Thesis for the Degree of Doctor of Philosophy

Peroxiredoxins in Redox Signaling and Aging

Friederike Roger

Department of Chemistry and Molecular Biology

Cover illustration: Image created by Deep Dream Generator based on a microscopy image of Saccharomyces cerevisiae combined with Wassily Kandinskys Improvisation 28. ISBN: 978-91-629-0329-9 (print) ISBN: 978-91-629-0328-2 (online) Available at http://handle.net/2077/53655 Printed in Gothenburg, Sweden 2017 By BrandFactory AB

Nothing in biology makes sense except in the light of evolution.

- Theodosius Dobzhansky, 1973

AbstractPeroxiredoxins have emerged as conserved modulators of the rate of aging in yeast and multicellular organisms and play a role in lifespan extension through the anti-aging intervention caloric restriction. Yet, it is not clear through what mechanism peroxiredoxins extend lifespan. First discovered as hydrogen peroxide scavengers, peroxiredoxins have been shown to have a genome protective function, to act as chaperones, to play a role in circadian rhythms and to be involved in redox signaling. In this thesis, I tried to identify the underlying mechanisms for peroxiredoxin mediated lifespan extension and its role in redox signaling. Using the yeast Saccharomyces cerevisiae as a model organism, we could show that the lifespan extension by the peroxiredoxin Tsa1 is not linked to increased genome stability. Our data indicate that Tsa1 recruits molecular chaperones to protein aggregates formed during oxidative stress and reduces the number of protein aggregates that accumulate during aging. Surprisingly, this contributes just to a limited extend to lifespan extension, as a mutant not able to form a chaperone still has a normal lifespan. Instead, redox-signaling that reduces protein kinase A (PKA) activity through Tsa1 mediated oxidation seems to be preeminently responsible for lifespan extension. Interestingly, the same signaling pathway is used in yeast to react to light stress. Hydrogen peroxide formed upon illumination by a conserved peroxisomal oxidase leads to increased redox cycling of Tsa1. Tsa1 then reduces PKA activity allowing the subsequent nuclear localization of the transcription factors Msn2 and Msn4 that induce transcription of stress-related genes. Our data thus clarify an important aspect of the role of peroxiredoxins in circadian rhythms, namely they mediate an organismal light response. Keywords: Aging, caloric restriction, peroxiredoxins, protein aggregates, redox signaling, PKA signaling, light stress

SammanfattningPeroxiredoxiner har nyligen uppmärksammats för att de reglerar åldrandet i såväl jäst som flercelliga organismer och eftersom de även spelar en roll i mekanismerna för hur kalori-restriktion bromsar åldrandet. Trots detta är det inte klart exakt genom vilka mekanismer peroxiredoxiner förlänger livslängden. Först upptäckta och beskrivna som väteperoxid-nedbrytande anti-oxidanter har roller för peroxiredoxiner nu även framkommit i att skydda genomet mot skador, som molekylära chaperoner, i cirkadiska rytmer och i redox-signalering. I den här avhandlingen har jag försökt identifiera mekanismer för hur peroxiredoxiner stimulerar långt liv och deras roll i redox signalering. Genom att använda jästen Saccharomyces cerevisiae som modellorganism har vi kunnat visa att förlängt liv via peroxiredoxinet Tsa1 inte kan kopplas till ökad genom-stabilitet. Våra data pekar vidare på att Tsa1 rekryterar molekylära chaperoner till protein aggregat som bildas då cellerna åldras. Förvånansvärt nog verkar denna roll enbart i begränsad utsträckning bidra till Tsa1s förmåga att förlänga cellernas liv eftersom en mutant som inte kan bilda chaperon-formen åldras normalt. Å andra sidan pekar mina data på att redox-signalering som minskar protein kinas A (PKA) aktivitet via Tsa1-katalyserad oxidering i högsta grad ansvarig för Tsa1s förmåga att bromsa åldrandet. Intressant nog används samma signalväg när jästen svarar på ljus stress. Väteperoxid som bildas via ett konserverat peroxisomalt oxidas då jästen träffas av ljus leder till ökad Tsa1-beroende redox-aktivitet. Tsa1 minskar PKA aktiviteten vilket möjliggör transkriptionsfaktorerna Msn2 och Msn4, som transkriberar stress-relaterade gener, att koncentreras i kärnan. Våra data klargör därmed en viktig aspekt i peroxiredoxiners roll i cirkadiska rytmer, nämligen genom att visa att de direkt kan styra hur celler svarar på ljus.

AcknowledgementsIt has been an incredible 6 years in Gothenburg and the Lundberg Lab with so many people who made this a really fun and interesting time. My biggest thanks go to my supervisor Mikael who hired me as a Ph.D. student on Christmas 2011. At first, I was even hesitant to apply because I feared that nobody would hire a 7-month pregnant woman. But you did and I got the chance to work on a really interesting project for 4 years. Thanks for all the support, for always being available to answer the countless questions I had and still have and for having such a contagious passion for research in general and peroxiredoxin in particular. Thanks also to my co-supervisor Thomas for hiring me as a project student in 2011 when I couldn’t wait to get back to the lab after my Master. Thanks for all the input and the ideas during numerous lab meetings and for giving me the opportunity to go to the yeast genetics and genomics course in Cold Spring Harbor without even blinking on the price tag. Thank you, Markus, for being my examiner and for quickly doing all the necessary paperwork and taking the time to discuss my Ph.D. planning. Also, I was really happy to have all my teaching duties in well-organized and really interesting courses. Anne, you are an inspiration as an outstanding teacher and I had quite some fun during my lab assistant duties. Thanks also to Ingrid, the good fairy of the course lab, for all the help and the nice chats. Danke, Frederik, für 4 Jahre Schreibtischstuhl-Pogo in unserem kleinen Office. Es war wirklich sehr praktisch eine direkte Quelle an Hefe-Weisheit zu haben und natürlich Fahrrad- und Marvelcomic-Diskussionen. Und wenn ich mal wieder Fragen dazu hatte, wie ein Experiment richtig gemacht wird, war ich froh, dass du, Anja, immer genau wusstest, welche Sachen es zu beachten gilt. It was really fun to work together in our big 3rd-floor lab with you guys, Sarah, Sandra, Lisa, Katarina, Rebecca A and J, Veronica,

Malin, Bertil , Per, Kanika, Anna, Roja and Doryaneh. Although I still don’t understand why one would eat lunch at 11 o’clock, I really enjoyed working together with all of you. In all of those years, there have been a lot of goodbyes but at least with memorable Ph.D. parties and awesome spexs! Thanks also to the Beidong group, with Xinxin, Jungsheng, Lihua, Qian Liu and Zheng Ju, and special thanks to Xiuling who save me from the ginormous spider in the corridor. I am also really thankful for all the work that the Master students have contributed to the project and the nice time we had in the lab: Thanks, Sara, Chikako, Banu, and Svenja! Und war ich froh, dass ich moralische Unterstützung hatte, bei den endlosen Stunden am Micromanipulator - danke Kathi. I was also really glad for all the microscopy wisdom, Kristofer, that came in really handy throughout my Ph.D. time. The same goes for the PKA knowledge, Ken, although I still think there could have been more Belgium beer during your stay here (and yes, it is sweet and not with more flavor). To my new homies on the 4th floor after an awkward time on the empty 3rd floor: I will really miss the long lunches and coffee breaks and I hope we will manage to have one more movie night before I will be mammaledig! Thanks, Sansan, Davide, Martin, Fabrice, Carolina, Christiana, Hanna, and Karl. Stefanie, ich hoffe wir schaffen es einen Tag mal ganz schwedisch einen auf Latte-Mamas zu machen! Also I think I am really lucky with my roommates, I really enjoyed all those long procrastination chats, Michelle. Thanks for the fun time in CSHL, Simon, I am still not sure how I have not seen you for 2 years before that. Ich bin wahnsinnig froh, kleiner Stinker, dass ich so eine liebe kleine Schwester habe. Danke, dass du dich um meine kleinen Stinker gekümmert hast, während ich länger gearbeitet habe oder einfach eine Pause brauchte. Ich hoffe, dass wir es auch in Zukunft schaffen, wenigsten ein wenig Lästerschwester-Zeit zu haben.

