C. elegans screening strategies to identify pro-longevity interventions

Mechanisms of Ageing and Development 157 (2016) 60–69

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Mechanisms of Ageing and Development journal homepage: www.elsevier.com/locate/mechagedev

C. elegans screening strategies to identify pro-longevity interventions Silvia Maglioni b , Nayna Arsalan b , Natascia Ventura a,b,∗ a b

Institute for Clinical Chemistry and Laboratory Diagnostic, Medical Faculty of the Heinrich Heine University, 40225 Duesseldorf, Germany Leibniz Research Institute for Environmental Medicine, Duesseldorf, Germany

a r t i c l e

i n f o

Article history: Received 6 April 2016 Received in revised form 22 July 2016 Accepted 25 July 2016 Available online 26 July 2016 Keywords: Aging C. elegans Development In vivo drug screening Mitochondria

a b s t r a c t Drugs screenings in search of enhancers or suppressors of selected readout(s) are nowadays mainly carried out in single cells systems. These approaches are however limited when searching for compounds with effects at the organismal level. To overcome this drawback the use of different model organisms to carry out modifier screenings has exponentially grown in the past decade. Unique characteristics such as easy manageability, low cost, fast reproductive cycle, short lifespan, simple anatomy and genetic amenability, make the nematode Caenorhabditis elegans especially suitable for this purpose. Here we briefly review the different high-throughput and high-content screenings which exploited the nematode to identify new compounds extending healthy lifespan. In this context, we describe our recently developed screening strategy to search for pro-longevity interventions taking advantage of the very reproducible phenotypes observed in C. elegans upon different degrees of mitochondrial stress. Indeed, in Mitochondrial mutants, the processes induced to cope with mild mitochondrial alterations during development, and ultimately extending animal lifespan, lead to reduced size and induction of specific stress responses. Instead, upon strong mitochondrial dysfunction, worms arrest their development. Exploiting these automatically quantifiable phenotypic readouts, we developed a new screening approach using the Cellomics ArrayScanVTI-HCS Reader and identified a new pro-longevity drug. © 2016 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2.

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Compound screens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 C. elegans drug screenings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 2.1. Advantages of C. elegans as an in vivo screening tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 2.2. C. elegans screenings to identify pro-longevity interventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Development of a new C. elegans screening strategy to identify pro-longevity interventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 3.1. Mitochondria as a target hub integrating different signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 3.2. C. elegans phenotypes in response to mitochondrial targeting interventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 3.3. A new phenotype-based screening to identify mitochondrial-targeting interventions acting from development to promote longevity . . . . 66 Conclusions and perspectives: from developmental effects to healthy aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

1. Compound screens During the last few decades, the continuous discovery of new chemicals and small molecules (Szymanski et al., 2012) lead to the

∗ Corresponding author at: Institute for Clinical Chemistry and Laboratory Diagnostic, Medical Faculty of the Heinrich Heine University, Duesseldorf 40225, Germany. E-mail address: [email protected] (N. Ventura). http://dx.doi.org/10.1016/j.mad.2016.07.010 0047-6374/© 2016 Elsevier Ireland Ltd. All rights reserved.

development of automated platforms to screen large number of compounds and quickly assess their biological or biochemical activity. These platforms can be mainly divided into high-throughput screening (HTS) and high content screening (HCS) strategies. In HTS, robotics liquid handling devices and data processing are required to simultaneously screen a large number of compounds (Liu et al., 2004). Around two decades ago the first libraries were screened on 96-well plates, with platforms able to process 10,000 compounds at the same time with a volume of 100–200 ␮l per well. Nowadays the miniaturization of the systems enables to screen from 40,000 to 200,000 compounds at once, using very small vol-

S. Maglioni et al. / Mechanisms of Ageing and Development 157 (2016) 60–69

ume ranges (from 2.5 to 10 ␮l), in 384-well to 1536-well plates, with a continuous trend towards further miniaturization of plates (Mayr and Fuerst, 2008; Brandish et al., 2006; Lee et al., 2008). In the context of drug discovery HCS can be seen as an extension of HTS which, combined with automated imaging platforms, is able to give multiple biological readouts at the same time (Maglioni et al., 2015; Buchser et al., 2004). The higher number of quantifiable parameters however lowers the number of screenable compounds compared to HTS. HCS approaches are used in drug discovery screening for the primary identification of lead compounds but they can also give a picture of chemicals activity, such as interaction with the cellular components, alteration of cellular morphology or localization within the cell (Buchser et al., 2004; Giacomotto and Segalat, 2010). Importantly, in the fields of pharmacology and clinical biology HCS became a powerful tool to identify drugs molecular targets and their side effects. These screening techniques are easy and straightforward in single cells systems such as bacterial, yeast or mammalian cells. However the use of single cells approaches in drug screening still presents some limitations, especially when searching for modifiers of human diseases (Segalat, 2007a). Indeed, most diseases affect organs as a whole, and sometimes more than one organ, and although technologies are being developed to reconstitute organs in vitro, for most cases (e.g. neuromuscular disorders) this is still not possible and we are far to be able to exploit them for screening purposes. Moreover, the complexity of an in vivo biological system with cells, tissues and organs being interconnected at different levels (e.g. physically through direct contacts or vases, or physiologically through hormones or cytokines), which in most cases is critical for disease development and progression, can in many cases limit the applicability of the in vitro models (Giacomotto and Segalat, 2010). Finally, the timing of disease development and progression can rarely be represented in in vitro single cells systems. These limitations become even more obvious when searching for interventions with potential anti-aging effects since aging can be seen as a multi-organ failure disease and is specifically associated with a progressive systemic deterioration in multiple tissues, organs and physiological functions. The use of model organisms in drug screenings looking for diseases or aging modifier is therefore preferable. Mouse models are frequently used in medical research because of the physiological similarities with human (West et al., 2000). However, mice lifespan is about three years and experiments are very laborious, expensive and time consuming (Alberts et al., 2002). To overcome these drawbacks the use of different invertebrate animals as model organisms to carry out drug screenings, such as Caenorhabditis elegans and Drosophila melanogaster, has exponentially grown in the past decade and has brought several advantages (Liu et al., 2004; Maglioni et al., 2015; Segalat, 2007b): the smallsized animals requires less space and allow the use of automated pipetting system at any developmental stages, the short life cycle accelerates the identification of new drugs, and real-time imaging of whole animal, also modeling human disorders, permit in vivo identification of compounds biological targets, activity and side effects (Giacomotto and Segalat, 2010; Segalat, 2007b).

