Withanolide A offers neuroprotection, ameliorates stress resistance and
prolongs the life expectancy of Caenorhabditis elegans
Bashir Akhlaq Akhoon, Swapnil Pandey, Sudeep Tiwari, Rakesh Pandey ⁎
Microbial Technology and Nematology Department, CSIR —Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, India
Received 9 September 2015
Received in revised form 15 February 2016
Accepted 2 March 2016
Available online 5 March 2016
Section Editor: Holly M. Brown-Borg
Withanolide A (steroidal lactone) forms the major constituent of the most popular herbal drug in Ayurvedic
medicine, Ashwagandha. It has been used since ancient times as an alternative medicine for the treatment of a
variety of age related disorders. Here we provide multiple lines of evidence indicating that Withanolide A im-
proves healthspan, delays age-associated physiological changes and also extends the lifespan of Caenorhabditis
elegans. We also report several neuroprotective beneﬁts of this natural product, including its anti-
amyloidogenic effects, alleviation of α-synuclein aggregation and neuroprotection through modulation of neural
mediators like acetylcholine. We observed that Withanolide A mediates lifespan extension and promotes stress
resistance via insulin/insulin-like growth factor signaling pathway. Such ﬁndings could be helpful to develop a
therapeutic medicine from this natural product for the prevention or reversal of age-related ailments and to
improve the survival of patients suffering from Alzheimer's or Parkinson's disease.
© 2016 Elsevier Inc. All rights reserved.
Aging is a challenge to every living organism and all human beings
have to encounter it. It has been considered as the main risk factor for
other most prevalent diseases, including several neurological disorders
(Niccoli and Partridge, 2012). In fact, numerous genetic pathways
such as the insulin/IGF-1 pathway that inﬂuence aging also provides
neuroprotection and surprisingly such pathways are evolutionarily
conserved (Kenyon, 2005, 2010; de la Monte and Wands, 2005;
There is now growing evidence that both healthspan and/or lifespan
can be prolonged by genetic and/or dietary interventions (Chen et al.,
2013a; Lucanic et al., 2013; Argyropoulou et al., 2013) and phytochem-
icals seem promising in this endeavor. Phytochemicals are non-
nutritive components that occur naturally in plants and possess sub-
stantial biological activities. Fruits and vegetables contain thousands of
biologically active phytochemicals that are likely to interact in a number
of ways to prevent disease and promote health (Surh, 2003;
Argyropoulou et al., 2013). Withanolide A (WA), a steroidal lactone, is
the major constituent of Indian herbal drug Ashwagandha (root of
Withania somnifera)(Baitharu et al., 2014). WA has been reported to
have potential therapeutic value for several neurological disorders,
including Alzheimer's disease-associated amyloid pathology, regenera-
tion of neuritis, recovery of damaged synapses, axonal outgrowth etc.
(Zhao et al., 2002; Kuboyama et al., 2002, 2005; Baitharu et al., 2014;
Kurapati et al., 2013). Also, Ashwagandha has been found to have a
remarkable area of applications in Ayurvedic medicine for a variety of
age related ailments, including its use as an adaptogen to help the
body cope with daily stress (Winston and Maimes, 2007). In the
Ayurvedic literature, Ashwagandha is known as Avarada that means
rejuvenation or youthfulness. While WA has been reported by several
researchers to have neuromodulatory effects, there is no scientiﬁcvali-
dation for its use as an anti-aging agent to delay aging and or increase
Caenorhabditis elegans, a free living soil nematode has contributed
enormously to our understanding of several neurological disorders
and aging (Kenyon, 2005, 2010; Markaki and Tavernarakis, 2010; Li
and Le, 2013). It is an appealing model for aging neuroscience owing
to its short life-cycle, fully sequenced genome, 60–80% human gene
counterparts, and amenability to classical and reverse genetics. Such
features of C. elegans make it a powerful platform for the discovery of
novel anti-aging and neuroprotective compounds. The C. elegans
model system has been successfully exploited by numerous investiga-
tors to discover compounds that impact neurobiology of aging
(Marvanova and Nichols, 2007; Petrascheck et al., 2007; Cho et al.,
2010; Argyropoulou et al., 2013; Lucanic et al., 2013; Fu et al., 2014).
The present study used this multifaceted animal model system
C. elegans to explore neuroprotective and anti-aging health beneﬁts of
WA and to shed some insights into its underlying mechanism.
Experimental Gerontology 78 (2016) 47–56
⁎Corresponding author at: Microbial T echnology and Nematology, CSIR–Central
Institute of Medicinal and Aromatic Plants (CSIR–CIMAP), Lucknow 226015, India.
E-mail address: firstname.lastname@example.org (R. Pandey).
0531-5565/© 2016 Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/expgero
2. Experimental procedures
2.1. Nematodes and pharmacological compound
The nematode strains used in this study were wild-type Bristol N2,
TK22: mev-1 (kn1), DA1116: eat-2 (ad1116), VC345: sgk-1 (ok538),
PS3551: hsf-1 (sy441), GR1307: daf-16 (mgDf50), EU1: skn-1 (zu67),
TJ356: zIs356 (daf-16::gfp +rol-6), CL2166: dvIs19 (gst-4p::GFP),
CF1553: muIs84 (sod-3p::GFP +rol-6). All the strains were procured
from the Caenorhabditis Genetics Center at the University of Minnesota.