Danke, Mama und Papa, dass ihr damals gesagt habt, dass das Gauß-Gymnasium genau das Richtige für mich wäre. Danke für eure Unterstützung. 10 Jahre, mon Schatz, und du kennst mich leider so gut, dass du genau weißt, wann ich prokrastiniere, wann ich mir zu viele Sorgen mache und warum ich mir eigentlich keine Sorgen machen müsste. Ich bin so froh dich immer an meiner Seite zu haben. Für meine kleinen Stinker, Emilian und Léon: Ich bin so unglaublich stolz auf euch und ich hoffe, dass ihr immer so neugierig und interessiert bleibt wie jetzt, dann kann ich euch noch viel mehr über kleine Hefezellen und winzig kleine Bakterien erzählen. Und den kleinsten Stinker, der ganz unermüdlich den Laptop getreten hat, der während des Doktorarbeitschreibens immer wieder in den Bauch gepiekt hat – wir freuen uns auf dich. I would also like to thank all those people that I shamefully forgot to mention and since I always have an excuse, I would like to blame it on being 9 month pregnant and suffering from pregnancy brain…

AbbreviationsROS Reactive oxygen species

SOD Superoxide dismutase

CuZnSODs copper and zinc containing SODs

FeSODs iron containing SODs

MnSODs manganese containing SODs

NiSODs nickel containing SODs

Pox Peroxisomal acyl-CoA oxidase

Ccp1 Cytochrome c peroxidase

Prx Peroxiredoxins

Cys Cysteine

Srx Sulfiredoxin

Trx Thioredoxin

RNR Ribonucleotide reductase

TrxR/Trr Thioredoxin reductase

Grx Glutaredoxins

GSH Glutathione (γ-glutamyl-cysteinyl-glycine)

GSSG Glutathione disulfide

GR Glutathione reductase

NADPH Nicotinamide adenine dinucleotide phosphate

PKA Protein kinase A

HSP Heat shock protein

CR caloric restriction

IGF insulin-like growth factor

TOR target of rapamycin

AC Adenylate cyclase

PapersincludedinthisThesisPaper I Lifespan control by redox-dependent recruitment of chaperones to misfolded proteins Hanzén, S., Vielfort, K., Yang, J., Roger, F., Andersson, V., Zamarbide-Forés, S., Andersson, R., Malm, L., Palais, G., Biteau, B., Liu, B., Toledano, M.B., Molin, M., Nyström, T. (2016). Cell, 166(1), 140-151. Paper II Light-sensing via hydrogen peroxide and a peroxiredoxin Bodvard, K.*, Peeters, K.*, Roger, F.*, Romanov, N., Igbaria, A., Welkenhuysen, N., Palais, G., Reiter, W., Toledano, M.B., Käll, M. & Molin, M. (2017).. Nature Communications, 8, 14791. * These authors contributed equally to this work Paper III Redox signaling via the yeast peroxiredoxin Tsa1 promotes longevity by inhibiting nutrient signaling Roger, F., Asami, C., Hanzén, S., Lagniel, G., Labarre, J., Nyström, T., and Molin, M. (2017) Manuscript

TableofContents…ANDTHENTHEREWASOXYGEN....................................................................1SOURCESOFREACTIVEOXYGENSPECIESANDTHEIRCELLULARDAMAGE...................1

CELLULARDEFENSESAGAINSTOXIDATIVEDAMAGE................................6SUPEROXIDEDISMUTASES....................................................................................................6CATALASE................................................................................................................................7PEROXIREDOXINS,THIOREDOXINS,GLUTAREDOXINSANDREDOXSIGNALING.........8YEASTASAMODELSYSTEM................................................................................................14

AGING........................................................................................................................16PROGRAMMEDAGING..........................................................................................................17EVOLUTIONARYAGINGTHEORIES....................................................................................18MutationAccumulationTheory...............................................................................18AntagonisticPleiotropyTheory...............................................................................19TheDisposableSomaTheory....................................................................................20

MOLECULARMECHANISMSOFAGING..............................................................................21Proteostasis......................................................................................................................21Genomestability.............................................................................................................24TheFreeRadicalTheoryofAging...........................................................................25

ANTI-AGINGINTERVENTIONS..........................................................................27CALORICRESTRICTION........................................................................................................27Sirtuins................................................................................................................................28Autophagy.........................................................................................................................28Nutrientsignaling..........................................................................................................29TheRedoxLink................................................................................................................32

MAINFINDINGS.....................................................................................................34DISCUSSIONANDOUTLOOK..............................................................................36REFERENCES...........................................................................................................39

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…AndthentherewasoxygenAround 2.45 billion years ago the rise in atmospheric oxygen, known as the ‘Great Oxidation Event’, completely changed the biosphere and geosphere of the earth (Sessions et al., 2009). The increase in atmospheric oxygen from less than 0.1% of the present atmospheric level to levels slightly higher than the current levels was a driving force in the evolution of both defense systems against reactive oxygen and respiration using oxygen (Schirrmeister et al., 2013). On the one hand, the oxygen produced by cyanobacteria caused a mass extinction of obligate anaerobic life forms due to the inherent oxygen sensitivity of many proteins. On the other hand, oxygen gave rise to a metabolic breakthrough, with a respiratory chain based on oxygen as a terminal electron acceptor generating about 16 times more efficient ATP than anaerobic fermentation. This increase in energy availability marks the rise of multicellularity, paving the way for the evolution of plants, animals, and fungi (Raymond and Segrè, 2006). The ambivalent nature of oxygen led to the conclusion that: “The aerobic life-style offers great advantages, but is fraught with danger.” (Fridovich, 1978). Mapping out the cellular damage caused by reactive oxygen species and identifying cellular defense systems was one of the first priorities in the field of redox biology.

SourcesofreactiveoxygenspeciesandtheircellulardamageMolecular oxygen has the rare property of being a stable molecule with 2 unpaired electrons. This limits the number of electrons that can be transferred by organic molecules to one electron at a time, protecting organic molecules like amino acids and nucleic acids from direct

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oxidation. However, organic radicals and metals with unpaired electrons readily interact with oxygen.

Figure 1: Oxygen is reduced to water via superoxide, hydrogen peroxide and the hydroxyl radical. An estimated 90% of cellular reactive oxygen species (ROS) can be traced back to the mitochondria. The respiratory chain includes enzymes that are able to transfer electrons to oxygen in univalent redox reactions. So far 11 different sites have been identified that produce superoxide and/or hydrogen peroxide during substrate catabolism, electron transport or oxidative phosphorylation (Brand, 2016). Their individual contribution to the overall ROS production varies depending on the available substrates and the activity level (Goncalves et al., 2015). Contrary to initial suggestions, these species do not escape as intermediates of oxygen reduction but are primarily formed by autoxidation of the respiratory dehydrogenases leading to electrons leaking prematurely to oxygen (Messner and Imlay, 2002). In vitro studies suggest that approximately 0.2% of the consumed oxygen is released as O2

.- and about 0.4% is released as H2O2 (St-Pierre et al., 2002). Interestingly the rate at which superoxide or hydrogen peroxide are generated does not depend on oxygen concentration (in the physiological range), respiration rate or the rate of electron flow in the electron transport chain (Brand, 2016). These observations have lead to the hypothesis that ROS production is not a malfunction of mitochondria but rather a way of signaling. Besides the dominant mitochondrial ROS production, there is a growing number of other cellular sources of ROS such as NADPH oxidases, lipid metabolism within the peroxisomes and cytosolic enzymes such as cyclooxygenases (Balaban et al., 2005).

O2 O2- H2O2 HO H2O

e- e-+2H+ e-+H+ e-+H+

+H2O

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In addition, reactive nitrogen species like nitric oxide (NO) and peroxynitrite (ONOO−) are produced by mitochondria but this overview will only focus on ROS. Due to their varying reactivity and chemical properties different ROS affect cellular components in distinctive ways. Superoxide is electrostatically attracted to the catalytic iron atom in iron-sulfur cluster containing proteins. Oxidation through superoxide destabilizes the iron-sulfur cluster leading to the loss of the catalytic iron atom (Flint et al., 1993). Interestingly, both Complex I and Complex II of the mitochondrial electron transport chain have multiple Fe–S clusters but protect them from direct superoxide damage by burying them structurally (Imlay, 2003). Superoxide and hydrogen peroxide do not react with DNA bases (Halliwell and Aruoma, 1991) but the iron released from iron-sulfur clusters, as well as trace amounts of free Fe2+ present in the cell facilitate the Fenton reaction (Keyer and Imlay, 1996): H2O2 + Fe2+ Fe3+ + OH- + HO.

