH, which is modified by many metabolic perturbations (Berg et al., 2009). Yet, this could potentially be solved by correcting for pH effect on the Perceval signal by concurrent measurements with the pH indicator dye SNARF-5F, which fluoresces in the red area of the spectrum (Berg et al., 2009). Although we found, by using HPLC, that the ADP/ATP ratio in wildtype worms shows a limited gradual increase over age, it might be interesting, for future studies, to analyze the ADP/ATP over age tissue-specifically by using Perceval. Also the (tissue-specific) effect of RNAi-mediated downregulation of AAK-2 on energy levels might be discovered using Perceval.
Finally, it might be interesting to use Perceval for the investigation of tissue- or cell-specific differences in energy levels in specific conditions, such as dietary restriction, dauer entry or exit, hypoxia or the presence of environmental stressors.
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Conclusion
In this master dissertation the relationship between cellular energy levels and aging in
Caenorhabditis elegans was investigated. Based on the results of this research and the literature the following can be concluded:
1.
ATP, ADP and AMP in worm samples can be accurately measured using the HPLCfluorometric assay. Extra work is needed to find out the optimal sampling method and to improve the robustness of the assay in order to obtain reliable and accurate measurements.
2.
We confirm that, unlike wild-type worms, longlived daf-2(e1370) animals are able to attenuate the age-related decline in standing levels of both ATP and ADP. A possible mechanism underlying the preservation of this condition is elevated AAK-2 activation as a consequence of reduced insulin/IGF-1 signaling in this insulin/IGF-1 receptor mutant.
3.
Fluorescence spectroscopy of a suspension of Perceval transgenic worms revealed that a changing ADP/ATP ratio caused by sodium azide treatment alters the fluorescent properties of the genetically encoded energy sensor Perceval with fast kinetics. This proves that Perceval can be used for non-disruptive detection of alterations in the ADP/ATP ratio in real-time in living
Caenorhabditis elegans
.
4.
By means of confocal laser scanning microscopy, Perceval can be used for reporting tissuespecific alterations in ADP/ATP ratios.
5.
In the future it will be important to obtain a stable line of Perceval transgenic worms to facilitate further research.
6.
More research is necessary to overcome limitations such as pH sensitivity of Perceval and to optimize its use as an accurate and reliable sensor of cellular energetics.
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