2. C. elegans drug screenings 2.1. Advantages of C. elegans as an in vivo screening tool The microscopic nematode C. elegans already proved to be one of the most suitable model organisms to study different biological processes ranging from cell differentiation and death to nervous system development and deterioration (Antoshechkin and Sternberg, 2007). Its applicability is due to a unique combination of features: small size (∼1 mm), body transparency, simple

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and inexpensive maintenance and cultivation, self-fertilization and ability to produce around 300 progeny in just three days, complete genome sequence with the possibility of easily achieve gene suppression through double stranded RNA-mediated interference (RNAi) (Wood, 2016; Riddle, 2016; Altun et al., 2016; Silverman et al., 2009). C. elegans genome presents more than 60% homology with the human one (Kaletta and Hengartner, 2006a) and several physiological processes are conserved (Leung et al., 2008) allowing to model complex human diseases like Parkinson’s (Braungart et al., 2004) and Alzheimer’s disease (Levitan et al., 1996). Thanks to its short lifespan, C. elegans is also a powerful model organism for aging studies as underlined by the fact that most evolutionarily conserved processes which modulate aging (e.g. reduced insulin-like signaling, mitochondrial disturbance, caloric restriction) have been originally discovered in this system (Torgovnick et al., 2013; Gruber et al., 2015). Moreover, many age-associated features described in mammals such as neuromuscular deterioration, decline of physiological functions and of resistance to stress and infections, and increase mortality rate over time, are similarly observed during C. elegans aging (Torgovnick et al., 2013). Notably, C. elegans small size and body transparency, short life cycle (about 3 days) and genetic tractability, make this animal a unique tool for whole organism-based HTS or HCS to search for therapeutic drugs (Wood, 2016; Riddle, 2016; Altun et al., 2016; Silverman et al., 2009; Kaletta and Hengartner, 2006a; Leung et al., 2008; O’Reilly et al., 2014; Artal-Sanz et al., 2006; Kaletta and Hengartner, 2006b). Absorption, distribution, metabolism, excretion and toxicity (ADMET), are the most important initial processes to be assessed upon drug administration, and in C. elegans these parameters can be monitored simultaneously. Furthermore, the use of RNAi is one of the most powerful and easily available genetic tools to identify drugs’ target (Giacomotto and Segalat, 2010; Kaletta and Hengartner, 2006a; O’Reilly et al., 2014; Segalat, 2006). The first high-throughput drug screenings using C. elegans (Tables 1 and 2) (RNAi-based screens are only briefly mentioned in this review, Table 3) were carried out using 24-well agar plates (Kwok et al., 2006; Gaud et al., 2004; Giacomotto et al., 2009, 2012; Rauthan and Pilon, 2015; Gill et al., 2003) in the attempt to minimize the amount of compounds used but at the same time keeping the traditional laboratory culture conditions (NGM agar plates with a bacterial lawn as nematode food source). Clearly this method is very laborious and time consuming and as such not in line with the HTS principles (Brenner, 1974). In the first large scale screening, worms were transferred using a BIOSORT (COPASTM , Union Biometrica) and 308 bioactive compounds were identified by visual observation of different phenotypes, with the help of a semi-automated imaging acquisition system (Kwok et al., 2006). This work represents a very straightforward example of the use of C. elegans for in vivo screenings to discover new bioactive compounds and identify their molecular targets. Indeed, in a first phase, the authors screened a compound library on wild-type animals looking for those able to alter normal animal phenotype. Then, to elucidate molecular targets of relevant compounds, they carried out a genetic suppressor screen using 180,000 randomly mutated wild-type genomes and look for dominant genetic suppressors of the phenotype induced by the previously identified drug. Importantly, HCS have been also carried out to search for diseases modifiers. In 2010 Gosai and collaborators identified compounds altering the intracellular accumulation of a mutant form of the human secreted serpin (Z-mutation of the ␣1-antitrypsin), which causes liver disease in ␣1-antitrypsin deficiency. In this study, for the first time, a HCS using a transgenic C. elegans (expressing a GFP tagged misfolded protein) was established (Gosai et al., 2010). The authors used 384-well plates, transferred the animals using the COPASTM BIOSORT and acquired the images by an

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Table 1 Selected works describing C. elegans compound screenings to identify phenotypic modulators (not related to aging). First author (year)

Compounds/genes screened

Phenotype(s) screened

Findings

Methods

Kwok et al. (2006)

14,100 small molecules for bioactivity in wild-type worms. 180,000 randomly mutated wild-type genomes to identify candidate targets of nemadipine-A,

308 compounds that induce a variety of phenotypes (defects in wild-type worms). 5 mutations that suppress nemadipine-A-induced defects.