This reaction creates hydroxyl radicals that oxidize most organic molecules at diffusion-limited rates. Hydroxyl radicals produced by Fenton reaction have been linked to protein carbonylation, membrane peroxidation, and DNA damage. The most frequent DNA modification is the conversion of guanine to 8-hydroxy guanine. Even if nearby bases have been transformed the low reduction potential of guanine facilitates electron movement leaving guanine with an unpaired electron that is commonly resolved as an 8-hydroxyguanine lesion (Giese, 2002). 8-hydroxyguanine can introduce replication errors and alter the enzyme-catalyzed methylation of adjacent cytosines thus providing a link between oxidative DNA damage and altered methylation patterns (Weitzman et al., 1994). This modification seems to occur in low levels in nuclear DNA but

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is found in notably higher concentrations in mitochondrial DNA due to the proximity to ROS production and the lack of protective histones covering the DNA(Richter et al., 1988; Hartman et al., 2004) Additionally, oxidative damage can cause structural rearrangements such as deletions, insertions and sequence amplification as well as single- and double-strand breaks (Wiseman and Halliwell, 1996). Cells try to maintain low steady-state levels of damage by constantly repairing oxidative DNA damage and imbalance in the system caused by increased oxidative stress or declining repair is associated with both, cancer and aging (Maynard et al., 2009). Hydroxyl radicals also react with unsaturated fatty acids in membranes, creating a lipid radical that upon reaction with oxygen triggers a chain reaction fueling the production of lipid peroxides. If the propagation of lipid peroxides continues long enough it can disturb central membrane functions. The strong increase of the leak conductance contributes to the depolarization of the membrane which if strong enough can trigger cell death through increased cytoplasmic Ca2+ concentration (Stark, 2005). Depending on the ROS, protein damage leads to the fragmentation of the polypeptide chain, oxidation of amino acid side chains or the generation of protein-protein cross-linkages. Similar to fatty acid peroxidation, the hydroxyl radical can react with the α-hydrogen atom of polypeptide backbone creating a radical at the carbon atom that readily reacts with oxygen to form a protein peroxide. Ultimately this modification leads to the cleavage of the polypeptide at the site of modification (Stadtman, 2006). Similarly, amino acid side chains can react with the hydroxyl radical leading to cleavage of the polypeptide and protein carbonylation. Protein carbonylation, the introduction of reactive aldehyde- or ketone-groups to four susceptible amino acid residues: proline, arginine, lysine, and threonine, can disrupt protein function and structure. This disruption can lead to protein aggregation, which has been linked to both, disease and

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aging (Nyström, 2005). Recently, however, it has been hypothesized that this oxidative modification could be reversible and play a role in redox signaling (Wong et al., 2012). H2O2 does not only oxidize protein amino-acid residues through the Fenton reaction but also directly, namely at cysteine and methionine residues. The oxidation of cysteine creates sulfenic acid adducts (-SOH) that can either form disulfide cross-links with other cysteines or be further oxidized to sulfinic acid moieties (-SOOH) or sulfonic acid (-SO3H). The reversible formation of sulfenic acid is a slow reaction that is greatly influenced by the surrounding amino acids and the presence of the thiolate anion (-S-) at a higher pH (Imlay, 2003). This property is exploited by the cellular defense against H2O2 with cysteine dependent enzymes displaying up to 106 times higher rate constants, something which I will discuss in detail in the following chapter. Physiological concentrations of ROS occurring in aerobic organisms are not always harmful but can be beneficial through involvement in cell signaling pathways and in the defense against invading pathogens. An imbalance between oxidants and antioxidant defenses favoring oxidants, termed ‘oxidative stress’, potentially leads to damage and may contribute to the development of various diseases, including cancer, hypertension, diabetes, atherosclerosis and premature aging (Sies, 2017).

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CellulardefensesagainstOxidativedamageThis chapter will focus on antioxidants that are synthesized in vivo, whereas dietary-derived antioxidants, like tocopherol or carotenoids, will not be considered. While the hydroxyl radical is so highly reactive that it is virtually impossible to scavenge since it will react with whatever cellular component it meets first, there are numerous components and enzymes present that scavenge superoxide and hydrogen peroxide.

SuperoxideDismutasesThe superoxide dismutases (SODs) are oxidoreductases that catalyze the conversion of superoxide to hydrogen peroxide and oxygen. Thereby SODs prevent damage from superoxide itself as well as a chain reaction with Fe2+

released from iron-sulfur clusters fueling the production of hydroxyl radicals through the Fenton reaction. This was confirmed by studies in model organisms were SOD knockouts showed greatly increased oxidative damage, reduced lifespan and in some cases lethality (Gregory and Fridovich, 1973; Williams et al., 1998; Duttaroy et al., 2003). Equally, damage from superoxide has been linked to a number of human diseases, with some directly linked to mutations in a SOD as in the case of familial amyotrophic lateral sclerosis (fALS) (Bryngelson and Maroney, 2007). There are three major classes of superoxide dismutase classified by the metals present in the catalytic center:

- copper and zinc containing SODs (CuZnSODs), - iron or manganese containing SODs (FeSODs/ MnSODs) - nickel containing SODs (NiSODs)

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All three classes use electron transfer to and from their active site metals to catalyze the reaction of 2 molecules of O2

.- to O2 and H2O2 but are found in different organisms and vary in their subcellular location (Fridovich, 1995). CuZnSODs are found in the periplasm of some bacteria and in the cytoplasm of eukaryotes with one isoform secreted to extracellular spaces (Zelko et al., 2002). Nickel and iron-containing SODs are mainly found in bacteria and plants. The most extensively studied SOD is MnSOD since it is found in bacteria and mitochondria, the primary source of ROS in eukaryotes. The enzymatic activity MnSOD can be enhanced via post-translational modifications in response to oxidative stress (Candas and Li, 2014) and it has been shown to play a major role in promoting cellular differentiation and tumorgenesis (Clair et al., 1994). The scavenging of superoxide leaves the cell with hydrogen peroxide, a less damaging molecule but nevertheless a potentially harmful substance that typically needs to be scavenged, too.

CatalaseSimilarly to SODs, catalases use transition metals to catalyze the degradation of two molecules of hydrogen peroxide to water and oxygen. Based on their sequence and structure three classes of proteins with significant catalase activity have been identified. The most prevalent class are monofunctional, heme-containing enzymes, which have been extensively studied. Less widespread are the bifunctional, heme-containing catalase-peroxidases and the third class are the Mn-containing catalases. Catalases are ubiquitously present in aerobic cells, are structurally very stable and display incredibly fast reaction rates and some catalases even defy Michaelis-Menten kinetics by being resistant to inhibition at very high substrate rates (Chelikani et al., 2004). Counterintuitively, catalases are rarely localized to mitochondria (Radi et al., 1991; Petrova et al., 2004; Salvi et al., 2007) where one would expect a

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potent hydrogen peroxide scavenger since this compound is produced by the respiratory chain and the superoxide dismutases. Studies in Drosophila and with human cell lines expressing catalase targeted to mitochondria showed that while the oxidative damage was reduced, there was no effect on the lifespan of Drosophila (Mockett et al., 2003) and the cell lines were more susceptible to tumor necrosis factor-α-induced apoptosis (Bai et al., 1999). This stands in contrast to a study in mice where mitochondrial targeted human catalase did indeed extant lifespan (Schriner et al., 2005). Catalase is present in the cytosol and in peroxisomes where it breaks down hydrogen peroxide formed by acyl-CoA oxidase during fatty acid β-oxidation (Hiltunen et al., 2003). Peroxisomal acyl-CoA oxidase (Pox1) can produce significant amounts of hydrogen peroxide, a property that in yeast is exploited to enable light sensing without dedicated photoreceptors (Paper II). Yeast also expresses another heme peroxidase, cytochrome c peroxidase (Ccp1), that is targeted to the mitochondrial intermembrane space and that protects mitochondria from ROS damage especially when challenged with a bolus of exogenous H2O2 (Martins et al., 2013). These heme-based scavengers are not the only enzymes reducing hydrogen peroxide; a number of enzymes and small molecules are responsible for thiol-dependent scavenging.