Withanolide A (procured from Natural Remedies, India) was
dissolved in dimethyl sulfoxide (DMSO) to prepare 1 mM stock solution.
Worms were treated with various concentrations of WA viz., 2 μM, 5 μM,
25 μM, and 50 μM to examine the effect of WA on the lifespan of
C. elegans. We used 0.1% DMSO as a vehicle control in all our experiments.
2.2. Lifespan analysis
Lifespan analysis was performed at 20 °C on triplicate NGM plates
(50 worms/plate). Late L4 worms were transferred to NGM plates spot-
ted with Escherichia coli OP50 and containing 5 μM, 25 μM and 50 μM
WA concentrations spread uniformly over the OP50. 50 μM 5-Fluoro-
2′deoxyuridine (FUdR) was also added to the NGM plates to inhibit
the progeny growth. Worms were transferred to new NGM plates
every 2–3 days and scored for survival using touch provoked
2.3. Measurement of body size and body movement
Worms were cultured at 20 °C similar to lifespan analysis, however
FudR was not added to the NGM plates. For body size measurement,
the pictures of 10 randomly selected worms were taken on days 2, 5
and 10 using Leica Application Suite V3 software (version 3.4.0). The
thrashing rate was determined as previously described (Vilchez et al.,
2012). In Brief, ten days 2, 5 and 10 worms were transferred to a drop
of M9 buffer, allowed to adapt for 30 s, and then the number of body
bends were recorded for 30 s.
2.4. Pharyngeal pumping and chemotaxis
For pumping assay, theage-synchronized worms werebred on NGM
plates and the movement of pharynx terminal bulb in worms (n = 10)
was recorded on days 2, 5 and 10 for 20-s intervals at room
Chemotaxis assay was performed using a 9 cm agar plate containing
1.5% agar, 1 mM CaCl
, 1 mM MgSO
NaCl salt solution (at pH 6.0) was added to the attractant spot and 10 μl
of double-distilled H
(Rabinowitch et al., 2014). Sodium azide (10 μl of 1 M) was added
10 min before the assay at the gradient peak to immobilize the worms
once they reach their targeted spot. Age-synchronized 50 day 5 adult
worms were washed in M9 buffer and placed in the center of agar
plates. Worms were allowed to move over the agar surface for a one-
and-a-half hours before the chemotaxis index (CI) was calculated as
CI = (A
) / 50, where A is the number of worms at the attractant
(NaCl) location and B is the number of worms on the control (H
2.5. Fecundity assay
The fecundity assay was performed as described (Schulz et al.,
2007). Brieﬂy, 10 worms were cultured on NGM plates with or without
WA and allowed to lay eggs for 12 h. Parent worms were removed and
the progeny was allowed to develop for 48 h before quantiﬁcation of
2.6. Aβ-induced paralysis assay
For paralysis assay, we used a CL4176 strain of C. elegans.Thistrans-
genic strain has been widely used to examine Aβtoxicity. Worms were
raised at 15 °C and after 48 h the temperature was upshifted to 25 °C for
induction of transgenic expression of Aβ. Paralyzed worms were scored
using touch provoked movement. The experiment was repeated twice
with each group containing 30–40 worms.
2.7. Relative quantiﬁcation of acetylcholine and acetylcholinesterase levels
Age-synchronized worms were added to treatment plates and incu-
bated at 22 °C for 48 h. The worms were then washed thrice using M9
buffer to clear off any adhering bacteria and sonicated in 1 × reaction buff-
er for 3 min. Worm suspension was then centrifuged at 7000 g for 7 min
and subsequently, 100 μl of supernatant was assayed for acetylcholine
(ACh) and acetylcholinesterase (AChE) levels using the Amplex Red
ACh/AChE Assay Kit (Cat No. A12217; Invitrogen) according to the
manufacturer's protocol. Fluorescence measurements were performed
using 96 well plate ﬂuorimeter (BMG Polarstar Galaxy) at excitation
544 nm and emission at 590 nm and the relative ﬂuorescence was nor-
malized with respect to protein content.
2.8. Stress resistance assays
Resistance to thermal stress was determined with minor modiﬁca-
tions as previously described (Vilchez et al., 2012). As described for
lifespan analysis, worms were cultured at 20 °C on WA and control
NGM plates. Heat-shock was given to day 3 adults for three and a half
hours at 35 °C, followed by a daily examination of the survival rate
until all nematodes were dead.
For oxidative stress assay, nematodes were maintained on WA or con-
trol plates at 20 °C until their progeny production ceased. On the sixth day
(after L4) worms were transferred to fresh NGM plates containing 10 mM
paraquat and checked daily for viability (Zarse et al., 2012).
2.9. Quantiﬁcation of ROS formation
Reactive Oxygen Species (ROS) levels were quantiﬁed with minor
modiﬁcations as described earlier (Shukla et al., 2012). Approximately,
100 young (day 3) adults were used for the assay. Fluorescence spectral
measurements were captured by Cary Eclipse ﬂuorescence spectropho-
tometer (Agilent Technologies) at the excitation wavelength of 485 nm
and the emission wavelength of 535 nm.
