Author's Accepted Manuscript

S-linolenoyl-glutathione intake extends life-span and stress resistance via SIR-2.1 upregu-lation in Caenorhabditis elegans

Roberta Cascella, Elisa Evangelisti, MariagioiaZampagni, Matteo Becatti, Giampiero D'Ada-mio, Andrea Goti, Gianfranco Liguri, ClaudiaFiorillo, Cristina Cecchi

PII: S0891-5849(14)00220-2DOI: http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.004Reference: FRB12013

To appear in: Free Radical Biology and Medicine

Received date: 28 November 2013Revised date: 18 April 2014Accepted date: 9 May 2014

Cite this article as: Roberta Cascella, Elisa Evangelisti, Mariagioia Zampagni,Matteo Becatti, Giampiero D'Adamio, Andrea Goti, Gianfranco Liguri, ClaudiaFiorillo, Cristina Cecchi, S-linolenoyl-glutathione intake extends lifespan andstress resistance via SIR-2.1 upregulation in Caenorhabditis elegans, FreeRadical Biology and Medicine, http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.004

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biomed

S-linolenoyl-glutathione intake extends lifespan and stress resistance

via SIR-2.1 upregulation in Caenorhabditis elegans

Roberta Cascella1, Elisa Evangelisti1, Mariagioia Zampagni1, Matteo Becatti1, Giampiero

D'Adamio2, Andrea Goti2, Gianfranco Liguri1, Claudia Fiorillo1, Cristina Cecchi*1

1Department of Experimental and Clinical Biomedical Sciences, University of Florence, V.le GB

Morgagni 50, 50134 , Italy

2 Department of Chemistry “Ugo Schiff”, University of Florence, 50019 Sesto Fiorentino, Florence,

Italy

Running title: S-acyl glutathione thioester promotes longevity

* Correspondence to: Cristina Cecchi,

Department of Experimental and Clinical Biomedical Sciences, University of Florence,

V.le GB Morgagni 50, 50134 Florence, Italy

Tel: +39-055-2751222, Fax: +39-055-7830303, e-mail: cristina.cecchi@unifi.it

2

Abstract

Oxidative stress has a prominent role in lifespan regulation of the living organisms. One of the

endogenous free radical scavenger systems is associated with GSH, the most abundant nonprotein

thiol in mammalian cells, acting as a major reducing agent and antioxidant defence by maintaining a

tight control of the redox status. We have recently designed a series of novel S-acyl-GSH

derivatives capable to prevent amyloid oxidative stress and cholinergic dysfunction in Alzheimer

disease models, upon the increase of GSH intake. In this study we show that the longevity of wild-

type N2 Caenorhabditis elegans strain was significantly enhanced by dietary supplementation with

linolenoyl-SG (lin-SG) thioester with respect to ethyl ester of GSH, linolenic acid or vitamin E.

RNA interference analysis and activity inhibition assay indicate that lifespan extension was

mediated by the upregulation of SIR-2.1, a NAD-dependent histone deacetylase ortholog of

mammalian SIRT1. In particular, lin-SG-mediated overexpression of sir-2.1 appears to be related to

the DAF-16 (FoxO) pathway. Moreover, lin-SG derivative protects N2 worms from the paralysis

and oxidative stress induced by Aβ/H2O2 exposure. Overall, our findings put forward lin-SG

thioester as an antioxidant supplement triggering sirtuin upregulation, thus opening new future

perspectives for healthy aging or delayed onset of oxidative-related diseases.

3

Highlights

- Linolenoyl-SG thioester extends C. elegans lifespan via SIR-2.1 upregulation through the DAF-16

(FoxO) pathway

- Linolenoyl-SG thioester shows a dual protective effect through GSH and linolenic acid

- Linolenoyl-SG thioester protects from Aβ/H2O2-induced paralysis and oxidative stress in C.

elegans

Keywords: Glutathione, longevity, SIR-2.1, DAF-16 (FoxO), oxidative stress, C. elegans.

Abbreviations: Alzheimer’s disease (AD), amyloid-beta peptide (Aβ), 1,3-bis-(2-chloroethyl)-1-

nitrosourea (BCNU), Caenorhabditis elegans (C. elegans), C-terminal glycine of GSH (GSH ethyl

ester), dimethylsulfoxide (DMSO), Dulbecco’s phosphate-buffered saline (D-PBS), 6-chloro-

2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide (EX-527), 5-Fluoro-2’-deoxyuridine (FUdR),

glutathione (GSH L-c-glutamyl-L-cysteinyl-glycine), linolenoyl-SG (lin-SG), hydroethidine (HE),

Nematode Growth Medium (NGM), oxidized glutathione (GSSG), polyunsaturated fatty acid

(PUFA), reactive oxygen species (ROS), RNA interference (RNAi), S-acyl-glutathione (acyl-SG),

vitamin E (vit. E).

4

Introduction

Sirtuins are a family of ubiquitous NAD(+)-dependent deacetylases that regulate various

cellular processes, such as metabolism and stress responses [1,2]. Activation of sirtuins is known to

extend lifespan by promoting healthy aging in a variety of species and by protecting crucial tissues

in the body, including those in the heart and brain [3]. In mammalian systems, sirtuin activators

protect against axonal degeneration, polyglutamine toxicity, and microglia-mediated Aβ toxicity,

suggesting the potential therapeutic value of sirtuin activation in Alzheimer’s disease (AD) patients

[4]. SIRT1 is also activated in response to reactive oxygen species (ROS) production, leading to a

significant decrease in ROS levels and promoting cell survival [5].