Solid medium, 24-well plates, COPAS BIOSORT worm sorter. Leica MZFLIII microscope and micrographs generated by a HiDI 1.0 high-throughput digital imager.

Gaud et al. (2004), Giacomotto et al. (2009, 2012)

∼1000 compounds from Prestwick Chemical Inc. and the French National Chemical Library.

Prednisone, ethazolamide (MTZ) and ichlorphenamide (DCPM).

Solid and liquid media, 24- and 96-well plates; two methods, one manual and the other semi-automated.

Gosai et al. (2010)

1280 compounds from the Library of Pharmacologically Active Compounds (LOPAC).

In wild-type worms: slow growth, lethality, uncoordinated movement and morphological defects In mutant worms: suppression of nemadipine-A-induced defects Impaired locomotion and reduced muscle degeneration in a model for Duchenne Muscular Dystrophy (phalloidin–rhodamine staining and GFP distribution in body wall muscle cells) Intracellular accumulation of the human aggregation prone mutant, which causes liver disease in ␣1-antitrypsin deficiency.

Liquid medium, 96- or 384-well plates, COPASTM BIOSORT worm sorter and ArrayScan VTI HCS Reader for imaging.

Peterson et al. (2008), Boyd et al. (2010) and Boyd et al. (2012)

1408 chemicals from the National Toxicology Program (NTP), including metals, pesticides, mutagens, and non-toxic agents. ∼364,000 compounds from Full Molecular Libraries Small Molecule Repository (MLSMR).

6 compounds inducing a concentration-dependent decrease in sGFP:ATZ accumulation, four ot them (cantharidin, tamoxifen, fluphenazine and pimozide) were previously identified as enhancers of autophagy. The toxicity of seven test toxicants on C. elegans reproduction was highly correlative with rodent lethality. Validated the platform for ultra HTS

Leung et al. (2011) and Leung et al. (2013)

Effects of chemicals on wild-type and myo-2:GFP C. elegans reproduction capacity.

SKN-1 activity by monitoring the GFP induction in a gst-4 (gene target of SKN-1) reporter strain. The fluorescence signal of a constitutively expressed RFP reporter (Pdop-3:RFP) was used to normalize for variation of worm number. Survival of the infected nematodes.

Zhou et al. (2011a,b)

1300 extracts (mainly secondary metabolites from endophytic fungi or medicinal plants)

Nayak et al. (2014)

16 novel small molecule proteasome inhibitors.

Germ line phenotypes characteristic of a reduction in proteasome function such as changes in polarity, aberrant nuclear morphology, and stimulation of apoptosis. (Paired DAPI staining and GLD-1:GFP reporter).

Rauthan and Pilon (2015)

Over 1200 (FDA approved) compounds from the Prestwick Chemical library.

Activation of hsp60:GFP reporters, with the oxidative agent paraquat used as a positive control.

Song et al. (2015)

Locomotion parameters (swimming frequency and bend amplitude) of the nematodes in response to chemical stimuli.

36 extract extend lifespan of nematodes infected by Pseudomonas aeruginosa, 4 with anti-multidrug resistant P. aeruginosa activity Optimization and validation of the screening procedure.

4 compounds that specifically activated the UPRmt (without also activating the UPRer). One of these compounds, methacycline hydrochloride (a tetracycline antibiotic) also protected C. elegans and mammalian cells from statin toxicity. Optimization and validation of a microfluidic device for chemical testing

96-well plates. Biomek 2000 Laboratory Automation Workstation and COPAS BIOSORT with REFLX option. Large-scale liquid worm culture, 384-well and 1536-well plates, EnVision MultiLabel® Plate reader for Fluorescence measurements.

Liquid and solid media, 96-well plates. Assessment of movement after high intensity light stimulation. Liquid medium, 96-well plates. DAPI staining and apoptotic nuclei scored after fixing the worms on agar pads on microscope slides. Epifluorescent images captured with a Nikon Eclipse E800 with ACT-1 (v2.62) software and processed with Pixelmator 1.4.1 (Pixelmator TeamLtd., London, UK). Solid medium, 24-wll plates. Images acquired using a Zeiss Axio Scope A1, the GFP intensity was measured with the Image J software (NIH, USA).

The microfluidic device. The layout of microfluidic channels includes three sections with different functions. An inlet channel network connecting two device inlets with eight nematode culture chambers for alternating the chemical solution or culture buffer supplied to the loaded nematodes. Eight circular chambers with separate nematode loading channels.