2.10. α-Synuclein protein/lipofuscin aggregation assay and visualization of
NL5901 (an integrated α-synuclein: YFP fusion construct driven by
the unc-54 promoter) strain of C. elegans was used to analyze the effect
of 5 μM WA on the aggregation of Parkinson's disease associated protein
α-synuclein. Worms were raised like the lifespan assay and after 72 h of
WA treatment from late L4, twenty randomly selected adult worms
were mounted on 3% agarose pads and anesthetized by 2% sodium
azide for visualization of autoﬂuorescence. Images were captured with
aﬂuorescence microscope (Leica, DMI3000) using GFP ﬁlter (with exci-
tation at 365 nm and emission at 420 nm). Quantiﬁcation of data was
performed by using ImageJ software. A similar protocol was followed
for Lipofuscin aggregation in N2 and other GFP ﬂuorescence measure-
ments carried out in this study.
2.11. Modeling of WA binding
The molecular docking approach wasusedtomodelWAintotheSGK-
1 binding site. For docking, the three-dimensional structure of WA was
retrieved from the PubChem database, one of the linked databases within
48 B.A. Akhoon et al. / Experimental Gerontology 78 (2016) 47–56
the NCBI's Entrez information retrieval system. The WA structure was
submitted to the idTarget server (Wang et al., 2012)foritspossiblepro-
tein target predictions using the Z-score (b0) as a ﬁlter. Other in silico
methods employed are described in the Results and discussion section.
2.12. Real-time RT-PCR analysis
The total RNA was harvested using RNAzol reagent (Invitrogen,
USA). cDNA was synthesized using a cDNA synthesis kit (Invitrogen)
and quantitative real-time PCR was carried out using 7900HT Fast
Real-Time PCR System (Applied Biosystems) with SYBR green dye ac-
cording to the manufacturer's instructions. Relative gene expression
was normalized to act-1 and the data was analyzed using the ΔΔCt rel-
ative quantitation method (Livak et al., 2001).
2.13. Statistical analyses
All the statistical calculations relevant to lifespan and stress resistance
assays were performed using MedCalc software (Log-rank signiﬁcance
test). Other statistical analyses were carried out using ANOVA in
ASSISTAT 7.7 beta statistical assistance software. In all experiments, a
P-value less than 0.05 was considered statistically signiﬁcant.
3. Results and discussion
3.1. WA treatment signiﬁcantly enhanced both lifespan and healthspan of
To investigate the impact of WA on the lifespan of a multicellular eu-
karyote, we exposed adult C. elegans to WA at 5 μM, 25 μM, and 50 μM
concentrations. When treated with the above-mentioned concentrations
of WA, the worm showed a signiﬁcant extension of lifespan as compared
with the negative control group (untreated) (Table 1). We noticed that
WA caused a signiﬁcant extension (P≤0.0001) of mean and median
lifespan by 29% and 21% respectively at 5 μM concentration (Fig. 1A).
We examined the lipofuscin (aging biomarker) appearance of day 10
worms to check whether the WA treatment delayed the initiation of
aging or just prolonged the aged stage and found that the appearance
of lipofuscin was signiﬁcantly reduced by approximately 27.54%
(Pb0.01 **) in worms treated with WA (Fig. 1B), showing that worms
treated with WA initiated the aging process more slowly than the control
worms. The rate of motor activity declines with age and has been report-
ed to be a physiological parameter of animal healthiness (Hsu et al.,
2009). Therefore, we were interested to check the effect of the optimal
concentration of WA (5 μM) on the motor activity and learning ability
of N2 worms. We found that the rate of the motor activity decay was
also reduced in WA treated animals (Fig. 2A). Also, it was observed that
C. elegans pre-exposed to WA-treatment showed increased levels of che-
motaxis (Fig. 2B) which is reported to decline with age (Glenn et al.,
2004). These results show that WA extend both healthspan and lifespan
of worms. Increased longevity has been noticed to be associated with re-
ductions in fecundity and growth and several dietary supplements that
extend adult lifespan has been reported to signiﬁcantly affect the progeny
production and body size in worms (Harrington and Harley, 1988;
Houthoofd et al., 2002; Liao et al., 2011). Therefore, we monitored the re-
productive capacity of both treated and control worms. In our study, we
observed 9% less progeny of WA-treated worms (12 h survey) in compar-
ison to untreated ones (Fig. 2C), showing that WA also modulates the re-
productive ability of worms that is associated with lifespan as reported by
earlier studies. Consequently, we measured the body size (length and
width) of wild-type adult animals pre-treated with WA. We found no-
ticeable variation in the body size of WA-treated worms on the day 2,
day 5 and day 10 of adulthood as the worms were longer and wider
than untreated animals (Fig. 2D). These results signify that WA mediated
lifespan extension is associated with body size of worms. Since WA has
been reported to have several neuromodulatory effects (as stated in the
Introduction section), we were interested to check if WA shows any neu-
roprotective effect in C. elegans.
α-Synuclein aggregation, a major culprit behind parkinsonism
culminates in neuronal cell death in the substantia nigra of Parkinson's
disease (PD) patients (Stefanis, 2012). We checked the effect of WA on
proteopathies by employing a C. elegans strain (NL5901) exhibiting mus-
cular expression of α-synuclein tagged with Yellow ﬂuorescence Protein
(YFP). We observed substantial decrease (38%) in α-synuclein levels
(compared to control group) in NL5901 worms treated with 5 μMof
WA (Fig. 3A), implicating beneﬁcial effects of WA for PD patients.