As with the orthologous genes in yeast, flies and mammals [6-9], the overexpression of sir-

2.1, the sirt1 homolog in Caenorhabditis elegans, promotes worm longevity, whereas deletion or

knockdown of the gene shortens lifespan [10-14]. In addition, the overexpression of the sir-2.1 gene

is necessary for the transcriptional activation of the FoxO transcription factor DAF-16 target genes

[15]. C. elegans is a useful model for understanding aging processes and age-related diseases for

several reasons: in spite of its great simplicity (it is made of 959 somatic cells, it lacks a circulatory

system and a central nervous system, but 302 of its cells are neurons), 60% of its genes are

homologous to human ones and 12 out of its 17 signal transduction pathways are conserved in

humans [16]. It has a very short generation time (from egg to egg in 3–4 days), thus producing

numerous clonal progeny, and a short lifespan (about two-three weeks).

Although radical production is part of normal cellular function, excess exposure of

macromolecules to oxidizing radicals play a main role in aging and in many diseases [17-19]. One

of the endogenous free radical scavenger systems is associated with glutathione (GSH, L-c-

glutamyl-L-cysteinyl-glycine), the most abundant nonprotein thiol in mammalian cells acting as a

major reducing agent and antioxidant defence by maintaining a tight control of the redox status

[20,21]. The impact of GSH deficiency in numerous pathologies has prompted several researchers

5

to investigate new alternative strategies for maintaining or restoring GSH levels in these patients

[22,23]. Currently, the use of GSH as a therapeutic agent is limited by its short half-life and low

plasma membrane permeability [23,24]. Much work has therefore focussed on developing carriers

and/or conjugates that permit cellular GSH uptake, with prodrugs, mimetics and analogs showing

particular promise [25-28]. Considering the importance of developing new antioxidant compounds

and their relevance in the promotion of healthy aging and in the treatment of oxidative stress-related

disorders, we have recently designed and synthesized a series of S-acyl-glutathione (acyl-SG)

thioesters capable of crossing plasma membranes and to be hydrolysed at their thioester moiety,

thereby increasing intracellular levels of the reduced form of GSH and polyunsaturated fatty acid

(PUFA) with antioxidant properties (PTC Patent WO/2009/047728; International application

PCT/IB2008/054146) [29]. In particular, linolenoyl-SG (lin-SG) thioester prevents amyloid

oxidative stress and cholinergic dysfunction by increasing cellular GSH uptake in Alzheimer’s

disease models [30]. In this study we show that dietary supplementation with lin-SG thioester significantly enhances

the longevity of wild-type N2 C. elegans strain with respect to ethyl ester of C-terminal glycine of

GSH (GSH ethyl ester), linolenic acid and vitamin E, via intracellular increase of GSH reductive

capacity. Accordingly, lin-SG derivative protects C. elegans from the paralysis and oxidative stress

induced by Aβ/H2O2 exposure. We show that a moderate intake of lin-SG in N2 worms promotes

SIR-2.1 upregulation. On the contrary, both sir-2.1 RNA interference (RNAi) and EX-527, a

specific sirtuin inhibitor, prevent lin-SG-mediated longevity. In particular, this effect seems to be

related to the longevity pathway of the FoxO transcription factor DAF-16.

Materials and Methods

Synthesis of linolenoyl-SG and evaluation of its stability

The linolenoyl-SG (lin-SG) thioester was synthesized according to the method reported by

Zampagni et al. [30]. Its stability in aqueous solutions was checked by NMR. A 0.5 ml of an 8 mM

6

solution of the thioester in DMSO was added to 3 ml of a PBS solution and kept for 7 days at 20 °C.

Aliquots of the solution were taken after 0, 3, and 7 days. After lyophilization of each sample, the

collected residue was dissolved in d6-DMSO and subjected to NMR analysis. Signals in the 5.6-3.8

ppm region were selected for the estimation of the percentage of unaltered thioester.

Nematode growth

N2 ancestral strain was obtained from the Caenorhabditis Genetic Center (University of

Minnesota, USA) and was propagated at 20 °C on solid Nematode Growth Medium (NGM) seeded

with E. coli (OP50) for food. To prepare age-synchronized animals, the nematodes were transferred

to fresh NGM plates on reaching maturity at 3 days of age and allowed to lay eggs overnight.

Isolated hatchlings from the synchronized eggs (day 1) were cultured on fresh NGM plates at 20 °C.

RNAi induced gene silencing

RNAi induced gene silencing was achieved by raising worms on E. Coli expressing dsRNA

corresponding to the target gene as previously described [31]. Briefly, the bacteria clones

expressing dsRNA for sir-2.1 or daf-16 (Ahringer RNAi library, SA Biosciences, Cambridge, UK)

were cultured for 6–8 h in LB containing 25 µg/ml carbenicillin, then seeded directly onto NGM

plates with 1 mM IPTG and 25 µg/ml carbenicillin and incubated overnight at 37 °C to induce

expression. Before usage, plates were cooled down to 20 °C and the synchronous aged embryos

were transferred onto NGM plates containing specific gene-of-interest seeded bacteria. Worms were

maintained on RNAi feeding strains for two generations prior to lifespan and dot-blot experiments.

Lifespan assay

Lifespan assays were performed at 20 °C as previously described [32]. Briefly, the NGM plates

(35×10 mm culture plates) were prepared under sterile conditions; 60 µl of a concentrated

suspension of E. coli OP50 was spotted to form a circular lawn on the centre of each plate.