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automated microscopy platform, the Thermo Scientific Cellomics ArrayScan VTI. Through a small molecule screen they identified compounds affecting the intracellular accumulation of the mutated misfolded secreted protein serpin, providing the proof of principal that live animal HCS for disease modifiers is feasible. Of note, many misfolded protein disorders have been already modeled in the nematode and could be thus used to identify novel therapeutics (Link, 1995; Kraemer et al., 2003; Lakso et al., 2003; Faber et al., 1999; Oeda et al., 2001). Another example of the possibility to exploit C. elegans to discover new therapeutic drugs is the use of worm models for neuromuscular disorders, such as Duchenne Muscular Dystrophy (Giacomotto et al., 2009, 2012). In these works the authors used the nematode in a drug screen to rescue the neuromuscular degeneration observed in the dys-1(cx18);hlh-1(cc561ts) double mutants (strain LS587) showing a dystrophic-like phenotype and identified molecules that have later been proved to be efficient also in mammalian models (Giacomotto et al., 2009, 2012). The screening of live animals for complex disease traits on highcontent imaging platforms clearly underscores the advantages of using the nematode C. elegans for preclinical drug discovery. In 2013 an ultra-high-throughput screening (uHTS) was also reported for the first time (Leung et al., 2013). This work took advantage of a C. elegans transgenic strain to screen SKN-1 inhibitors. SKN-1 is the ortholog of the mammalian Nrf (Nuclear factor E2related factor) and mainly responsible for response to oxidants and electrophilic xenobiotics and for the multi drug resistance in parasitic nematodes (Park et al., 2009). SKN-1 is activated upon oxidative stress and controls the induction of antioxidant response triggering Phase II enzymes such as gst-4 (Kahn et al., 2008). For this screening 1536-well plates were used and among 364,000 compounds 125 were identified which specifically inhibited the gst-4:GFP expression in worms. GFP fluorescence was measured through a fluorimeter without imaging acquisition. In the past decade other studies used C. elegans to screen big libraries of compounds such as antimicrobial and antifungal compounds (Moy et al., 2006; Breger et al., 2007), host-pathogens interactions (Zhou et al., 2011a), and toxicants (especially neurotoxic compounds) (Peterson et al., 2008; Boyd et al., 2010, 2012; Avila et al., 2012; Jung et al., 2015). Additional promising strategies to identify novel diseases or phenotypes suppressors/inducers are represented by the development of microfluidic device-based platforms (Song et al., 2015). It is therefore undeniable that C. elegans based HTS strategies (more than HCS) are greatly helping to gain insight into chemicals biological processes and possible therapeutic exploitations, but although these techniques rapidly improved in little more than a decade (Kwok et al., 2006; Gosai et al., 2010) more sophistication is still possible and desirable. 2.2. C. elegans screenings to identify pro-longevity interventions Thanks to its short lifespan, stereotyped phenotypes and behaviors, and easy maintenance in laboratory conditions, C. elegans is an optimal model organism for studying drugs that influence aging and age-associated phenotypes. Worm’s lifespan can be assessed reproducibly and robustly, and its efficacy as a pharmacological tool has been demonstrated by the recent identification of small molecules with pro-longevity effect, representing possible strategies to delay aging and the onset of age-associated diseases in mammals (Lucanic et al., 2013) (Table 2). Already in 2003, starting from the assumption that genetic or environmental manipulations that extend lifespan in the nematode also enhance survival following acute stresses (e.g. oxidative damage and thermal stress) (Lithgow et al., 1995), a screening strategy was optimized to potentially identify pharmacological agents extending lifespan through their ability to enhance resistance to oxygen radicals or other stressors (Gill et al., 2003). In this work, the authors developed a sur-

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vival assay that used the uptake of the fluorescent dye SYTOX green as a marker for nematode death. They combined an automated worm-handling technology with automated real-time fluorescence detection. Their proposed fluorescent method for survival analysis improved in throughput compared with the conventional survival assay and was potentially able to accelerate the discovery of new pharmacological interventions against aging and oxidative stress (Gill et al., 2003). In 2005 a small compound screen led to the identification of a pro-longevity effect of anticonvulsant drugs (Evason et al., 2005). A much larger screen was carried out in 2007, when 88000 molecules were tested in liquid for their ability to extend C. elegans lifespan and a drug used in human as antidepressant (mianserin) was identified (Petrascheck et al., 2007). The lifespan was assessed in 96or 384-well plates supplemented with 5-fluoro-2 -deoxyuridine to avoid self-fertilization and the fraction of animal alive was scored by eye based on body movement induced by shaking or application of light. A second screen of 1083 molecules, carried out in the same way, led to the identification of 115 chemicals that produced statistically significant extension of lifespan (Petrascheck et al., 2009). Similarly, the same authors recently identified 60 compounds extending lifespan, many of them being compounds already approved for human administration (Ye et al., 2014). A very recent screen also identified new pro-longevity drugs by combining manual and automated assays to test the effects of 30000 chemicals (Lucanic et al., 2016). Finally, highly promising strategies for the identification of new lifespan extending interventions are nowadays represented by the development of microfluidic devises-based platforms (Xian et al., 2013). All the above-mentioned works clearly prove the validity of the nematode as an efficient model to identify pro-longevity interventions. Although the automated systems for preparation and imaging of the multi-well plates greatly increases the throughput of the possible screening, the need to score animals survival for the entire lifespan is still very time-consuming and represents a bottleneck for the fast discovery of pro-longevity interventions. The identification of surrogate markers for life extension that allow the operator to shorten the experimental time is therefore very desirable and would lead to a drastic acceleration of discovery of new life extending interventions. One example in this direction, as described above, was the use of increased stress resistance as a phenotype often concurrently segregating with extension of lifespan. However, it is becoming clearer that this is not always true and screening for stress resistance, although it might be useful, it might also lead to false positive or negative (Zhou et al., 2011b). Large-scale RNAi C. elegans screenings, of single chromosomes or genome-wide, have been also conducted to identify crucial genes and key pathways involved in lifespan extension (Table 3). The first two systematic RNAi screen were carried out in 2002 and 2003 and led to the identification of the critical role played by mitochondria in C. elegans longevity (Dillin et al., 2002; Lee et al., 2003). In 2005 two genome-wide screen identified 89 and 23 new genes which, when inactivated, extend the worm lifespan (Hamilton et al., 2005; Hansen et al., 2005). Moreover, in 2007, 2700 genes, previously identified as essential for C. elegans development, were silenced after larval development and led to the identification of 64 genes able to extend lifespan (Curran and Ruvkun, 2007). During the past two decades these RNAi screens permitted a great improvement of our understanding of the genetic mechanisms of organismal aging. Notably, almost all of the known C. elegans longlived mutants show concurrent phenotypes besides the prolonged longevity, and most of these longevity genes were initially identified through their associated phenotypes, and only subsequently have been recognized to also modulate longevity (Ni and Lee, 2010). Therefore the establishment of surrogate markers/phenotypes for extended longevity represents the best option to quickly identify