Peroxiredoxins,Thioredoxins,GlutaredoxinsandRedoxSignalingThe following chapter will give an overview of the peroxiredoxin/ thioredoxin – system and glutaredoxin system as antioxidants and outline their role in the defense against oxidative stress. This role in oxidative stress defense is not limited to their ability to reduce hydrogen peroxide or oxidized cysteine residues but is inherently linked to their role in redox signaling. From the late 1980ies on, evidence started to accumulate that H2O2 was not only a harmful substance cells needed to detoxify but also a signaling molecule that modulates the activity of transcription factors and regulates

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signaling pathways (Marinho et al., 2014). This concept was very controversial due to the apparent paradox of a highly reactive molecule fulfilling the requirement of target specificity needed for signaling. One piece in this puzzle are the properties of cysteine residues that are ideally suited for reacting with H2O2. The reactivity of cysteine towards H2O2 varies greatly depending on the surrounding amino acids and the structure of the protein. This, combined with an ability of cysteines to cycle between different stable redox forms allows for the selectivity and specificity needed for redox signaling (D'Autréaux and Toledano, 2007). The catalytic centers of thiol-specific antioxidant proteins have evolved to allow for extremely rapid reactions with rate constants up to 108 M−1s−1 (Peskin et al., 2007). Peroxiredoxins (Prxs) have either one (1-Cys) or two (2-Cys) highly redox-active cysteines that are very efficient scavengers when H2O2 levels are low because of their much higher affinity for H2O2 compared to catalases that are only efficient when levels are high (Seaver and Imlay, 2001). Prxs are ubiquitous proteins that are found in very high abundance mainly in the cytosol, but also in mitochondria, chloroplasts, peroxisomes, the endoplasmic reticulum, and associated with nuclei and membranes (Wood et al., 2003). The high abundance combined with their high affinity make Prxs a major scavenger removing more than 90% of cellular peroxides in eukaryotes (Winterbourn, 2008). Although 3 major Prx families exist (1-Cys, typical 2-Cys, and atypical 2-Cys), I will only describe typical 2-Cys peroxiredoxins in further detail since Tsa1, central to all 3 papers presented in this thesis, is a member of this family. Typical 2-Cys Prxs are homodimers containing two identical active sites with a peroxidatic cysteine and a resolving cysteine. The peroxidatic cysteine reacts with H2O2 due to its very low pKa value. The sulfenic acid form (Cys – SPOH) of one subunit is then attacked by the resolving cysteine (Cys – SRH) located in the C-terminus of the other subunit forming a disulfide bridge between the two subunits. This step requires the α-helix where the

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Figure 2: Representation of the structure the typical 2-Cys peroxiredoxin, Prx IV, around Cys-124 and Cys-245 with different chains colored blue and red. The cysteine side chains are shown as yellow sticks, and sulfur atoms are shown as yellow balls (Without modification from Cao et al., 2011). sulfenylated peroxidatic cysteine is situated in to partially unwind (See Figure 2). The kinetics of this locally unfolded state vary between different Prxs and slow unfolding makes the sulfenylated peroxidatic cysteine susceptible to additional oxidation by H2O2 forming a sulfinic acid (Cao and Lindsay, 2017). There are two motifs that help stabilize the fully folded state and favor hyperoxidation, the GGLG sequence and the YF motif in the C-terminus (Wood et al., 2003a). First dismissed as accidental inactivation of the enzyme, hyperoxidation was shown to enable the formation of decamer/dodecamer forms that carry chaperone function (Jang et al., 2004). In Paper I, we show that hyperoxidation of Tsa1 acts as a redox switch enabling the molecular chaperones Hsp70 and Hsp104 to be recruited to protein aggregates during oxidative stress and aging. It is striking that hyperoxidation stabilizes a do-decameric, chaperone active form of Tsa1 (Noichri et al., 2015; Jang et al 2004) suggesting that chaperone-active Tsa1 binds to aggregated proteins and recruits the other chaperones there.

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This redox switch is reversible through the ATP-dependent activity of sulfiredoxin (Srx) that reduces the sulfinic acid to sulfenic acid (Biteau et al., 2003). At this point, Prx could enter a new redox cycle forming an intermolecular disulfide bridge that can be reduced by thioredoxin (Trx) (See figure 3). Thioredoxin is a small dithiol protein with a Cys-Gly-Pro-Cys active site motif that acts as a general disulfide reductase to maintain a reducing intracellular redox state (Arnér and Holmgren, 2000). Trxs are used as hydrogen donors in the mechanisms of essential enzymes such as ribonucleotide reductase (RNR) that catalyzes the formation of deoxyribonucleotides or methionine sulfoxide reductases that repair oxidized proteins. Oxidized Trx is reduced by thioredoxin reductase (TrxR or Trr), an enzyme that differs fundamentally between prokaryotes and eukaryotes. In eukaryotes, TrxR is a NADPH-dependent homodimeric flavoprotein with an additional active site that contains seleno-cysteine in mammals and cysteine in other eukaryotes (Lee et al., 2000; Zhong et al., 2000). The thioredoxin/thioredoxin reductase system is involved in numerous processes including redox signaling, immune response, cancer and cell death not further discussed here (For a recent review see: Lu and Holmgren, 2014).

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Figure 3: Redox-reaction steps of the peroxiredoxin/thioredoxin system. The peroxidatic cysteine (small red circle) of peroxiredoxins can be sequentially oxidized to sulfenic acid and sulfinic acid. The resolving cysteine (small yellow circles) forms intermolecular disulfide bridges with the sulfenic acid. Sulfiredoxin can reduce sulfinic acid in an ATP-dependent manner. Thioredoxin catalyzes the reduction of the disulfide bridge (S-S) thereby enabling the redox cycling of Prx. In this process, Trx becomes oxidized and is in return reduced by thioredoxin reductase (TrxR or Trr) at the expense of NADPH. A partially overlapping function as high-capacity hydrogen donor system for reductive enzymes is shared by the glutathione-glutaredoxin (Grx) system. Glutaredoxins (Grxs) are structurally very similar to Trx containing a dithiol active site motif with some exceptions containing a monothiol active site (Eklund et al., 1984). Grxs also catalyze the reversible reduction of protein disulfides, act as hydrogen donors to RNR but contribute to redox signaling in a different way by facilitating the glutathionylation of cysteine residues (mixed disulfides between thiols and glutathione) (Lillig et al., 2008). The tripeptide glutathione (γ-glutamyl-cysteinyl-glycine, GSH) is a thiol compound present in millimolar concentrations in the cell that plays an important role as a redox buffer keeping a high free thiol level (Meister, 1994). Glutathione reduces Grx and its oxidized form, glutathione disulfide

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Figure 4: The glutathione-glutaredoxin system: Glutaredoxin (Grx) catalyzes the reversible reduction of protein disulfides. Grx is reduced by glutathione (GSH) that in turn is reduced by glutathione reductase (GR) at the expense of NADPH. (GSSG), can be reduced with the help of the dimeric flavoenzyme glutathione reductase (GR) at the expense of NADPH (See figure 4). With such a complex system dedicated to scavenge ROS and keeping a reducing cellular environment the question arose how exactly basal cytosolic H2O2 concentrations of approximately 1–10 nM can contribute to redox signaling (Sies, 2017). With peroxiredoxins by far outnumbering targets of redox regulation and having a higher affinity towards H2O2, how do cysteines, e.g. of transcription factors, become oxidized (Stone and Yang, 2006)? One proposition was the ‘floodgate’ model, in which transient local increases in H2O2 were proposed to result in temporary Prx inhibition that allows H2O2 to build up and directly oxidize downstream targets (Wood et al., 2003a). There are a few studies showing this mechanism for signaling (Kil et al., 2012). One example is growth factor signaling that stimulates NADPH oxidase and hence increased H2O2 production leading to inactivation of Prx, but interestingly, through phosphorylation and not through hyperoxidation (Woo et al., 2010). Another mechanism that has been proposed are redox relays or redox switches, where Prxs transfer oxidizing equivalents directly or indirectly to key regulatory components, e.g. transcription factors or kinases/phosphatases (Delaunay et al., 2002; Sobotta et al., 2015; Stöcker et al., 2017, Travasso et al., 2017). In Paper III we propose a mechanism

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that could be a redox relay with the peroxiredoxin, Tsa1, regulating nutrient signaling through the oxidation of conserved cysteines present in protein kinase A (PKA). With an ever-expanding landscape of redox regulation it will be interesting to see what other mechanisms have evolved to use a seemingly unfitted molecule for signaling and what role peroxiredoxins play in these processes (Marinho et al., 2014; Flohé, 2016; Antunes and Brito, 2017).