Amyloid β(Aβ) is an obligate player behind Alzheimer's disease
(AD) and its excessive accumulation is toxic to the cells (Karran et al.,
2011; Villemagne et al., 2013). In order to detect the effect of 5 μM
WA on Aβaggregation, a paralysis assay was conducted using a trans-
genic CL4176 strain of C. elegans. The employed strain expresses muscu-
lar Aβupon temperature upshift. In comparison to control worms
(52.00 ± 2.30), a signiﬁcant decrease (Pb0.001) in the percentage of
paralyzed CL4176 worms (27.33 ± 1.76) was noticed after WA treat-
ment (Fig. 3B). Such results highlight the anti-amyloidogenic effects of
WA in C. elegans as large numbers of worms paralyzed indicates
increased toxicity of Aβ.
Impaired neurotransmission and cholinergic abnormalities/deple-
tion are one of the marked symptoms of AD patients (Perry et al.,
2000). Moreover, the degeneration of the cholinergic system is also a
contributing factor to the learning and memory deﬁcits observed in
AD patients or during normal aging. In order to determine the effect of
WA on ACh levels, the total ACh was estimated through the Amplex
Red assay kit. In comparison to the relative ACh levels in a control
group (1.00 ± 0.1), a signiﬁcant increase (Pb0.05) in gross ACh levels
(1.78 ± 0.01, P-value: 0.05) was observed in 5 μM WA treated group
(Fig. 3C). We were also interested to check the effect of WA on AChE
activity. We used an Amplex red AChE kit for this purposeand observed
that AChE activity as observed in control group (1.00 ± 0.01), was also
slightly increased after WA treatment (1.34 ± 0.09), however, the in-
crease was insigniﬁcant (Fig. 3D). A signiﬁcant increase in Ach levels
without alteration in Ache levels is of particular interest in the context
of the deﬁcits prevalent in AD. Our results showed the beneﬁcial effect
of WA in curtailing the cholinergic dysfunction through a possibly
enhanced synthesis of Ach, without affecting the gross Ache activity.
3.2. WA-mediated lifespan extension is independent of calorie restriction
Restricting dietary consumption of compounds without malnutri-
tion extends lifespan and attenuates age-related declines/diseases in
multiple species (Masoro, 2005), thereby suggesting a conserved un-
derlying mechanism from nematodes to humans (McCay et al., 1935;
Lifespan analysis of wild-type worms cultured at 20 °C.
Strains Treatment (μM) Mean lifespan SE No. of worms (N) % change Pvalue Median lifespan Max. mean lifespan (±SE)
N2 Control 13.700 0.475 120 Pb0.0001 14 17.66 ± 0.13
2μM 14.293 0.561 117 4.32 Pb0.0001 14 19 ± 0.33
5μM 17.763 0.593 121 29.65 Pb0.0001 17 25.66 ± 0.17
25 μM 15.864 0.659 128 15.79 Pb0.0001 16 22 ± 0.15
50 μM 15.182 0.615 152 10.81 Pb0.0001 15 20.33 ± 0.26
49B.A. Akhoon et al. / Experimental Gerontology 78 (2016) 47–56
Fontana et al., 2010). Since many external molecules extend lifespan by
dietary restriction phenomenon (Bass et al., 2007; Pallauf et al., 2013),
we asked whether WA acts as a dietary restriction mimetic. We tested
this hypothesis by prospecting the rate of pharyngeal pumping in
worms. We did not notice any signiﬁcant effect on the rate of pharyn-
geal pumping (Fig. 4A). We also found that the lifespan of eat-2
(ad1116) mutant animals, which exhibit the phenomenon of calorie re-
striction because of slower pumping (Lakowski and Hekimi, 1998), was
extended by WA treatment (Fig. 4B). Additionally, we examined the ef-
fects of WA treatment on the eat-2 gene expression of wild-type ani-
mals. WA treatment failed to affect the eat-2 expression of N2 animals.
These results suggest that calorie restriction per se is unlikely to be
the cause of the lifespan extension observed in WA-treated worms.
Therefore, to predict the possible targets of WA, we performed large
scale in silico protein screening using the idTarget. Our docking results
showed that WA recognizes human serine/threonine-protein kinase
SGK1 (PDB ID: 2R5T) with a good binding afﬁnity (−9.63 kcal/mol).
3.3. sgk-1 is required for lifespan extension by WA
The insulin/IGF-1 signaling (IIS) pathway has been implicated in lon-
gevity in various organisms and its reduction leads to lifespan extension
Fig. 1. WithanolideA (WA) extends C. eleganslifespan. (A) Dose–responseKaplan–Meier survival curves of wild-type (N2) populations exposedto 0 μM (control),2, 5, 25 and 50 μMWAat
20 °C. Treatmentwith 5 μM, 25 μMand50μM WA signiﬁcantly (Pb0.0001) extendedthe mean lifespanof worms, whereastreatment with 2 μMWAdidnotcauseasigniﬁcant increasein
lifespan. The maximum mean and median lifespan extension by 29% and 21% respectively was seen in worms treated with 5 μM WA. (B) WA delays the progression of aging. Higher
accumulation of lipofuscin was seen in control worms compared with their respective 5 μM WA treated animals. Representative images show intestinal lipofuscin autoﬂuorescence of
untreated (Control) and WA-treated (5 μM) day 10 N2 animals (n = 20). Images were quantiﬁed using the ImageJ software. Scale bar, 30 μm.