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Table 2 Works describing C. elegans screenings for lifespan extending compounds. First author, year

Compounds screened

Gill et al. (2003)

Evason et al. (2005)

19 drugs from a variety of functional or structural classes

Petrascheck et al. (2007) and Petrascheck et al. (2009) Xian et al. (2013)

88,000 chemicals

Ye et al. (2014)

1280 compounds from LOPAC library

Chin et al. (2014)

normal metabolites and aberrant disease-associated metabolites

Maglioni et al. (2015)

5 recently developed ATPase modulators with anti-inflammatory properties (form Lycera Corporation, Ann Arbor, US) 33,000 synthetic, diverse drug-like chemicals

Lucanic et al. (2016)

Findings

Methods

Set up and validation of the screening strategy.

Liquid medium, 24-well plates. Technologies: (i) the COPAS BIOSORT worm sorting and dispensing platform, (ii) SYTOX green fluorescent dye, and (iii) a microplate fluorometer. For the Microplate thermotolerance assay 384-well plates. The uptake of the fluorescent dye SYTOX green as a marker for nematode death due to oxidative stress. For the oxidative stress resistance test: plate-reading fluorometer to quantify the fluorescence of individual nematodes over time. Solid medium, Petri dishes.

Anticonvulsant Medications (ethosuximide, trimethadione, and 3,3-diethyl-2-pyrrolidinone). Mianserin, mirtazapine, methiothepin and cyproheptadine (serotonin receptor antagonist used as an antidepressant in humans). Set up and validation of the screening strategy.

60 compounds that increase longevity in C. elegans, 33 of which also increased resistance to oxidative stress. Many of these compounds are drugs approved for human use. The tricarboxylic acid (TCA) cycle intermediate a-KG (but not isocitrate or citrate) delays ageing and extends the lifespan by 50%. One ATPase modulator (LYC-30904) extending lifespan.

57 chemicals that reproducibly induced lifespan, Three of them were closely related, containing a nitrophenyl piperazine backbone and they were therefore named nitrophenyl piperazine-containing compounds 1–3 (NP1-3).

and unravel new potential interventions and pathways involved in extension of healthy lifespan in C. elegans and, hopefully, thanks to the high homology between nematode and mammalian genes, conserved through the species. 3. Development of a new C. elegans screening strategy to identify pro-longevity interventions 3.1. Mitochondria as a target hub integrating different signals Mitochondria are far away from being the static and isolated, kidney-shaped organelles that the scientific literature described in

Liquid medium, 384- or 96-well plates. The fraction of animals alive was scored on the basis of body movement. FUDR was included to prevent progeny production. WormFarm: integrated microfluidic device for culturing nematodes. 30–50 animals was maintained throughout their lifespan in each of eight separate chambers on a single chip. Automated analysis of videos was performed to quantitate survival and other phenotypes (body size and motility). GFP intensity in worms expressing GFP tagged with a mitochondrial import signal under the control of the myo-3 promoter was used as biomarker of biological age. Liquid medium, 384- or 96-well plates. The fraction of animals alive was scored on the basis of body movement. FUDR was included to prevent progeny production. Solid medium, FUDR was included to prevent progeny production.