YeastasamodelsystemThe evident connection of reactive oxygen species and of the cellular defense system to numerous diseases and phenotypes of aging raises the question how reduced or increased activity of these components influence the redox balance in the cell and thereby disease and aging phenotypes. One well-established model organism is the yeast Saccharomyces cerevisiae that is easily cultured and fast growing with an approximate generation time of 90 minutes. Very valuable tools for yeast research are the extensive strain collections among others with GFP-labeled genes or knockout collections of non-essential genes that enable genome-wide screens for genetic interactions (Tong et al., 2001). These genetic interactions are currently exhaustively analyzed in an effort to find connections within and between protein complexes and pathways on the basis of which genotype-to-phenotype relationships might be identified not just for yeast but also in regard to human diseases (Costanzo et al., 2016). A rather surprising finding was made nearly 60 years ago when it was discovered that yeast undergoes a finite number of divisions (Mortimer and Johnston, 1959). This discovery paved the way for yeast as a model organism for aging research. The lifespan of yeast determined by the number of daughter cells produced by one mother cell is called replicative lifespan with common laboratory strains averaging around 25 divisions. With an increasing number of division yeast cells are also displaying signs of aging including an increase in size, prolonged generation times and nuclear fragmentation.

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This technique as an approximation for aging in higher organisms has come under critique, as most cells are non-dividing, post-mitotic cells. While replicative lifespan might rather provide clues to the aging of mitotic cells or stem cell populations another technique termed chronological aging has been proposed. Chronological lifespan measures the length of time that a non-dividing yeast cell survives (For a recent review see Longo et al., 2012). Both techniques have been used to find genes modulating lifespan that were termed gerontogenes (Rattan, 1995). An increasing number of genes linked to aging are being discovered fueling a debate on the causes of aging described in the next chapter (Kenyon, 2010).

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AgingAging, or senescence, is characterized by a progressive loss of physiological integrity over time (Rose, 1991) and is an almost universal feature of all living organisms. Aging is highly complex and affects numerous processes and cellular components at the same time. Recently 9 hallmarks of aging have been defined in an effort to further advance the field of aging research (See figure 5; López-Otín et al., 2013).

Figure 5: The Hallmarks of Aging (With permission from López-Otín et al., 2013).

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The universality of aging combined with the very broad range of species-specific lifespans drives an ongoing debate about the underlying causes and the driving forces of aging. One of the biggest divides remains the question whether aging is programmed or non-programmed although some argue that both forms could occur in nature in different species (Trindade et al., 2013).

ProgrammedAgingThe underlying idea of programmed aging is that a genetic program shaped by natural selection determines and controls the gradual decline of vital functions and limits the lifespan of species (Libertini, 2015). The theory assumes an evolutionary force limiting life after optimum lifespan while non-programmed aging theories rather argue that there is no evolutionary driving force to prolong lifespan after a reproductive period (Goldsmith, 2012). The evolutionary driving force of programmed aging would need to be selection at the group level rather than the individual level. Such a mechanism could have evolutionary benefits since longevity contrary to reproduction is detrimental for adaptability (Martins, 2011) and longevity could be traded-off to enhance reproduction of the kin. One argument against such an evolutionary force is that “there is scant evidence that senescence contributes significantly to mortality in the wild ... As a rule, wild animals simply do not live long enough to grow old ... Therefore, natural selection has limited opportunity to exert a direct influence over the process of senescence” (Kirkwood and Austad, 2000). Although this paradigm has been put into perspective by a later study showing widespread senescence in wild population (Nussey et al., 2013) further issues remain. Despite the advances in the understanding of genetic regulation and cellular signaling no clock mechanisms, as predicted by programmed aging, has been found yet. Recent advances in aging research have uncovered a number of conserved genes and pathways that influence aging as would be expected from programmed aging but they do so in a very inconsistent manner with great variations between genetically identical individuals as

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shown in model organisms and studies with monozygotic human twins (Finch and Kirkwood, 2000, Kirkwood, 2005). This lack of indication of a stable and controlled aging process has a majority of researchers and research favor non-programmed aging theories discussed below.

EvolutionaryAgingTheoriesThe different theories presented here show the ongoing effort to identify the evolutionary origins of aging and as well as the causes of the variety of age-related phenotypes affecting an organism on a molecular, cellular and tissue level.

MutationAccumulationTheoryThe mutation accumulation theory, first proposed by Medawar in 1952, has as its underlying assumption that in natural populations individuals hardly ever reach senescence due to strong extrinsic mortality e.g. through predation. Since only very few individuals reach old age germline mutations affecting late-acting alleles could only be weakly acted against by natural selection and accumulate in the genome. The consequence is that the accumulated mutations manifest as aging in the individuals that have escaped extrinsic death long enough. Although the argumentation is plausible the underlying assumption of the theory has come under increased scrutiny. The assumption that death is supposed to occur before senescence has an effect, thus senescence not necessarily affecting the probability of death, is not reflected in mortality rates of real species (Libertini, 2015). Even more importantly “That senescence is rarely, if ever, observed in natural populations is an oft-quoted fallacy within bio-gerontology.” (Nussey et al., 2013).

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In light of recent research contradicting the idea that senescence is absent in natural populations, the mutation accumulation theory might be rather seen as a historic attempt to explain aging than a basis for further research.

AntagonisticPleiotropyTheoryAnother theory that assumes different evolutionary forces on young versus old individuals is the antagonistic pleiotropy theory (Williams, 1957). Here the idea is that organisms have genes, termed antagonistic pleiotropic, that increase the odds of successful reproduction early in life but have deleterious effects later in life. So from an evolutionary point of view, the advantage of higher reproduction rates would outweigh consequences on mortality due to senescence caused by the same genes. This idea gained new traction when a study found that a delay in reproduction resulted in an increased lifespan in fruit flies while at the same time reducing early life fitness (Sgrò and Partridge, 1999). Given the diverse effects and phenotypes of aging one would expect to find a high number of genes that are beneficial early in life but contribute to senescence later. Studies that tried to identify genes having an antagonistic pleiotropic effect found that although there are a number of such examples like p53, Insulin-like growth factor signaling, or Hsp70s, the effects on aging are often not as clear as predicted by the model and strongly interwoven with other genes and pathways (Leroi et al., 2005; Goto, 2008; Ungewitter and Scrable 2009). A comprehensive study concluded that the evidence for antagonistic pleiotropic effects in the wild becomes negligible if natural polymorphism is considered and the remaining evidence from Drosophila genes is excluded (Leroi et al., 2005). More research is needed on the early-life fitness effects of conserved longevity genes and pathways, particularly on nutrient signaling pathways, which are beneficial in early stages of life but a reduction of nutrient signaling has been linked to longevity (Discussed in the chapter Anti-aging Interventions)

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TheDisposableSomaTheoryThe disposable soma theory proposed by Kirkwood in 1977 is based on the concept that resources are limited under natural conditions forcing an organism to prioritize energy expenditure. As a consequence somatic maintenance and repair is limited to a level only good enough to keep the organism in sound physiological condition for as long as it has a reasonable chance of survival and reproduction (Kirkwood, 2005). The model predicts the accumulation of cellular and molecular damage as the cause of senescence, hence genes that regulate the levels of somatic maintenance and repair determine longevity. This theory has the advantage that accumulation of different types of damage is strongly influenced by chance and thus a high variability in the aging phenotype would be expected, an observation the above-discussed theories could not sufficiently explain. Equally a number of genes that promote longevity in different model organisms can be categorized as having a maintenance and repair function (See Anti-aging Interventions). The theory reaches its limitations with semelparous organisms that die quickly or directly after reproduction and with programmed cell death and aging in unicellular organisms (Longo et al., 2005; Fabrizio et al., 2004). The most commonly used example is Pacific salmon that dies very quickly after spawning. Although some argue that the energy expenditure during the swim against the rivers current makes it an extreme example of the disposable soma theory (Kirkwood, 2005), studies showed that sudden death occurred independent of the river’s length or current and depended on the presence of the gonads and adrenal glands (Robertson and Wexler, 1962). Many more evolutionary aging theories have been proposed throughout the years but a major divide remains the question whether aging is programmed or not. Although the question is often approached as one or the other both theories might be valid depending on the ecology of the species. Depending on the number of offspring, the degree of parental investment, social

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behavior and many other factors that have evolved as survival strategies, different forms of aging might have evolved accordingly, making it all the more difficult to develop one unifying theory of aging.

MolecularMechanismsofAgingWhile the previous sections focused more on the question why we age the following part will further discuss different theories about the question how we age, particularly on a molecular level.