Fig. 2. WA effects on the healthspan of C. elegans. (A) Effect of 5 μM WA on the motility of N2 worms evaluated as the mean numberof body bends in a 30-s period in 10 individual worms
on days 1, 3 and 5 adulthood. WAtreatment delays the age-relateddecline in body movement. Data represent the average number ofbends. (B) Salt chemotaxis in wild-type (day 5) N2
animals. Worms pretreated with WA showed improved levels of chemotaxis. (C) Average fertility of C. elegans treated with 5 μM WA. Worms when maintained on WA (72 h) showed
decrease in total progeny production (12 h survey). Plot is a representative of 2 independent experiments with a total of 10 nematodes per group. (D) Quantiﬁcation of body size
measurements. The body length and width of each worm at days 2, 5 and 10 was measured. Values represent mean size of worms (n = 10). Body size of WA treated worms was
signiﬁcantly increased in comparison to control animals. Asterisks (** and *) indicate signiﬁcant changes at Pb0.01 and Pb0.05.
50 B.A. Akhoon et al. / Experimental Gerontology 78 (2016) 47–56
(Tazearslan et al., 2011). The DAF-2 insulin receptor-like signaling path-
way in C. elegans also controls its lifespan by phosphorylating and
inhibiting the nuclear translocation of DAF-16/FoxO or SKN-1
accumulation in intestinal nuclei, where SGK-1 (C. elegans homolog
of SGK), functions in parallel to AKT-1 and AKT-2 to mediate this sig-
naling. SGK-1 contains a kinase domain which is 78% similar and 67%
identical to the human SGK kinase domain (Hertweck et al., 2004).
sgk-1 gain-of-function mutation enhances the lifespan in C. elegans
(Chen et al., 2013b). To analyze in vivo whether WA has any effect
on the sgk-1 of C. elegans, we assessed the impact of WA on the
lifespan of sgk-1 (ok538) null mutant. sgk-1 (ok538) harbors an
852 bp deletion that eliminates the seventh and most of the eighth
exon that forms the major part of the kinase domain, critical for
SGK-1 activity (Hertweck et al., 2004). Incubation with WA has no
inﬂuence on the lifespan of sgk-1 mutant (Table 2; Fig. S1A), showing
that sgk-1 is required for the lifespan extension observed in WA-
Moreover, our bioinformatics analysis showed that SGK-1 kinase do-
main is conserved in many species, from C. elegans to Homo sapiens
(Fig. S2). We aligned the kinase domain of 2R5T (human SGK-1) with
the SGK-1 of C. elegans and found 62.8% identity and 79.1% similarity
among them at sequence level (Fig. 5A). Such results reveal that SGK-
1ofC. elegans shares a similar kinase domain organization as of
human and have an evolutionary and likely functional relationship. To
examine the structural differences, we modeled the SGK-1 of
C. elegans using the I-TASSER (Roy et al., 2010). The program uses
Fig. 3. WA shows multifunctional neuroprotective roles in C. elegans.(A)αsynuclein levels in NL5901 worms. A substantial decrease in alphasynuclein level was seen in WA treated
worms, compared to control. Fluorescence intensity was quantiﬁed by ImageJ software . (B) Graphical representation of Aβ-induced toxicity in CL4176 worms. The worms were
cultivated at 15 °C fo r 48 h. At the 48 h time point, the temperature was up-shifted to 25 °C. The worms were scored at 18 h after the initiation of upshift and the sc oring was
continued in 1 h increments until all worms were paralyzed. WA signiﬁcantly reduces the percentage of CL4176 worms paralyzed as a result of Aβtoxicity. (C, D) Effect of WA on
neurotransmitter acetylcholine and acetylcholinesterase in wild-type worms. WA elevates both ACh (C) and AChE (D) levels however the acetylcholinesterase increase was found
insigniﬁcant. ***Pb0.001, **Pb0.01, *Pb0.05, ns not signiﬁcant. Scale bar, 30 μm.
Fig. 4. WA-mediated lifespan extension is independent of calorie restriction in C. elegans. (A) Effect of WA on pharyngeal pumping. WA failed to affect the rate of pharyngeal pumping.
(B) Survival curves of eat-2 mutant animals (n = 100) either untreated or treated with 5 μM WA throughout their adult life. WA augments the lifespan of eat-2 mutants.
51B.A. Akhoon et al. / Experimental Gerontology 78 (2016) 47–56
restraints from templates identiﬁed by multiple threading programs to
build full-length model using replica-exchange Monte-Carlo simula-
tions and reports a conﬁdence score (C-score) of the resultant model
to estimate the quality of the predicted models (Akhoon et al., 2014).