Liquid medium, 96-well plates; animal imaging, GFP and size quantification using the ArrayScan VTI HCS Reader. Solid medium, 96-well plates prepared with Multidrop 384 reagent dispenser (Thermo Scientific). Chemicals in each well to a final concentration of 50 lM dispensed with a Biomek FX Liquid Handler (Beckman Coulter). Bot manual and automated lifespan assays (for HTP lifespan a temperature sensitive sterile strain TJ1060 was used).

the past. They are highly dynamic organelles, which continuously undergo fusion and fission transforming their interconnections (Vafai and Mootha, 2012). These processes are vital in regulating mitochondrial morphology, biogenesis, transport and function. The primary function of mitochondria is to produce ATP through the process of oxidative phosphorylation but they also regulate a variety of different important cellular processes, such as production of reactive oxygen species (Murphy et al., 2011), adaptive thermogenesis (Azzu and Brand, 2010), iron homeostasis (Cardoso et al., 2010) and programmed cell death (Spencer and Sorger, 2011). Moreover, mitochondria coordinate different intracellular signaling networks in physiological conditions and integrate them in response to exter-

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Table 3 Works describing C. elegans screenings for lifespan extending genes. First author, year

Genes screened

Findings

Dillin et al. (2002)

Nearly all of the genes on Chromosomes I

Lee et al. (2003)

Nearly all of the genes on Chromosomes I and II (5690 genes)

Hamilton et al. (2005)

16,475 RNAi agains > 80% of the ∼19,000 C. elegans open reading frames

Hansen et al. (2005)

16,757 RNAi aginst 87% of the C. elegans open reading frames

Curran and Ruvkun (2007)

2700 essential for normal development excluded from previous screening analysis. 57 genes that cause developmental arrest RNAi library along chromosomes III and IV for genes that, when silenced, confer paraquat resistance

Genes important for mitochondrial (daf-2;daf-16; nuo-2, cyc-1, cco-1, and atp-3) IIS independent. Genes important for mitochondrial carrier gene F13G3.7 and phosphoglycerate mutase gene F57B10.3, IIS dependent. 89 genes extend C. elegans lifespan. Components of the daf-2/insulin-like signaling pathway are recovered, as well as genes that regulate metabolism, signal transduction, protein turnover, and gene expression. 23 new longevity genes affecting signal transduction, the stress response, gene expression, and metabolism. 64 genes that extend lifespan when inactivated post-developmental.

Chen et al. (2007) Kim and Sun (2007)

Walter et al. (2011)

Transcription Factor RNAi Library (Gene Service Inc.) covering 41% of the predicted transcription factors in C. elegans

Sinha and Rae (2014)

RNAi clones, from the Ahringer library, against a subset of 150 candidate genes selected from microarray results

nal cues such as stressors or nutritional factors (Yin and Cadenas, 2015). Mitochondrial function decline during aging and this is associated with the appearance of alterations in their morphology. The respiratory chain capacity has been observed to decline with age in human liver, heart, and skeletal muscle (Bratic and Larsson, 2013). And the number of mitochondria was shown to decrease with age in mice, rats and humans together with a decrease in mtDNA copy number and mitochondrial protein levels. Mitochondrial deterioration is not only a central feature of normal aging, but also a crucial condition associated with a wide variety of common illness ranging from neurodegenerative disorders such as Parkinson’s disease to cancer and diabetes (Johannsen and Ravussin, 2009). Several genetic diseases also exist that are directly ascribed to mutations in nuclear- or mitochondrial- encoded proteins mostly involved in the functionality of the mitochondrial respiratory chain (Maglioni and Ventura, 2016; McInnes, 2013). Given the fundamental importance of mitochondria for cellular homeostasis and organismal health it is not surprising that a number of mitochondrial quality control pathways exist (Schiavi and Ventura, 2014; Ni et al., 2015) to finely regulate and maintain their integrity and functions and to adapt to cellular metabolic changes under pathophysiological conditions. 3.2. C. elegans phenotypes in response to mitochondrial targeting interventions In humans signs and symptoms of mitochondrial alteration only appear when their dysfunction is very severe while mild mitochon-

4 genes that extend lifespan in Caenorhabditis elegans when inactivated during adulthood. 84 genes that, when inactivated by RNAi, lead to significant increases in animal lifespan. Among the 84 genes, 29 were found toact in a manner dependent on daf-16. 17 RNAi consistently shortened the lifespan of the isp-1;ctb-1 long-lived mutant by more than 10%. Among these RNAi 13 candidate transcription factors not previously implicated in directly affecting C. elegans longevity. Particularly ceh-23 novel longevity determinant that specifically responds to altered mitochondrial function to affect lifespan in a non-cell autonomous manner. Genes essential for both lifespan and immunity of germline deficient animal. For example transcription factor DAF-16/FOXO, the PTEN homolog lipid phosphatase DAF-18 and several components of the proteasome complex (rpn-6.1, rpn-7, rpn-9, rpn-10, rpt-6, pbs-3 and pbs-6). Novel role for genes including par-5 and T12G3.6 in both lifespan-extension and increased survival on X. nematophila.