ProteostasisProteins play a central role in many cellular functions and thanks to their prominent role proteins have even been suspected to be the source of genetic inheritance before the discovery of DNA. Unsurprisingly their homeostasis (proteostasis) is tightly managed with a complex molecular chaperone system for folding, re-folding, transport and several pathways for degradation. In aging cells, the ability to preserve proteostasis under resting and stress conditions gradually declines (Labbadia and Morimoto, 2015). The accumulation of protein aggregates as a consequence of proteostasis failure is linked to senescence as well as to pathologies, like the neurodegenerative diseases, Parkinson’s disease and Alzheimer’s disease (Kaushik and Cuervo, 2015). The molecular chaperone system is involved in every step of a proteins path from synthesis to folding and subcellular targeting until degradation. A central role plays the constitutively expressed but also stress-inducible chaperone heat shock protein (Hsp) 70 that assists protein folding in an ATP-dependent manner. ATP binding and hydrolysis changes the affinity for polypeptides and is regulated by nucleotide exchange factors and J-domain proteins of the Hsp40 family. The conformational change driven by ATP hydrolysis is the basis for Hsp70s role in folding, disaggregation of

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protein aggregates and translocation of polypeptides (Mayer, 2013; Hartl et al., 2011). For aggregate clearance, Hsp70s interact with Hsp104, a disaggregase that becomes essential under heat-stress conditions (Parsell et al., 1994). In yeast Hsp104 is also involved in the retention of oxidatively damaged proteins in the mother cell (Erjavec et al., 2007; Tessarz et al., 2009), disaggregation of aggregates formed during oxidative stress (Paper I) and formation or inhibition of prions and amyloids depending on the cellular environment (Grimminger‐Marquardt and Lashuel, 2010). In Paper I, we show that the recruitment of Hsp70s and Hsp104 facilitates the lifespan extension by the peroxidase, Tsa1, through the clearance of age-induced protein aggregates. Since this system and other chaperones are ATP-dependent, it is suspected that the reduced energy availability in senescent cells could be a cause for failing proteostasis (Brehme et al., 2014). The collapse of proteostasis as an early event of cellular senescence is not only depending on the molecular chaperone system but also on the different degradation pathways (Ben-Zvi et al., 2009). Malfunctions in proteolytic systems such as the ubiquitin-proteasome system, autophagy and mitochondrial autophagy (mitophagy) are associated with accelerated aging and neurodegenerative diseases (Kaushik and Cuervo, 2015). Increased activity in the proteostasis systems and proteome stability has been associated with longevity as in the case of the naked mole rat (Pérez et al., 2009a) but the complexity of the system impedes the development of proteostasis-targeted interventions. With the ubiquitin-proteasome system alone having over 700 components and the molecular chaperone system not lacking in complexity as well, more research is needed to understand the mechanistic basis for proteostasis collapse and the different health consequences with regard to aging.

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Figure 6: Overview of cellular proteostasis: Chaperones and two proteolytic systems, the ubiquitin proteasome system (UPS) and autophagy, maintain intracellular proteostasis. Chaperones (blue and purple circles) assist de novo synthesized proteins and unfolded proteins to reach their folded stable status and help to disaggregate protein aggregates. If folding is not possible, chaperones target the unfolded protein for degradation by the proteasome (often after ubiquitination-green circles).

Ribosome

FoldedProtein

UnfoldedProtein

Autophagy

ProteosomalDegrada:on

Hsp70Hsp104

Hsp70

Aggregate

Ubiqui:n

Proteasome

Autophagosome

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GenomestabilityStudies focusing on conditions that accelerate human aging, such as Werner syndrome (WS), Hutchinson Gilford progeria syndrome (HGPS) or Cockayne syndrome (CS), have found that the vast majority share mutations that affect nuclear DNA metabolism or more specifically genome maintenance (Schuhmacher et al., 2008). Combined with the observation that the continued exposure to extrinsic and intrinsic sources of DNA damage, like ionizing radiation, cellular ROS or double strand breaks, leads to accumulation of mutations in cellular DNA, maintenance of genome stability has been proposed as a cause for cellular senescence (Vijg, 2000). This cellular senescence has been suggested as a tumor suppressor mechanism, whereby cells that have accumulated mutations enter a state of irreversible cell cycle arrest (Krtolica and Campisi, 2002). Another mechanism in line with this idea is cell cycle arrest triggered by “uncapped” telomeres. These are telomeres that through repeated replication have been shortened to the point where they lost their structural features as well as protective proteins (Ben-Porath and Weinberg, 2004). These senescent cells that eventually undergo apoptosis, will lead to a depletion of stem cells and thereby to the aging of the entire organism. Most evidence for the importance of DNA repair for cellular senescence stems from human disease or mouse models, which exhibit impaired DNA repair (Lombart et al., 2005). A more convincing argument to support this theory might be gained from model organisms that possess enhanced repair. Some experimental support comes from yeast where increased genome stability, particularly in the rDNA locus, through overexpression of Sir2 extends lifespan (discussed in Anti-aging Interventions; Kaeberlein et al., 1999). DNA as an essential and non-replaceable component of the cell undoubtedly plays a role in aging, in how far DNA repair plays a central role has to be determined by future research.

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TheFreeRadicalTheoryofAgingThe free radical theory of aging proposes that the reactive oxygen species, produced as part of normal metabolism, cause aging through accumulated oxidative damage to macromolecules, organelles, and cells (Harman, 1956). Considering the diverse damage caused by ROS in the cell, discussed in chapter 1, as well as the observation that ROS and their damage increase in an aging cell, it seems only consequent to consider ROS as a cause of aging (Stadtman, 1992, Hamilton et al, 2001). In line with this observation was the discovery that antioxidants can prolong lifespan. Peroxiredoxins extend lifespan in yeast (Molin et al., 2011; Paper I), C. elegans (Oláhová et al., 2008) and Drosophila (Lee et al., 2009). In studies focusing on the overexpression of superoxide dismutase (SOD) lifespan extension was found in Drosophila melanogaster (Orr and Sohal, 1994; Phillips et al., 2000; Sun et al., 2002), C. elegans (Doonan et al., 2009) and yeast chronological lifespan (Harris et al., 2003). Catalase overexpression was found to prolong life in Drosophila as well as in mice (Orr and Sohal, 1994; Schriner et al., 2005). These observations were challenged by the critique that the Drosophila strains used in the above-mentioned experiments were short-lived and that similar experiments in long-lived strains showed no significant effect on lifespan (Orr et al., 2003). Equally studies in mice found that the overexpression of SOD had no effect on lifespan (Pérez et al., 2009b; Huang et al., 2000) and that lifespan extension in C. elegans overexpressing the major SOD, sod-1, was not due to decreased oxidative damage (Cabreiro et al., 2011). This critique might not undermine the oxidative damage theory since SOD, transforming O2

•− to H2O2, can lead to increased H2O2 levels in the cell, which can be both beneficial or deleterious for lifespan (Buettner et al., 2006). More strikingly the lifespan in knockout experiments mostly remains normal in non-lethal antioxidant enzyme-deficient mice despite the fact that increased tumor burden was observed (Muller et al., 2007). Although not true for all antioxidant enzymes and all model organisms, this stands in

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direct contradiction with the predictions of ROS and ROS damage being a cause of aging. Another conflicting observation is the increasing evidence that ROS, particularly H2O2, may actually work as signaling molecules potentially increasing lifespan. One example is the action of the antidiabetic drug metformin that was shown to extend lifespan by increasing the production of ROS and signaling via peroxiredoxin (DeHaes et al., 2014). This is an example of mitochondrial hormesis, a specific form of hormesis, whereby moderate oxidative stress indirectly proves beneficial (Goulev et al., 2017). Proponents of mitochondrial hormesis argue that mildly increased ROS production is health promoting and increases the lifespan rather than accelerating senescence (Ristow and Zarse, 2010). Some authors argue that our limited understanding of the complex cellular processes of ROS production and the molecular defense system should stop us from disregarding the theory in the face of these challenges (Kirkwood and Kowald, 2012). Other authors think that it is time to progress to a more open damage theory of aging or straight out declare the theory dead (Gladyshev, 2014; Speakman et al., 2011, Salmon et al., 2010). While the free radical theory of aging, especially with its later extension as a mitochondrial theory of aging (Harman, 1972), does cover some phenotypes that are associated with senescence, such as mitochondrial dysfunction, oxidative DNA damage and protein aggregation, it appears too narrow to explain damage from other processes, such as metabolite damage, translational errors, transcriptional heterogeneity, mistargeting of proteins to cellular compartments (Gladyshev, 2014). In our research, we see that while the loss of the peroxiredoxin Tsa1 increases the mutation rate, the lifespan extension caused by an extra-copy of Tsa1 is not due to reduced DNA damage but rather due to reduced nutrient signaling (Paper III) and the decreased accumulation of protein aggregates (Paper I). In summary, I think that a deeper understanding of H2O2 signaling and its cellular implications will demand to further distinguish the contribution of ROS to senescence from damage or deregulated signaling.