7

Populations of N2 worms, after egg synchronization, were placed at 20 °C on fresh NGM small

plates (100 worms/plate) seeded with E. coli. At L4 larval stage, the worms were transferred to new

plates and treated with 100 µl of vehicle (Control), 20 µM GSH, 20 µM linolenic acid, linolenoyl-

SG (lin-SG) thioester at three different concentrations (1, 5, 20 µM) and 20 µM GSH ethyl ester, in

the presence of 75 µM 5-Fluoro-2′-deoxyuridine (FUdR) to prevent offspring from nematodes

under study from reaching adulthood. The worms were also treated with 5 µM lin-SG plus 25 µM

1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), a selective inhibitor of the glutathione reductase, 20

µM vitamin E (vit. E), 5 µM lin-SG plus 1 µM 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-

carboxamide (EX-527), a selective inhibitor of sirtuin, and 5 µM lin-SG thioester in the presence of

sir-2.1 or daf-16 RNAi feedings. All conditions contained 20 µM DMSO, as the stock solution of

lin-SG was prepared at 4 mM in 100% DMSO. The number of worms paralyzed (considered dead)

was scored starting from 24 h after treatment (day 1), and for each consecutive day until all worms

were dead. Nematodes that failed to display touch-provoked movement were scored as dead.

Nematodes that died from causes other than aging, such as sticking to the plate walls, internal

hatching of eggs (‘bagging’) or gonadal extrusion were censored as were lost worms.

Determination of intracellular GSH intake

Populations of N2 worms, after egg synchronization, were placed (400 worms/plate) at 20 °C

on fresh NGM plates seeded with E. coli. At L3 larval stage, the worms were fed with vehicle

(Control), 5 µM lin-SG thioester, 5 µM GSH, 5 µM linolenic acid, 5 µM GSH ethyl ester, 5 µM lin-

SG plus 25 µM BCNU, 25 µM BCNU, 5 µM lin-SG plus 1 µM EX-527, 1 µM EX-527 and 20 µM

vit. E (Fig. 3A). Worm lysates were prepared at L4 larval stage, as previously reported [33], upon

minor modifications. Briefly, worms were washed off to remove bacteria, collected by

centrifugation, and the pellet was resuspended in 400 µl of lysis buffer containing 20 mM HEPES

(pH 7.6), 10% (v/v) glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA supplemented with 0.2

8

mM PMSF, 10 mg/ml leupeptin and aprotinin. After three freeze–thaw cycles, the worms were

homogenized by sonication for 15 min, cycling on and off for 30 s intervals, and centrifuged at

2,000 g for 5 min at 4 °C. The supernatant was retained and protein concentration determined

according to the Bradford method [34]. Levels of reduced GSH and oxidized glutathione (GSSG)

were measured in 40 µg of worm protein extracts according to glutathione assay kit (Cayman

chemical, Ann Arbor, MI), a spectrophotometric method developed by Tietze as previously

described [35-37]. This method is an enzymatic recycling procedure which offers a high sensitivity

rate. Total GSH is assayed by a system in which it is readily oxidized by DTNB [5,5’-dithiobis(2-

nitrobenzoic acid)] and reduced by glutathione reductase in the presence of NADPH. The rate of 2-

nitro-5-thiobenzoic acid formation is monitored and the level of total intracellular GSH equivalents

in worms is determined by the comparison of the result with a standard curve of GSH. GSSG levels

were determined by the same method in the presence of 2-vinylpyridine, and reduced GSH was

calculated as the difference between total GSH and GSSG [36,38].

Dot-blot analysis of SIR-2.1

The levels of SIR-2.1 were assessed in worms treated with vehicle (Control), 5 µM lin-SG

thioester, 5 µM lin-SG thioester in the presence or absence of sir-2.1 RNAi feeding, 5 µM GSH, 5

µM GSH ethyl ester, 5 µM linolenic acid 5 µM lin-SG plus 1 µM EX-527, 1 µM EX-527, 5 µM lin-

SG plus 25 µM BCNU, 25 µM BCNU and 20 µM vit. E. Equal amounts of worm protein extracts

(1 µg) were spotted onto PVDF Hybond membranes, which were then incubated overnight at 4 °C

with 1:1,000 diluted rabbit polyclonal anti-SIRT1 antibodies (Santa Cruz Biothecnologies, USA).

After washing, the membranes were incubated with 1:1,000 diluted peroxidase-conjugated anti-

rabbit secondary antibodies (Pierce, Rockford, IL, USA) for 1 h. Immunolabeled spots were

detected using a supersignal west dura (Pierce, Rockford, IL, USA) and quantified using the Image

J software for image analysis.

9

Paralysis assay

The paralysis phenotype of the worms can be easily and clearly scored by paralysis, pharyngeal

pumping and body bend assays [32]. To this aim, populations of N2 worms, after egg

synchronization, were placed at 20 °C on fresh NGM plates seeded with E. coli. Amyloid-derived

diffusible ligands (ADDLs) were prepared incubating Aβ42 peptide (Sigma-Aldrich, St. Louis, MO)

as previously described [30]. At L3 larval stage, the worms were fed with vehicle (Control), 10 µM

Aβ42 oligomers, 10 µM Aβ42 oligomers plus 5 µM lin-SG thioester, 10 µM Aβ42 oligomers plus 5

µM GSH, 10 µM Aβ42 oligomers plus 5 µM linolenic acid, 5 µM lin-SG, 5 µM GSH, 5 µM

linolenic acid, 5 µM lin-SG plus 25 µM BCNU, 25 µM BCNU, 5 µM lin-SG thioester plus 1 µM

EX-527 and 1 µM EX-527 (100 µl/plate), all containing 20 µM DMSO. Paralysis was evaluated at

L4 larval stage (Fig. 3A). The worms that did not move or only moved their head when gently

touched with a platinum loop were scored as paralyzed.