drial deficiency does not lead to any obvious phenotypic outcome. However, interestingly, in the nematode C. elegans, the disruption of different mitochondrial genes can result in the appearance of very distinct phenotypic features depending on the degree of gene disruption (Rea et al., 2007; Feng et al., 2001; Tsang et al., 2001; Zarse et al., 2007; Wong et al., 1995). Paradoxically, partial suppression of numerous genes encoding mitochondrial proteins has been shown to extend lifespan in C. elegans (Lee et al., 2003; Dillin et al., 2002; Ventura et al., 2006; Munkacsy and Rea, 2014; Dancy et al., 2014), as well as in Drosophila (Sun et al., 2002) and in mice (Dell’agnello et al., 2007; Liu et al., 2005; Houtkooper et al., 2013). This class of long-lived nematodes with genetic or RNAi-mediated suppression of different mitochondrial proteins is generally indicated with the name of C. elegans Mitochondrial mutants (Rea, 2005). Similar pro-longevity effects can be achieved also by low doses of different mitochondrial targeting drugs (Dillin et al., 2002; Schmeisser et al., 2013a; Lee et al., 2010; Yang and Hekimi, 2010; Maglioni et al., 2015). These animals are characterized not only by an extended lifespan but also by other typical phenotypes, such as slow development, reduced size and extension of the fertile period (Rea et al., 2007; Feng et al., 2001; Tsang et al., 2001; Zarse et al., 2007; Wong et al., 1995). The majority of mitochondrial downregulated genes in these mutants are involved directly or indirectly in the functionality of the respiratory chain, playing an important role in the organelle function (Hamilton et al., 2005; Hansen et al., 2007). On the other hand, as expected, a strong suppression of the same mitochondrial proteins, which are often associated

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with diseases in humans (Ventura and Rea, 2007), leads in the nematode to dramatic consequences, like arrested development, sterility and eventually lethality (Rea et al., 2007; Tsang et al., 2001; Ventura and Rea, 2007; Ventura et al., 2009). This phenomenon of opposite responses to low and high doses of a certain stress is a well-established concept in the toxicology field named hormesis (or double-dose response) (Calabrese, 2004). In this context the different adaptive responses to mild and strong degree of mitochondrial stress have been named mitohormesis (Ristow and Zarse, 2010; Ristow and Schmeisser, 2014; Yun and Finkel, 2014; Tapia, 2006) and are can be responsible for the opposite effects on animal lifespan, and most likely also for the other observed differential animal phenotypes (Ventura and Rea, 2007; Ventura et al., 2009). The opposite phenotypes that appear in C. elegans upon mild or strong mitochondrial protein suppression represent a very useful tool to mimic the progressive chronic course of many mitochondrial associated diseases, and thus to investigate the organismal response in the window of partial mitochondrial suppression, which in human is asymptomatic, while in these models is associated with very specific and recognizable phenotypes (Ventura and Rea, 2007). Understanding the underlying molecular mechanisms associated with longevity in response to mild mitochondrial dysfunction may thus not only shed light on healthy aging mechanisms, but it may also lead to the identification of novel preventive therapeutic targets for mitochondrial associated diseases. Many efforts indeed aimed at unraveling the cellular adaptive responses to mitochondrial stress, ranging from targeted approaches, which identified p53, mitophagy and neuronal proteins as being causally involved in Mit mutants longevity (Ventura et al., 2009; Torgovnick et al., 2010; Schiavi et al., 2013, 2015; Maglioni et al., 2014), to more unbiased approaches such as metabolomics or genetic screening, which identified mitochondrial-derived ␣-ketoacids and ␣-hydroxyacids (Butler et al., 2013; Mishur et al., 2016) and different transcription factors (Khan et al., 2013; Walter et al., 2011) respectively, as novel mediators of Mit mutants longevity. Of note, the different phenotypic features, which results in response to increasing levels of mitochondrial alteration (i.e. reduced adult size and fertility upon mild stress, and arrested development or sterility upon severe mitochondrial stress), represent very distinct and reproducible phenotypes amenable for automatic quantification in high content screening in search of interventions targeting mitochondria (Maglioni et al., 2015). 3.3. A new phenotype-based screening to identify mitochondrial-targeting interventions acting from development to promote longevity We recently exploited the differential C. elegans phenotypic and biochemical changes associated with mild and strong degree of mitochondrial stress, as surrogate markers to establish a phenotypic-based screening (Maglioni et al., 2015) to search for pro-longevity interventions targeting mitochondria (Fig. 1). To this end we optimized different automated microscopy protocols utilizing the Cellomics ArrayScan® VTI HCS Reader (Thermo Scientific) imaging platform and one of its BioApplications, the SpotDetector. This HCS instrument has different BioApplications, which facilitate the counting and detection of animals showing a specific phenotype and can be utilized to measure their size in terms of area or length. The use of the fluorescent channel allows at the same time to quantify the expression of specific fluorescently tagged proteins. In the optimization phase of our screening strategy we used worm liquid cultures in 96-well plate, and first established the maximum number of adult animals recognized as valid objects in each well. We then established the protocol parameters required to identify worms treated with a mild or a strong dose of RNAi against different nuclear-encoded mitochondrial proteins. Animals left untreated,