A C-score that ranges from −5 to 2 has been extensively tested in
large-scale benchmarking tests (Zhang and Skolnick, 2004; Zhang,
2008). In our case, we observed that C. elegans SGK-1 model was built
by I-TASSER using restraints from PDB templates viz., 2R5T, 4CRS,
3PFQ, 4L9I, 3A8X with a better C-score of −1.38. I-TASSER has another
scoring function, TM-score, to assess the structural similarity of model
and template protein structures. The score has been reported to over-
come the problem of RMSD which is sensitive to the local error. Irre-
spective of the protein length, a TM-score N0.5 indicates a model of
correct topology and a TM-score b0.17 means a random similarity.
The modeled protein (Fig. 5B) was found to have TM-score of 0.54 ±
0.15, indicating a better structural match of the C. elegans SGK-1 with
the templates. We also applied the ProSA-web service to check the over-
all model quality and to compare it with other experimentally deter-
mined protein structures of the same size. The z-score (a statistical
score for overall model quality) (Wiederstein and Sippl, 2007) of the
modeled SGK-1 protein was found to be −8.02, a value too close to
the experimentally resolved structures (Fig. 5C). Furthermore, overlap-
ping of the kinase domain of crystal structure 2R5T and the modeled
SGK-1 protein from C. elegans by CLICK (Nguyen et al., 2011) revealed
that these proteins were substantially similar in structure with a Cα
RMSD deviation of 1.64 A° (Fig. 5D). Such information was in favor as
signiﬁcant structural deviations could affect or modify the activity of
the protein (Srivastava et al., 2010). Many researchers have implement-
ed molecular docking approaches for the computational prediction of
receptor-ligand binding interactions/afﬁnities (Pant et al., 2014;
Baloria et al., 2012; Akhoon et al., 2010, 2011). To get additional in-
sights, we modeled the docking of WA into the SGK-1 model through
an automated molecular-docking procedure using the web-based
SwissDock program (Grosdidier et al., 2011). Docking carried out in
SwissDock using the ‘Accurate’parameter in otherwise default parame-
ters, with no region of interest deﬁned (blind docking) produced 30
clusters (docking poses) for WA. Analysis of all clusters showed a single
cluster (cluster no. 20) with the binding mode of WA similar to the ref-
erence structure (2R5T-WA complex modeled by idTarget) (Fig. 6),
showing that WA has preferred interaction (−8.09 kcal/mol) towards
C. elegans SGK-1 and form 3 hydrogen bonds with LYS143, SER145,
PHE146 amino acid residues.
Fig. 5. Modeling of Serum/glucocorticoid-regulated kinase 1 in C. elegans. (A) A kinasedomain sequencealignment ofSGKs from human andC. elegans. The conserved (identical and similar
amino acid residues) are highlighted in black and gray colors. (B) 3D structure of the modeled kinase domain of SGK-1 protein in C. elegans. (C) Investigation of the modeled SGK-1
structure using the ProSA-web service. The z-score of this model is −8.02, a value too close to the experimentally resolvedstructures. (D) Structural overlay of the homology model of
SGK-1 protein of C. elegans (yellow) with the SGK of H. sapiens (cyan) (PDB ID: 2R5T).
Lifespan of C. elegans mutants treated with Withanolide A.
Strains Treatment (μM) Mean lifespan (±SE) No. of worms (N) % change Pvalue Max. mean lifespan (±SE)
sgk-1 (ok538) Control 17.603 ± 0.500 126 22.23 ± 1.4
5μM 18.075 ± 0.455 119 2.68 Pb0.0001 23.66. ± 1.1
daf-16 (mgDf50) Control 11.098 ± 0.381 106 15.5 ± 1.3
5μM 10.849 ± 0.418 108 2.24 Pb0.0001 15 ± 1.1
skn-1 (zu67) Control 11.693 ± 0.326 114 16.5 ± 0.21
5μM 11.254 ± 0.306 113 3.75 P= 0.0001 16 ± 0.43
hsf-1 (sy441) Control 10.205 ± 0.256 105 15 ± 0.13
5μM 10.667 ± 0.305 111 4.5 Pb0.0001 15 ± 0.17
mev-1 (kn1) Control 9.151 ± 0.342 84 12.66 ± 0.26
5μM 10.571 ± 0.290 86 15.51 Pb0.0001 15.66 ± 0.53
52 B.A. Akhoon et al. / Experimental Gerontology 78 (2016) 47–56
3.4. WA-mediated lifespan extension is DAF-16 dependent
DAF-16/FoxO, a major transcriptional output of IIS pathway, is a pri-
mary mediator of increased longevity and stress resistance in C. elegans.
The negative regulation of DAF-16 (responsible for increased longevity
and stress resistance) is controlled by DAF-2 via PI3K-AKT/SGK signal-
ing pathway. SGK-1 acts parallel to AKT-1 and promotes C. elegans lon-
gevity in a DAF-16 dependent manner (Chen et al., 2013b). We asked
whether WA treatment has any effect on the lifespan of DAF-16 and
to address this possibility, we assayed the lifespan of worms harboring
the daf-16 (mgDf50) mutation. WA did not extend the lifespan of daf-
16 animals (Table 2; Fig. S1B), indicating that WA promotes longevity
in an IIS-dependent manner.