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Anti-agingInterventionsThe free radical theory of aging derived from the rate-of-living hypothesis that was proposed in 1908 (Rubner, 1908). The rate-of-living hypothesis proposed that the lifespan of an organism depends on the rate of its metabolism – a concept that was overly simplistic and was later refuted (Magalhães et al., 2007). Nevertheless, this idea has lead to one of the most successful anti-aging interventions, caloric restriction (CR) (McCay et al., 1935).

CaloricrestrictionCaloric restriction (CR) or dietary restriction is the reduction of caloric intake without malnutrition, historically developed to reduce metabolism and thereby extend lifespan. CR has been adapted successfully for many model organisms ranging from yeast to rhesus monkeys and has been shown to extend lifespan in most organisms where it has been studied (Lin et al., 2000; Mattison et al., 2017). However, it was not through the originally anticipated mechanism. In yeast, experiments showed that CR actually increased respiration through a metabolic shift from fermentation to respiration instead of lowering it (Lin et al., 2002). Equally an increase in mitochondrial respiration has been noted in C. elegans (Schulz et al., 2007). In recent years four major areas have been investigated as the underlying cause of CR:

• Gene silencing and signaling through sirtuins • Induction of autophagy • Reduction of nutrient signaling • Induction of mitochondrial function and oxidative stress resistance

These 4 mechanisms are heavily intertwined and each of the four effectors has been shown to prolong the lifespan in model organisms on its own. It

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seems highly likely that not one but all of the above-mentioned factors are necessary for CR mediated lifespan extension.

SirtuinsSirtuins have been identified as longevity genes in a number of model organisms and have been shown to be necessary for lifespan extension through CR (Guarente, 2013). Sirtuins regulate a number of processes either through direct deacetylation of regulatory proteins or through histone deacetylation-induced gene silencing. A number of sirtuins have been shown to promote a metabolic shift away from glycolysis and toward mitochondria thereby increasing oxidative stress resistance (Hallows et al., 2012; Zhong et al., 2010; Tao et al., 2010). Suppression of NF-kB signaling is also mediated by several sirtuins and may be an explanation for the general anti-inflammatory effect of CR (Yeung et al., 2004; Rothgiesser et al., 2010). Additionally, the sirtuin SIRT1 activates autophagy through deacetylation of FOXO3, a transcription factor itself associated with longevity (Lee et al., 2008; Morris et al., 2015).

AutophagyCR is an efficient inducer of autophagy, a process where portions of the cytoplasm are sequestered within autophagosomes that fuse with lysosomes for digestion of their content. The process is important for the clearance of protein aggregates and dysfunctional organelles and reduced autophagy activity has been linked to neurodegenerative disease (Simonsen et al., 2008), arteriosclerosis, and type 2 diabetes (Levine and Kroemer, 2008). Caloric restriction-induced autophagy appears to be mediated mainly through Sirtuin-1. The same pathway is also triggered by resveratrol, a proposed anti-aging supplement (Morselli et al., 2010). Interestingly, another anti-aging supplement, spermidine, also induces autophagy but in a sirtuin-independent way (Eisenberg et al., 2009). Autophagy is also linked

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to nutrient signaling through the insulin/IGF-1 (insulin-like growth factor 1) pathway connected to one of the key autophagy regulators, the target of rapamycin (TOR) kinase (Madeo et al., 2010).

NutrientsignalingAn effect of caloric restriction that has been widely reported is the repression of nutrient signaling and it has been clearly linked to the promotion of longevity. Several pathways have been identified including insulin/insulin-like growth factor (IGF), TOR and RAS–PKA signaling. The prolonged health- and lifespan through reduced nutrient signaling are attributed to several down-stream effects including slower growth, increased insulin sensitivity and a general increase in stress resistance. Caloric restriction decreases the level of insulin/IGF signaling affecting downstream factors such as PI3K/Akt/Ras involved in cell proliferation and the repression of the forkhead box O (FOXO) transcription factor, which regulates stress response genes (Salih and Brunet, 2008). Interventions and mutation modulating insulin/IGF signaling have been shown to affect lifespan from worms to mammals (Cohen and Dillin, 2008; Kenyon, 2010; Bartke, 2016). In accordance with the idea that CR simultaneously affects several systems, research showed that CR had an additive effect in long-lived Ames dwarf mice that through mutation are deficient for growth hormone (Alderman et al., 2010). Similarly, lifespan extension by CR was still observed in Drosophila with mutated FOXO (Min et al., 2008). Connected to insulin/IGF signaling is the target of rapamycin (TOR) pathway that plays a central role in growth regulation in response to available nutrients, both in proliferating and non-proliferating eukaryotic cells (Schmelzle and Hall, 2000). TOR is a highly conserved serine/threonine protein kinase and several components of the TOR pathway have been linked to longevity (McCormick et al., 2011). It has also

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been linked directly to CR in yeast (Kaeberlein et al., 2005) and mice (Ma et al., 2015). While reduction of TOR signaling can prolong lifespan there are adverse side effects depending on the severity of TOR repression. In C. elegans strong reduction of TOR activity resulted among others in reduced fertility and developmental arrest at the L3 larval stage (Jia et al., 2004). The TOR pathway has received much attention in aging research since the known inhibitor rapamycin has been proposed as an anticancer drug (Sabatini, 2006) and as a pharmacological intervention to extend lifespan (Fang et al., 2013). In yeast the TOR pathway is connected to the Ras-cAMP-PKA pathway through shared downstream effectors, the transcription factors Msn2 and Msn4 that regulate stress genes (Medvedik et al., 2007). The PKA pathway is a glucose-responsive signaling pathway promoting fermentative growth in yeast while inhibiting stress resistance characteristics (Figure 7). Reduced PKA activity has been shown to extend lifespan in yeast and mice (Lin et al., 2000; Enns et al., 2009; Cheng et al., 2014) and genetic interventions that reduce signaling through the cAMP-PKA pathway have been used to mimic caloric restriction (Kaeberlein, 2010). To induce CR in yeast, the glucose present in the medium is reduced from 2% to 0.5%. Glucose availability is signaled through a G-protein-coupled receptor (Gpr1) that activates adenylate cyclase (AC, named Cyr1 or Cdc35 in yeast) via the G protein Gpa2. Different to higher eukaryotes, yeast also regulates AC activity through Ras proteins. The glucose signal is integrated through the guanine exchange factor Cdc25 that activates Ras while the GTPase-activating proteins Ira1 and Ira2 inhibit Ras activity. Adenylate cyclase regulates the cAMP pool together with the cyclic AMP phosphodiesterases Pde1 and Pde2 that are responsible for degradation. Finally, cAMP regulates protein kinase A (PKA) activity by binding to the regulatory subunit (Bcy1) that then dissociates from the catalytic subunits, a dimer of Tpk1, 2 or 3 (Rolland et al., 2002).

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Figure 7: The Ras-cAMP-PKA pathway with stimulatory components in green and inhibitory components in red. Active PKA phosphorylates downstream targets like the transcription factors Msn2/Msn4 that are shuttled out of the nucleus upon phosphorylation (Görner et al., 1998). The actual downstream effectors of reduced PKA activity are less clear. One proposition is that reduced PKA activity could act through an increased stress resistance as demonstrated by an increased resistance to hydrogen peroxide (Molin et al., 2011). Molin and collaborators showed that CR reduced PKA activity in yeast, which induced the expression of the sulfiredoxin Srx1 delaying the age-associated increase in hyperoxidation of the peroxiredoxin Tsa1. In Paper III we show that the catalytic cycle of Tsa1 is able to inhibit PKA activity possibly through redox-modification of conserved cysteines present

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in the catalytic subunit. This demonstrates the high interconnectedness of cellular pathways and establishes nutrient signaling as a central integrator of diverse signals.