representing our control, were identified as objects bigger than 300 pixels (relative units). Animal treated with a mild dose of RNAi had a body size between 300 and 150 pixels, while nematodes fed a strong dose of RNAi were identified as objects smaller than 150 pixels. Using mild and strong RNAi against different mitochondrial proteins already known to lead to discrete animal phenotypes (Rea et al., 2007; Ventura and Rea, 2007; Simmer, 2003; Ventura et al., 2005; Fraser et al., 2000), we obtained highly reproducible results. These cut-off references could then be utilized to identify compounds that reduce animal size between 300 pixels and 150 pixels as a first indication for those acting through a mild mitochondrial stress, and consequently with potential pro-longevity effects. The Spot Detector can also quantify intensity and distribution of fluorescently tagged proteins in transgenic animals. Thus, the expression of two GFP reporter genes were monitored, the glutathione S-transferase 4 (gst-4) and the heat shock protein 6 (hsp-6), whose induction has been previously associated with lifespan-extension upon genetic or pharmacological inhibitors of the mitochondrial respiratory chain (Curran and Ruvkun, 2007; Ventura and Rea, 2007; Ventura et al., 2009; Torgovnick et al., 2010; Durieux et al., 2011; Yoneda et al., 2004; Liu et al., 2014). The RNAi against genes known to activate those reports were used to set up the fluorescent parameters. Then, as a proof of principle of the efficiency of our protocols, we tested different pharmacological interventions modulating mitochondrial function at concentrations known to extend C. elegans lifespan or to induce animal lethality (Houtkooper et al., 2013; Lee et al., 2010; Yang and Hekimi, 2010; Schmeisser et al., 2013b; Chin et al., 2014). Those drugs, fed continuously from embryos, mimicked the effects of mild versus severe mitochondrial stress. Examining animal development, size, fertility and GFP expression, for each of the four chemicals, two doses were established: a lower one, which reduced size, fertility and induced the expression of stress reporter genes (hsp-6 and gst-4) and a high dose, which arrested animals development at a larval stage (L2/L3) and differentially affected transgenes expression. Finally, we validated the screening platform by identifying a new small molecule targeting mitochondria and for which an effect on C. elegans lifespan was unknown. We found that an allosteric modulator of the mitochondrial F1F0-ATPase matched the screening criteria: at low dose reduced animal size and fertility and induced the expression of stress response genes, while at higher dose arrested animal development. The effect of this molecule on modulating mitochondrial functional parameters and its lifespan extending property were then validated directly (Maglioni et al., 2015), indicating that our screening strategy can effectively identify new pro-longevity interventions acting through mitochondria.

4. Conclusions and perspectives: from developmental effects to healthy aging We recently established an automated microscopy platform that will allow the identification of interventions acting during development to extend lifespan by inducing mild mitochondrial stress (Maglioni et al., 2015). In principle our surrogate markers for longevity extension could be induced by any interventions acting through mitochondria or triggering mitochondrial stress response pathway through a different route. In both cases this would lead to lifespan extension and therefore to the identification of prolongevity interventions potentially useful for healthy aging (e.g. nutrients or drugs). In light of the potential translational effect, the dosage of intervention promoting healthy-aging has to be carefully fine-tuned to identify the specific amount which is not detrimental to mitochondrial functionality and allows to trigger protective responses such as mitophagy (Schiavi et al., 2015) or neuronal pathways (Maglioni et al., 2014), ultimately leading to the beneficial

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Fig. 1. A) Different degrees of mitochondrial stress are associated with discrete and very reproducible phenotypic readouts. Animals with a mild reduction of mitochondrial functionality display a reduction in body size and live longer (highlighted in green) than wild-type (control) animals. On the contrary, severe mitochondrial dysfunction leads to developmental arrest and/or shortening of lifespan (highlighted in red). See text for details. B) Phenotypic-based screen flow chart. Candidate compounds were automatically screened through the Cellomics ArrayScan using fluorescence filters to identify those giving the expected phenotypes (see text for details). Fifty L1 worms (reporter strain for stress response genes) were dispensed in triplicate in 96-wells plates pre-seeded with chosen compounds concentrations and their effect on animal size and fluorescence expression were assessed on day 4. Drugs that are able to give the typical mild mitochondrial mutant phenotypes, indicating the activation of beneficial pathways, represent potentially pro-longevity interventions. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

consequences. Of note, in order to promote longevity, these interventions have to act during the pre-fertile age, as demonstrated by the fact that in most cases they do not extend lifespan when fed to adult nematodes. This effect is similar to that of RNAi-targeting mitochondrial genes (Maglioni et al., 2015; Dillin et al., 2002; Rea et al., 2007) and different from other pro-longevity interventions such as for instance caloric restriction. Interestingly, this early life requirement for the beneficial pro-longevity effect reminds the antagonistic pleiotropic theory of aging proposed by George Christopher Williams in 1957 (Williams, 1957). According to this theory a gene (e.g. p53, TOR) or one of its intracellular regulated processes (e.g. cellular senescence, protein synthesis), which in an organism can control both favorable and unfavorable phenotypic traits, can be evolutionarily selected if its positive effect appears early in life while its negative ones later in life (Campisi, 2005; Ungewitter and Scrable, 2009; Promislow, 2004; Kapahi, 2010). In other words what is good during development is bad later during aging and consequently, what is bad during development (e.g. a reduction in mitochondrial function), if appropriately modulated,

could be beneficial later in life and promote healthy aging. In conclusion, although HTS and HCS are quickly improving our insight on age-regulatory genes and compounds, the identification of early life markers for healthy-aging and extended lifespan is of high priority to speed up the discovery of new pro-longevity interventions through these screening platforms. Acknowledgments The authors gratefully acknowledge funding by the Deutsche Forschungsgemeinschaft (DFG VE663/3-1), the Strategic Research Funding of the Heinrich Heine University (SFF701301988), and the Start-up Competitive Research Funding of the Medical Faculty of the Heinrich Heine University (Forschungskommission 43/2013). References Alberts, B., Johnson, A., Lewis, J., et al., 2002. Molecular biology of the cell. In: The Mouse, 4th edition. Garland Science, New York.

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