3.5. WA induces sod-3 expression, but did not inﬂuence the DAF-16/FoxO
To further examine the idea that WA act on a component of the IIS
pathway, DAF-16, to inﬂuence longevity, we examined the nuclear lo-
calization of DAF-16 using a GFP reporter strain (TJ356). However, we
did not ﬁnd nuclear-localized DAF-16::GFP fusions in the body of
worms after 72 h of treatment with WA. Since, it has been reported
that sgk-1 gain-of-function mutations did not inﬂuence the DAF-16/
FoxO subcellular localization (Chen et al., 2013b) and had different ef-
fects on the expression of distinct DAF-16/FoxO target genes, therefore,
we monitored GFP ﬂuorescence in sod-3 transgenic strain CF1553 (SOD-
3∷GFP) of C. elegans following WA treatment (72 h). The level of sod-3:
GFP induction in WA-treated worms was signiﬁcantly higher (Pb0.01)
than control worms (Fig. 7A). Our results reveal that WA extends
lifespan, possibly through indirect regulation of a subset of DAF-16/
FoxO targets without affecting the subcellular localization of DAF-16.
3.6. WA activate SKN-1 and HSF-1 transcriptional activity
SKN-1, a C. elegans Nrf family transcription factor and a major com-
ponent of IIS, controls numerous biological processes including stress
resistance and longevity (Hsu et al., 2003). Earlier reports show that
SGK-1 may promote longevity by regulating other proteins that
functionally and/or physically interact with DAF-16/FoxO, such as
SKN-1 (Tullet et al., 2008)andHSF-1(Hsu et al., 2003). Heat shock
factor 1 (HSF-1) overexpression increases heat resistance and extends
lifespan in a DAF-16 dependent manner (Tullet et al., 2008). Additional-
ly, it has also been suggested that daf-16 and hsf-1 function in a common
pathway to regulate longevity (Morley and Morimoto, 2004). Based on
these reports, we tested the effect of WA on the lifespan of SKN-1 and
HSF-1 animals. We did not observe lifespan extension upon WA
treatment in skn- (zu67) (Fig. S1C) and hsf-1 (sy441)mutants
(Fig. S1D), suggesting that WA-mediated longevity is IIS pathway
dependent. Similar to the regulation of DAF-16 by insulin signaling,
the IIS kinases phosphorylate SKN-1 and reduces IIS, which leads to con-
stitutive SKN-1 accumulation in intestinal nuclei whereby its target
gene activation modulate worm longevity (Tullet et al., 2008; Choe
et al., 2009). SKN-1, expressed in the intestine and in the ASI neurons
of worms, mediates the phase 2 stress response (An and Blackwell,
2003). GST-4 is a phase II enzyme and is tightly regulated by the SKN-
1 transcription factor in response to oxidative stress (Choe et al., 2009;
Kell et al., 2007; Landis and Murphy, 2010). To gain insights into the
Fig. 6. Dockedconformationof WA with SGK of C. elegansan d H.sapiens. WA is indicatedin CPK, blue colored.The putative interacting residuesof C. elegans SGK-1forming hydrogen bonds
with WA are labeled and shown as sticks.
Fig. 7. WA up-regulates stress-responsive genes in C. elegans. Fluorescent photomicrographs of (A) SOD-3::GFP in CF1553 and (B) GST-4:GFP transgene in the CL2166 strains. GFP
expression in WA-treated worms is higher thanthat in control worms. Quantiﬁcation of images was performed by ImageJ software. **Pb0.01. Scale bar, 30 μm.
53B.A. Akhoon et al. / Experimental Gerontology 78 (2016) 47–56
effects of WA on gst-4 activity, we tested the expression of a gst-4p: GFP
transgene in the CL2166 (dvIs19 [pAF15] (gst-4::GFP::NLS)) transgenic
worm strain treated with WA. We observed increased ﬂuorescence in
the pharynx region of CL2166 worms (Fig. 7B). All these observations
show that WA acts through the IIS pathway.
3.7. WA increases stress resistance and lowers ROS generation
Besides longevity, these components (DAF-16, SKN-1, and SGK-1) of
IIS pathway also play a key role in the regulation of stress resistance in
C. elegans (Tatar et al., 2003; Murphy et al., 2003; Wolff et al., 2006;
Chen et al., 2013b). It has also been frequently demonstrated that
there is a strong correlation between lifespan extension and resistance
to multiple environmental stresses (Kenyon, 2010). Enhanced stress
tolerance is a common characteristic of long-lived mutants and over-
expression of some antioxidant enzyme genes also extends the lifespan
(Lithgow et al., 1995; Johnson et al., 2000; Kim et al., 2010; Zhang et al.,
2013). Therefore, to address this, we explored anti-stress effects of WA
in C. elegans.