Pharyngeal pumping and body bend assays

Populations of N2 worms, after egg synchronization, were placed at 20 °C on fresh NGM plates

seeded with E. coli. At L3 larval stage, the worms were fed with vehicle (Control), 10 µM Aβ42

oligomers, 10 µM Aβ42 oligomers plus 5 µM lin-SG thioester, 10 µM Aβ42 oligomers plus 5 µM

GSH, 10 µM Aβ42 oligomers plus 5 µM linolenic acid, 5 µM lin-SG, 5 µM GSH and 5 µM

linolenic acid (100 µl/plate), all containing 20 µM DMSO. Pharyngeal pumping and body bend

rates were evaluated at L4 larval stage (Fig. 3A). The pumping behaviour was scored by counting

the number of times the terminal bulb of the pharynx contracted over a 1-min interval under

inverted microscope. For the body bend assay, one worm for each condition was picked and

transferred into a 96-well microtiter plate containing 100 µl of double-distilled H2O. The number of

left-right movements over a 1-min interval was recorded under inverted microscope. The

10

experiments were replicated three times to obtain the average pharyngeal pumping and body bend

rates from at least 30 worms.

Analysis of protein carbonyl content and mitochondria superoxide production

Protein carbonyl content was determined by using the sensitive Protein Carbonyl Fluorimetric

Assay Kit (Cayman chemical, Ann Arbor, MI) according to the manufacturer’s instructions.

Synchronized N2 worms at L3 larval stage were incubated (400 worms/plate) with vehicle

(Control), 10 µM Aβ42, 10 µM Aβ42 plus 5 µM lin-SG thioester, 10 µM Aβ42 plus 5 µM GSH, 10

µM Aβ42 plus 5 µM linolenic acid, 10 µM Aβ42 plus 5 µM lin-SG thioester plus 25 µM BCNU (100

µl/plate), for 24 h at 20 °C. Worm protein extracts (50 µl of 5 mg/ml) were incubated overnight at

room temperature with 50 µl of PCF Fluorophore. After two cycles of centrifuge at 10,000 x g for

10 min, the excess of fluorophore was washed away and the fluorescence was estimated at an

excitation wavelength between 480-490 nm and an emission wavelength between 525-535 nm.

Mitochondria-specific oxidant levels were assessed by using the fluorescent probe MitoSOX

Red, a lipophilic hydroethidine (HE) derivative that accumulates 100- to 1000-fold within

mitochondria due to charge attraction of its triphenylphosphonium cation through the mitochondria

membrane bilayers into the negatively-charged mitochondria matrix [39]. Mitochondria-generated

oxidants react with MitoSOX to yield two primary fluorescent products, a 2-hydroxyethidium

derivative (2-OH-Mito-E+) resulting from superoxide oxidation, and Mito-E+, a non-specific

oxidized product [40]. Synchronized N2 worms at L3 larval stage were incubated (100

worms/condition) with vehicle (Control), 10 µM Aβ42, 10 µM Aβ42 plus 5 µM lin-SG thioester, 10

µM Aβ42 plus 5 µM GSH, 10 µM Aβ42 plus 5 µM linolenic acid, 5 µM lin-SG thioester, 5 µM

GSH, 5 µM linolenic acid, 25 µM BCNU and 5 µM lin-SG thioester plus 25 µM BCNU (100

µl/plate), for 1 h at 20 °C. In another series of experiments worms were fed with 150 µM H2O2, 150

11

µM H2O2 plus 5 µM lin-SG, 150 µM H2O2 plus 5 µM GSH (100 µl/plate). After treatment, the

worms were seeded on fresh NGM plates spread with OP50 E. coli and either 10 µM MitoSOX Red

(Molecular Probes, Eugene, OR). Following 24 h incubation, nematodes were collected by

centrifugation and the oxidant was washed away. The worms were seeded for 1 h on fresh

nematode growth medium plates to clear their guts of residual dye. Living nematodes were

collected and paralyzed by directly adding 4 % paraformaldehyde. Fluorescence microscopy

analysis was performed on confocal Leica TCS SP5 scanning microscope (Mannheim, Germany)

equipped with laser sources for fluorescence measurements at 594 nm and a Leica Plan Apo 40X oil

immersion objective. A series of optical sections (1024x1024 pixels), 10.0 µm in thickness, were

taken through the worm depth for each examined sample, maintaining constant setting.

Statistical analysis

All data are expressed as mean ± standard deviation (S.D.). Comparisons between different

groups were performed using ANOVA followed by Bonferroni’s post-comparison test. A p-value

less than 0.05 was accepted as statistically significant.

RESULTS

Linolenoyl-SG thioester extends lifespan upon the increase of intracellular GSH levels

We have previously shown that novel acyl-SG derivatives easily cross the plasma membrane

and are trapped in the cytosolic compartment by thioesterase-catalyzed hydrolysis, releasing the

reduced form of GSH and the parent carboxylic acid [29,30,41]. Here, we aim to investigate

whether dietary supplementation with linolenoyl-SG (lin-SG) thioester, the most effective acyl-SG,

can promote the survival of C. elegans. Indeed, this nematode represents a valuable model to study

the effect of intracellular antioxidant capacity on lifespan. We found that exposing synchronized

12

and sterilized wild-type N2 populations at L4 larval stage to 20 µM lin-SG thioester leads to an

increase in median lifespan (~30%) with respect to worms treated with the reduced form of

glutathione (GSH) or vehicle (Control) (Fig. 1A,C). In order to rule out quick hydrolysis or

decomposition of lin-SG thioester in aqueous media, its stability in PBS has been assessed. The