TheRedoxLinkWhile there is a consensus that CR affects nutrient signaling, the actual down-stream effectors of CR are less clear. There is extensive research connecting CR to ROS and antioxidants but these reports are often conflicting. While some studies find a reduction in ROS production and lower levels of ROS damage others report an increase in ROS production that has a hormetic effect thereby increasing ROS defenses. Although mitochondrial oxygen consumption remained unchanged during CR, a reduction in hydrogen peroxide production has been observed in mice (Gredilla et al., 2001; Lambert and Merry, 2004). The opposite has been reported in studies with Drosophila (Zid et al., 2009) and C. elegans where an increase in ROS production was reported and lifespan extension by CR was abolished by antioxidant supplements (Schulz et al., 2007; Zarse et al., 2012). Regardless if through increased or decreased ROS production, CR has been repeatedly linked to lower damage, especially in the genome. Assessing the age-related accumulation of 8-hydroxy guanine levels in nuclear DNA in different tissues in mice and rats, a significant reduction was found in animals that were subjected to CR (Hamilton et al., 2001). Equally, in mouse models with defective DNA repair, a model of Cockayne syndrome, CR reduced endogenous DNA damage (Vermeij et al., 2016). The authors hypothesize that not increased repair but reduced damage possibly through increased redox defense could be the cause. A potential downstream target of CR in mice could be increased de novo glutathione biosynthesis, which was shown to attenuate an age-related pro-oxidizing shift (Rebrin et al., 2011). It was also reported that induction of

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SOD expression (Qiu et al., 2010; Zarse et al., 2012) and a more reduced pool of peroxiredoxins through sulfiredoxin expression could be mediators of CR (Molin et al., 2011). Taken together there is a strong line of evidence for a prominent role of redox regulation and redox defense systems in aging and as mediators for caloric restriction. A better understanding of cellular processes in general, and of aging in particular, will be indispensable for the future. In a society challenged by an ever-increasing chance to become a centenarian but a health span that does not mirror this development, there is a yet unmet need for anti-aging interventions and drugs that improve health and slow down aging (De Cabo et al., 2014; Martinez et al., 2017).

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MainfindingsPaper I • An extra copy of the peroxiredoxin Tsa1 extends yeast lifespan by

ca. 40% • Tsa1 recruits the Hsp70s, Ssa1 and Ssa2, and subsequently Hsp104

to hydrogen peroxide induced aggregates • Hyperoxidation of the peroxidatic cysteine (C48) but not the

resolving cysteine was required for recruitment of chaperones to aggregates

• Sulfiredoxin-mediated reduction of hyperoxidized Tsa1 is required

for the resolution aggregates • An extra copy of Tsa1 also reduces age-associated accumulation of

aggregates Paper II • Despite the lack of dedicated photoreceptors yeast reacts to light by

shuttling the transcription factors Msn2 and Msn4 in and out of the nucleus

• The peroxisomal acyl CoA oxidase Pox1 produces H2O2 upon illumination

• Increased H2O2 levels trigger a redox relay from Tsa1 to Trx1/Trx2

impinging on the PKA pathway • Tsa1 affects the PKA catalytic subunits directly by promoting their

nuclear export • Decreased PKA activity leads to dephosphorylation of Msn2/Msn4

promoting nuclear localization of the transcription factors

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Paper III • Tsa1 mediated reduction of the rate of aging depends on both

cysteines • The shortened lifespan and H2O2 sensitivity of a tsa1∆ strain can be

counteracted by decreasing PKA activity • Lifespan extension and increased H2O2 resistance by an extra copy

of Tsa1 depends on reduced PKA activity • Tsa1 reduces PKA activity down-stream of Ras and cAMP

• Tpk1 is oxidized upon H2O2 treatment

• A conserved cysteine in the PKA subunit Tpk1 dictates cellular

H2O2 resistances

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DiscussionandOutlookPeroxiredoxins have been implicated in numerous cellular processes apart from their function in scavenging hydrogen peroxide, raising the question which function is responsible for their anti-aging properties. The primary scavenging function is important as it is linked to genome stability with the deletion of Prx leading to an increased mutation rate (Huang and Kolodner, 2005; Neumann et al., 2003). But the scavenging alone does not explain the increase in lifespan with an extra copy of Tsa1. The age-related accumulation of mutations in the genome does not decrease in the presence of twice as much scavenger (Paper I). In contrast, a clear reduction in age-associated protein aggregates can be seen when Tsa1 is overexpressed. The ability of hyperoxidized Prx to assemble to a high molecular weight form that acts as a chaperone seems to be beneficial during aging. Interestingly, Tsa1 seems to be essential for the recognition of protein aggregates formed during oxidative stress but plays no role in the recognition of heat-induced aggregates (Paper I). This would argue for the accumulation of oxidative protein damage during aging assuming, that similarly to the DNA damage, not increased scavenging but in this case an increased chaperone activity is responsible for the decrease in protein aggregates. With a still ongoing debate about the actual toxicity of protein aggregates, there remains the question how important the ability of Tsa1 to reduce protein aggregates is for lifespan extension. In a Tsa1 mutant lacking the YF motif that stabilizes the sulfenic acid form, Tsa1 forms disulfide bridges more readily and enters the catalytic cycle rather than getting hyperoxidized. In this mutant we only see a minor effect on lifespan (Paper III) compared to a mutant that lacks the resolving cysteine C171 which is as short-lived as the Tsa1 knockout mutant. In order to really separate the chaperone function from other functions of Tsa1, it would be interesting to test if the tsa1∆YF mutant indeed accumulates more protein aggregates, especially in aged cells. If this was the

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case it would be interesting to see how an extra copy of tsa1∆YF would affect the lifespan in a tsa1∆YF background. The equilibrium between oxidized Prx entering the redox-cycle and Prx becoming hyperoxidized seems to be important also for a proposed role in circadian rhythms. It was proposed that the endogenous rhythm in the generation of reactive oxygen species (ROS) could lead to circadian oscillations in the oxidation state of peroxiredoxin (Edgar et al., 2012). This oscillation between hyperoxidized and reduced Prx can also be observed in yeast where it influences yeast respiratory oscillations. Yet, these oscillations are maintained in knockout strains suggesting that Prxs are not required for circadian rhythms (Causton et al., 2015). Based on these observations disrupting the ability of Tsa1 to become hyperoxidized should not affect yeast metabolism but specifically disrupt chaperone function. However, it is unclear if local hyperoxidation of Tsa1 is needed for other signaling processes based on the floodgate model. Interestingly, the generation of ROS by light is exploited as a possibility to signal light stress in yeast (Paper II). But the signal transmission requires the catalytic cycle and accordingly the tsa1∆YF mutant showed an increased response to light as observed by increased Msn2/4 nuclear localization. The light-induced production of H2O2 through conserved peroxisomal acyl-CoA oxidase could be a mechanism by which Prx circadian rhythms are entrained by light. But this raises the question what kind of signal hyperoxidized Prxs that accumulate during the light phase transmit to putative downstream targets, as the signal transduction we see during light stress is exclusive to the catalytic cycle. The ability of Tsa1 to reduce PKA activity is also the basis for increased hydrogen peroxide resistance and lifespan extension. Counterintuitively, not the presence of more Tsa1 but the reduced PKA activity seems to be responsible for a higher H2O2 resistance on plate (Paper III). Likely effectors could be down-stream targets of PKA/Msn2/4 such as sulfiredoxin, catalase, cytochrome c peroxidase and glutaredoxin (Hasan et al., 2002)

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For lifespan extension, a similar mechanism emerges that is in accordance with hormesis research showing a beneficial effect of increased stress resistance (Goulev et al., 2017; Ristow and Zarse, 2010). However, in a strain with an extra copy of Tsa1 where the PKA regulated transcription factors Msn2 and Msn4 are knocked out, we still observe lifespan extension, although to a lesser extent (Paper I). In support of the notion that lifespan extension mediated by reduced PKA does not exclusively depend on Msn2/4 as downstream mediators, is the observation that a restored lifespan in a ras2∆,tsa1∆ strain does not correlate with increased Msn2,4 activity (Paper III). Still, the question remains how much the lifespan extension is mediated by reduced PKA signaling and how much should be contributed to the role of Tsa1 in proteostasis. Untangling these two effects would need further investigation on the downstream effectors of reduced nutrient signaling, a question which is equally interesting in regard of how caloric restriction slows down aging. Additionally, it remains to elucidate if PKA regulation through Tsa1 is a direct or indirect process and if this mechanism is exclusive to PKA or a general mechanism of hydrogen peroxide signaling with more downstream targets. In that regard, there is room for a lot more research to identify the cellular functions of peroxiredoxins and their involvement in cellular aging.

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