NMR spectra showed that the thioester was almost stable after 3 days (Fig. 2B) and still largely

unaffected (ca. 70%) after 7 days (Fig. 2C). Interestingly, lin-SG thioester significantly promoted

the worm survival even at low concentrations (1 µM and 5µM) (Fig. 1A,C). On the contrary, the

treatment with ethyl ester of GSH (GSH ethyl ester), which has been previously found to protect

against GSH deficiency in aged mice [42], or with linolenic acid showed only a slight protective

effect in the first week of the worm survival (Fig. 1A), resulting in a minor increase in median

lifespan (~8%) (Fig. 1C). To confirm that C. elegans life extension was mediated by the increase of

antioxidant intake, we evaluated intracellular GSH levels following worm administration with lin-

SG derivative (Fig. 3A). Unlike GSH, GSH ethyl ester or linolenic acid, lin-SG thioester

significantly increased intracellular GSH content (ca. 2,6 fold) with respect to worm treatment with

vehicle (Control) (Fig. 3B). The protective effect of lin-SG thioester on longevity was entirely

abolished in the presence of BCNU (Fig. 1B,C), a selective inhibitor of the glutathione reductase.

Indeed, the inhibition of glutathione reductase resulted in a significant increase in oxidized

glutathione (GSSG) levels, albeit maintaining the total glutathione content (GSH plus GSSG), in

worms exposed to lin-SG thioester (Fig. 3B). Accordingly, a minor change in GSH/GSSG ratio was

also evident in populations fed with BCNU without lin-SG (Fig. 3B). Various non-catalytic

antioxidants, such as vitamin E and C, trolox, α-tocopherol, N-acetylcysteine, and oleuropein

aglycone were also found to affect lifespan differently in distinct studies [43-46]. In our

experimental conditions, worms fed with vit. E showed no significant increase in median lifespan

(~6%), whereas a minor protective effect in the first week of the worm survival was observed (Fig.

1B,C). Overall, our data suggests a specific protective effect of GSH intake on worm lifespan.

13

Linolenoyl-SG thioester extends lifespan via SIR-2.1 upregulation through the DAF-16

pathway

SIRT1, an ubiquitous NAD(+)-dependent deacetylase, has been implicated in regulating

lifespan and aging through modulation of specific cellular pathways [47]. C. elegans Sir-2.1, the

homolog of mammal Sirt1, has also been found to regulate worm lifespan [12]. Thus, we next

assessed whether lin-SG thioester increases worm lifespan via a SIR-2.1-mediated mechanism. The

increase in worm longevity was completely prevented upon sir-2.1 silencing by worm feeding with

sir-2.1 RNA-interference (RNAi) or by the selective inhibition of SIR-2.1 activity with EX-527

(Fig. 1B,C), without a significant change in intracellular redox capacity (Fig. 3B). In order to

confirm the involvement of SIR-2.1 in thioester-mediated longevity, we evaluated by dot-blot

analysis SIR-2.1 expression in worm fed with 5 µM lin-SG derivative at L3 larval stage (Fig. 3A).

We found that dietary supplementation with lin-SG thioester significantly increased SIR-2.1 amount

in C. elegans with respect to worms fed with vehicle (Control) (Fig. 3C). We also showed that sir-

2.1 RNAi completely abolish SIR-2.1 expression, both in the absence and in the presence of lin-SG

thioester (Fig. 3C). SIR-2.1 levels were not modified by the treatment with GSH, GSH ethyl ester

or linolenic acid. When we treated worms with 5 µM lin-SG in the presence of the sirtuin inhibitor

EX-527 we found that SIR-2.1 expression significantly increased still upon enzyme activity

inhibition (Fig. 3C), suggesting that lin-SG thioester promotes the overexpression of sir-2.1 gene. In

contrast, SIR-2.1 expression was not modified by EX-527. Furthermore, SIR-2.1 levels were not

modified when worms were fed with lin-SG derivative in the presence of BCNU, with BCNU alone

or with vit. E (Fig. 3C). It has been demonstrated that DAF-16, a homolog to the mammalian

forkhead transcription factor FOXO [48,49], regulates lifespan in flies, worms, and mammals [50],

and its activation is promoted by SIR-2.1 [15,51]. Thus, we tested whether lin-SG-mediated worm

longevity upon SIR-2.1 upregulation was driven by the daf-16 pathway. Daf-16 silencing by worm

14

feeding with daf-16 RNAi prevented lin-SG-mediated worm longevity (Fig. 1B,C), suggesting a

mechanism by which lin-SG upregulates SIR-2.1 via the DAF-16 pathway. Accordingly, sir-2.1 or

daf-16 RNAi, without lin-SG thioester, displayed no significant influence on worm lifespan (Fig.

1C). Taken together these findings suggest a specific anti-aging effect of lin-SG derivative, which is

mediated by SIR-2.1 upregulation through the DAF-16 pathway upon the increase of intracellular

GSH redox capacity.

Linolenoyl-SG thioester protects from Aβ-induced paralysis phenotype

We have recently demonstrated that lin-SG thioester prevents intracellular lipid peroxidation and

mitochondrial dysfunction in primary fibroblasts from familial AD (FAD) patients and human SH-

SY5Y neuroblastoma cells experiencing oxidative injury, and protect cholinergic neurons and glial

cells against Aβ-induced damage in rat brains [30]. In this study we analyze whether the anti-

oxidant properties of lin-SG derivative is also effective against Aβ–induced toxicity in C. elegans

model. To this aim we investigated the paralysis phenotype of the worms fed with Aβ42 at L3 larval

stage (Fig. 3A) by paralysis, pharyngeal pumping and body bend assays at L4 larval stage (Fig.

4A,B). We found that lin-SG thioester significantly prevented Aβ42-induced paralysis (Fig. 4A). By

contrast, GSH did not show a significant protective effect against Aβ42 oligomer injury (Fig. 4A).

Interestingly, linolenic acid showed a significant protective effect against Aβ42-induced paralysis

(Fig. 4A), suggesting the dual role of the fatty acid portion such as an antioxidant and a GSH

carrier. Moreover, lin-SG thioester, GSH or linolenic acid supplementation, in the absence of Aβ42,

did not induce worm paralysis (Fig. 4A). In addition, dietary supplementation with lin-SG plus

BCNU or lin-SG plus EX-527 did not show any effect as well as BCNU or EX-527 alone (Fig. 4A).

Accordingly, lin-SG thioester and linolenic acid, through to a lesser extent, were also found to

significantly prevent Aβ42-induced decrease of pharyngeal pumping and body bend (Fig. 4B),

15

whereas GSH did not show any protective effect (Fig. 4B). Furthermore, lin-SG derivative, GSH or

linolenic acid supplementation, in the absence of Aβ42, did not modified worm phenotype (Fig. 4B).

These findings suggest that lin-SG thioester protects C. elegans against the amyloid–induced

paralysis by reducing the severity of symptoms. The innovative feature of lin-SG thioester is the

dual protective effect enclosed in a single molecule: free reduced GSH, which acts as a scavenger,

and a PUFA, which acts as a carrier and itself has inherent antioxidant properties.

Linolenoyl-SG thioester prevents Aβ42-induced oxidative stress

Finally we investigated the antioxidant properties of lin-SG against Aβ–induced oxidative stress

in C. elegans., by quantifying the protein carbonyl content, one of the most commonly used marker

of protein oxidation. Worms fed with Aβ42 oligomers showed a significant increase in protein

carbonyl content with respect to control worms (Control), suggesting high levels of oxidative stress

(Fig. 4C). Unlike GSH, lin-SG thioester and linolenic acid significantly protected worms from the

oxidation induced by Aβ42 exposure (Fig. 4C). In addition, worm treated with Aβ42 plus lin-SG plus

BCNU showed high protein carbonyl content, suggesting that the inhibition of the glutathione

reductase prevent the protective effect of lin-SG (Fig. 4C).

Finally, we investigated the antioxidant properties of lin-SG against Aβ–induced oxidative

stress in C. elegans by using the fluorescent probe MitoSOX Red, a lipophilic hydroethidine (HE)

derivative that accumulates 100- to 1000-fold within mitochondria [39]. C. elegans has a muscle-

tube-like continually pumping pharynx that is densely populated by mitochondria, that can be

readily labeled with targeted fluorescent dyes. Nematodes are optically transparent, which permits

precise assessment of tissue localization of the ingested fluorescent dyes. Worms fed with Aβ42

oligomers showed an increase in red fluorescence signal in their terminal pharyngeal bulbs with

respect to control worms (Control), suggesting high levels of oxidative stress (Fig. 5A,C). Unlike

16

GSH, lin-SG thioester significantly protected worms from the oxidation induced by Aβ42 exposure

(Fig. 5A,C). Moreover, linolenic acid showed a significant protective effect against Aβ42-induced

oxidative stress (Fig. 5B,C). Accordingly, a comparable and selective protective effect of lin-SG

derivative was observed in the presence of H2O2 (Fig. 5B,C). In addition, worm treatment with lin-

SG or BCNU or both, in the absence of oxidant inducers, resulted in a low mitochondrial

accumulation of oxidized products (Fig. 5B,C). Overall, our findings suggest that food

supplementation with lin-SG thioester can be beneficial against oxidative stress in C. elegans.

Discussion

Aging is a multi-factorial process influenced by environmental and genetic factors. Many

studies have indicated that oxidative damage limits longevity [52-56], although some have

questioned a causal role of oxidation in aging due to failure of antioxidant interventions to extend

life [57-59]. Anyway, great attention is being paid to the identification of dietary regimens that can

promote healthy aging. Our data show that dietary supplementation with lin-SG thioester promotes

lifespan in a short-term lived model such as C. elegans. The lipophilic nature of lin-SG derivative

allows it to be easily adsorbed and trapped in the cellular compartment and to release the free

reduced form of GSH and the parent linolenic acid via a thioesterase-catalyzed hydrolysis. Worm

life extension induced by lin-SG is entirely abolished in the presence of BCNU, a selective inhibitor

of the glutathione reductase. The longevity effect therefore results from the ability of lin-SG

thioester to significantly enhance the intracellular reductive capacity of GSH. Moreover, our results

suggest that the anti-aging effect is specific to the lin-SG derivative, relative to other antioxidant

compounds such as vitamin E. Indeed, vit. E does not promote worm longevity in our experimental

conditions. In this regard, its role on lifespan in model organisms, including single-cell organisms,

17

rotifers, C. elegans, Drosophila melanogaster and rodents, has long been studied with contradictory

results [60].

Among an array of GSH derivatives capable of increasing intracellular levels of the reduced

thiol [26,27,61], GSH ethyl ester has been previously found to protect against GSH deficiency due

to biological aging in mice [42]. On the contrary, C. elegans lifespan was not modified by GSH

ethyl ester or GSH in our experimental conditions, suggesting a higher value of linolenic acid with

respect to ethyl moiety as a GSH carrier. The lower anti-aging power of GSH ethyl ester appears to

be related to a partial oxidation of the thiol group of glutathione once released into the cells. On the

contrary, the conjugation of GSH, via the thiol group of cysteine, to an acyl chains appears a more

effective approach for the production of diffusible prodrugs and supplementation of reduced GSH

levels in C. elegans model. Our findings also support recent studies demonstrating that elevated

intake of polyunsaturated fatty acids (PUFAs) — and high levels of antioxidant compounds could

act synergistically in improving cognitive performance and possibly in preventing or delaying the

onset of dementia [62]. Accordingly, we show that lin-SG thioester protects N2 worms from the

three features of the paralysis phenotype induced by Aβ42 exposure, enhancing the vitality, the

pharyngeal pumping and the body bends of the worms. Finally, lin-SG conjugate prevents the

oxidative stress induced by both amyloid and H2O2, confirming a protective role against the

oxidative damage. By contrast, GSH alone did not show any protective effect, because of a lower

permeability of plasma membrane. In addition, it has been demonstrated that supplementing C.

elegans culture media with ω-6 PUFAs increases their resistance to starvation and extends their life

span in conditions of food abundance [63]. Accordingly, dietary supplementation with linolenic

acid leads a slight protective effect in the first week of worm survival and in median lifespan,

significantly preventing the Aβ42-induced paralysis and oxidative stress of the worms.

In recent years, a role for GSH in the modulation of signal transduction through direct

interaction with key cysteines located in the active site or modulator regions of kinases,

18

phosphatases, and transcription factors has been recognized [64-66]. Our data show a new

mechanism by which SIR-2.1 can be upregulated via GSH intracellular increase. Indeed, sir-2.1

RNAi completely abolish the extension of C. elegans median lifespan induced by lin-SG derivative.

In addition, lin-SG-mediated longevity was also prevented in the presence of the selective sirtuin-

inhibitor EX-527, indicating a prominent role for deacetylases activity of sirtuins in this process.

These results agree with recent findings indicating that sirtuins regulate metabolism and stress

responses, thus extending lifespan in yeast, worms and flies [51,67]. However, the mechanisms

underlying sirtuin-dependent longevity are still debated. We have recently demonstrated that SIRT1

activation, induced by resveratrol, modulates mitogen activated protein kinase (MAPK) pathway

[68]. Small molecules such as resveratrol are of great interest because they increase lifespan in

many species in a sirtuin-dependent manner [69,70].

It has been demonstrated that DAF-16, a homolog to the mammalian forkhead transcription

factor FOXO, regulates lifespan in flies, worms, and mammals [50], The overexpression of sir-2.1

gene in C. elegans was found to extend lifespan by activating daf-16 [15,67]. SIR-2.1 is likely to

activate DAF-16 directly, by deacetylation, as mammalian SIRT1 is known to deacetylate FOXO

proteins in response to oxidative stress [67], which, in turn, shifts their target specificity towards

genes involved in stress resistance. Oxidative stress may have similar effects in worms, as it

stimulates the binding of SIR-2.1 to DAF-16 [15] and can extend lifespan in a sir-2-dependent and

daf-16-dependent manner [71]. Accordingly, our daf-16 silencing data showed that lin-SG-mediated

overexpression of sir-2.1 extends worm lifespan through the daf-16 pathway. Thus, we can

hypothesize that, in our experimental model, GSH can modulate the DAF-16 pathway, via SIR-2.1

upregulation.

Overall, these findings show that dietary supplementation with a GSH carrier promotes

oxidative-stress resistance, thereby delaying the occurrence of aging. To our knowledge, our data

reveal for the first time a new mechanism to upregulate sirtuins and to promote longevity, just

19

enhancing the intracellular reductive capacity of GSH. Lin-SG thioester intake may therefore

represent a novel approach to alleviate age-associated diseases and possibly extend healthy lifespan.

Acknowledgements

We thank Luisa Diomede for the valuable discussion and Giulia Bruschi for technical advice.

This work was supported by the Regione Toscana [POR CRO FSE 2007-2013 “AMILOIDOSI”].

Author Disclosure Statement

No competing financial interests exist.

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FIGURE LEGENDS

Fig. 1 Lin-SG thioester extends C. elegans lifespan via a SIR-2.1-mediated mechanism. A-B,

Percent survival of wild-type (N2) worms. Populations of N2 worms, after egg synchronization,

were placed on fresh NGM small plates (100 worms/plate) seeded with E. coli for 48 h at 20 °C. L4

larvae growing at 20 °C were transferred to new plates in the presence of 75 µM fluorodeoxyuridine

(FUdR) to prevent offspring from nematodes under study from reaching adulthood, and then treated

with 100 µl of vehicle (Control), 20 µM GSH, 20 µM linolenic acid, 1 µM or 5 µM or 20 µM lin-

SG thioester, 20 µM GSH ethyl ester (A), or with 5 µM lin-SG thioester, 5 µM lin-SG thioester plus

25 µM BCNU, 20 µM vit. E, 5 µM lin-SG thioester plus 1 µM EX-527, 5 µM lin-SG thioester in

the presence of sir-2.1 or daf-16 RNAi feedings (B), all containing 20 µM DMSO. Nematodes were

scored as alive, dead or lost starting from 24 h after treatment (day 1 in survival curves). C, Percent

change in median lifespan of N2 populations exposed to the above reported conditions. Plots are

representative of three independent experiments. Survival curves were plotted and statistical

analyses were performed using Graphpad Software. The triple asterisk indicates significant

difference (p≤0.001) versus control worms.

Fig. 2 Control experiments for stability of lin-SG thioester in aqueous media. Insets of the 1H-

NMR spectrum of lin-SG (400 MHz, d6-DMSO) in the 5.60-3.80 ppm region during stability

control experiments in a PBS/DMSO solution at 20 °C after different times. A, 0 days; B, 3 days;

C, 7 days. D, full spectrum of pure lin-SG thioester.

Fig. 3 Lin-SG-mediated longevity relies for the activation of SIR-2.1 upon the increase of

intracellular redox potential. A, Diagram illustrating when the treatments were administered and

when the assays were scored. The wild-type N2 worms, synchronized and placed on E. coli at 20

°C, were treated as indicated (100 µl/plate) at L3 larval stage. Worm homogenates were rated 24 h

later, when the worms were at L4. B, Intracellular GSH and GSSG levels in worms fed with vehicle

28

(Control), 5 µM lin-SG thioester, 5 µM GSH, 5 µM GSH ethyl ester, 5 µM linolenic acid, 5 µM lin-

SG thioester plus 25 µM BCNU, 25 µM BCNU, 5 µM lin-SG thioester plus 1 µM EX-527, 1 µM

EX-527. C, Representative dot blot analysis and relative densitometric quantification of SIR-2.1

levels in worms treated with vehicle (Control), 5 µM lin-SG thioester, 5 µM lin-SG thioester in the

presence or absence of sir-2.1 RNAi feeding, 5 µM GSH, 5 µM GSH ethyl ester, 5 µM linolenic

acid, 5 µM lin-SG thioester plus 1 µM EX-527, 1 µM EX-527, 5 µM lin-SG thioester plus 25 µM

BCNU, 25 µM BCNU and 20 µM vit. E. Values are means ± S.D. of three independent

experiments, each performed in duplicate. The symbols *** and § indicate significant difference

(p≤0.001) versus control worms and worms treated with lin-SG, respectively..

Fig. 4 Lin-SG thioester prevents Aβ-induced paralysis phenotype. A, Percentages of paralysis

of worms fed with vehicle (Control), 10 µM Aβ42 oligomers, 10 µM Aβ42 oligomers plus 5 µM lin-

SG thioester, 10 µM Aβ42 oligomers plus 5 µM GSH, 10 µM Aβ42 oligomers plus 5 µM linolenic

acid, 5 µM lin-SG thioester, 5 µM GSH, 5 µM linolenic acid, 5 µM lin-SG thioester plus 25 µM

BCNU, 25 µM BCNU, 5 µM lin-SG thioester plus 1 µM EX-527, 1 µM EX-527. B, Percentages of

pharyngeal pumping and body bends of worms fed with vehicle (Control), 10 µM Aβ42 oligomers,

10 µM Aβ42 oligomers plus 5 µM lin-SG thioester, 10 µM Aβ42 oligomers plus 5 µM GSH, 10 µM

Aβ42 oligomers plus 5 µM linolenic acid, 5 µM lin-SG thioester, 5 µM GSH, and 5 µM linolenic

acid. All egg-synchronized N2 worms were placed for 72 h at 20 °C on fresh NGM plates, after that

they were treated for 24 h before the assays (100 μl/plate) (see Fig. 2A for treatment schedule). The

pumping and body bend behaviours were scored by counting the number of times the terminal bulb

of the pharynx contracted and the number of left-right movements, respectively, over a 1 min

interval, under an inverted microscope. C, Analysis of protein carbonyl content in worms fed with

vehicle (Control), 10 µM Aβ42, 10 µM Aβ42 plus 5 µM lin-SG thioester, 10 µM Aβ42 plus 5 µM

GSH, 10 µM Aβ42 plus 5 µM linolenic acid, 10 µM Aβ42 plus 5 µM lin-SG thioester plus 25 µM

BCNU (100 µl/plate), for 24 h at 20 °C. Data are shown as percentages ± S.D. with respect to

29

control worms (n=100, three independent assays). The triple asterisk indicates significant difference

(p≤0.001) versus Aβ42-treated worms.

Fig. 5 Lin-SG thioester prevents Aβ/H2O2-induced oxidative stress. A-B, Nematode terminal

pharyngeal bulbs labelling with mitochondria-targeted dye. Egg-synchronized N2 worms were

placed for 72 h at 20 °C on fresh NGM plates. Then, worms were fed with vehicle (Control), 10 µM

Aβ42 oligomers, 10 µM Aβ42 oligomers plus 5 µM lin-SG thioester, 10 µM Aβ42 oligomers plus 5

µM GSH, 10 µM Aβ42 oligomers plus 5 µM linolenic acid, 5 µM lin-SG thioester, 5 µM GSH, 5

µM linolenic acid, 25 µM BCNU, 5 µM lin-SG thioester plus 25 µM BCNU (A), or with 150 µM

H2O2, 150 µM H2O2 plus 5 µM lin-SG thioester and 150 µM H2O2 plus 5 µM GSH (B), all

containing 20 µM DMSO, for 24 h before the assays (100 μl/plate). MitoSOX fluorescence of N2

pharynx overlay with DIC image demonstrates preferential labelling in terminal pharyngeal bulbs.

C, Semi-quantitative analysis of the MitoSOX red fluorescence signal in the pharyngeal bulbs.

Values are means ± S.D. of three independent experiments. The triple asterisk indicates significant

difference (p≤0.001) versus Aβ42-treated or H2O2-treated worms.

Highlights

- Linolenoyl-SG thioester extends C. elegans lifespan via SIR-2.1 upregulation through the DAF-16

(FoxO) pathway

- Linolenoyl-SG thioester shows a dual protective effect through GSH and linolenic acid

- Linolenoyl-SG thioester protects from Aβ/H2O2-induced paralysis and oxidative stress in C.

elegans

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5