To examine whether WA could have any effect on antioxidant stress
resistance, we subjected N2 worms to oxidative stress induced by para-
quat, an intracellular reactive oxygen species (ROS) generator. We no-
ticed that WA-treated nematodes were more resistant than control
worms, to oxidative stress induced by growing the worms in the pres-
ence of paraquat, and lived markedly longer than control worms
(Fig. 8A). Since increased antioxidant activity can result from lower re-
active oxygen species (ROS) generation, also ROS impinges on so many
cellular processes, including lifespan; we chose to further study wheth-
er WA treatment has any effect on ROS levels. We assessed total ROS of
WA-treated and control worms, the observation showed that the total
ROS levels were signiﬁcantly decreased (Pb0.01) in treated worms
compared to control (Fig. 8B). Moreover, we also checked the effect of
WA on mev-1 (kn1) mutant that has a defective electron transport in
complex II. The mev-1 mutant is short lived and is highly susceptible to oxida-
tive damage due to higher levels of ROS (Adachi et al., 1998). WA treatment
extended the lifespan of mev-1 mutants by 15% (Fig. 8C), conﬁrming that the
antioxidant properties of WA are relevant in this context that explains the en-
hanced survival of WA-treated mev-1 mutants compared to control. Together,
these results indicate that it is the decreased level of ROS that consequently
lead to the prolonged survival of WA treated worms (Table 2).
It has been observed that many antioxidant compounds that
enhance oxidative stress resistance also confer thermotolerance. To ex-
plore whether WA has any effect on thermal resistance, we subjected
worms to a heat shock for three and a half hours at 35 °C. We observed
that treatment of adult C. elegans with WA lead to a high level of ther-
motolerance by 19% (Fig. 8D). Taken together, these results suggest
that WA reduces oxidative stress and render the worms resistant to
3.8. Genetic requirements for WA-mediated lifespan extension in C. elegans
In order to verify the ﬁndings further, we conducted gene expression
studies using quantitative real-time PCR after exposing the worms to
WA for 72 h. We found that WA enhances the expression of sgk-1 by
2.4 fold (Fig. 9). Our observations were in agreement with the recent
ﬁndings whereby sgk-1 gain-of-function promotes longevity (Chen
et al., 2013b) and contrast with the earlier reported lifespan extension
induced by sgk-1 RNAi (Hertweck et al., 2004). Since sgk-1 is a molecular
component of the C. elegans IIS pathway, we examined the effect of WA
on daf-16, a key component of the IIS cascade. From our qRT-PCR results,
we found that WA treatment leads to the induction of the daf-16 and en-
hances its expression by 1.9 fold (Fig. 9). We measured the expression
Fig. 8. WA increases stress resistance of C. elegans. (A) Oxidative stress induced by paraquat. The experiment was done in the no-FUdR condition and worms were exposed to paraquat
(10 mM) after their cease of progeny production (sixth day after L4). WA (5 μM) increases antioxidant defense in C. elegans and prolongs survival of worms under oxidative stress
conditions. (B) Relative formation of reactive oxygen species (ROS) after 72 h of exposure to 5 μM WA. Less ROS was produced in WA pretreated worms as assessed with the total ROS
dye indicator DCF-DA, compared to control. (C) WA increases the % mean survival (15%) of mev-1 (kn1) mutants that are highly susceptible to oxidative damage due to higher levels
of ROS generation. (D) Thermo survival of nematodes pre-exposed (72 h after L4) to 5 μM WA. WA enhances C. elegans resistance to thermal stress.
54 B.A. Akhoon et al. / Experimental Gerontology 78 (2016) 47–56
level of sod-3, a known DAF-16 target gene involved in stress responses.
Notably, our results data revealed that sod-3 expression levels were
almost 1.7 fold higher (Fig. 9) in WA-treated worms compared to con-
trol. To further ascertain whether DAF-16 is involved in WA-mediated
lifespan extension, the expression level of another direct target of
DAF-16 (fkh-9) was checked. We found higher fkh-9 expression levels
(2.3 fold) inWA-treated nematodes as compared to control worms. To-
gether, these ﬁndings support the notion that WA-mediated lifespan
extension is DAF-16 dependent. Our qRT-PCR experiments showed
that WA also upregulates the mRNA expression levels of another impor-
tant component of IIS, skn-1 and its target gene gst-4 by 2.7 and 3.1 folds
respectively. Moreover, we also found signiﬁcant upregulation of ther-
mal shock response factors, hsf-1 (2 fold) and its target gene hsp-16.2
(1.6 fold) in WA-treated N2 worms (Fig. 9).
In summary, our ﬁndings highlight the anti-aging and neuroprotec-
tive rolesof WA and demonstrate the involvement of IIS pathwayin WA
mediated lifespan extension in C. elegans. SGK1 activated by WA does
not affect DAF-16 translocation but the transcription of selective DAF-
16 targets. Further, WA enhances the transcriptional level of other im-
portant transcription factors suchas the antioxidant response transcrip-
tion factor SKN-1 and the heat-shock transcription factor HSF-1. Taken
together, these results suggest that SKN-1 and HSF-1 likely act in con-
cert with DAF-16 in WA-treated worms to increase neuron functionali-
ty, stress resistance, and lifespan. Our study demands more research on
this multifaceted natural product (WA) to gain a better understanding
of its therapeutic value for human beings.
The authors declare that they have no conﬂict of interest.
We are grateful to the Director, CSIR–CIMAP, Lucknow, India for his
kind support. BAA thanks Virendra Shukla, Shreesh Sammi and Laxmi
Rathor for their useful comments and assistance in the experimental
protocols. BAA also acknowledges useful discussions with Shailendra
K Gupta, Shishir K Gupta and Aakansha Pant. All the strains were obtain-
ed from the Caenorhabditis Genetic Center at the University of Minneso-
ta. BAA and ST were ﬁnancially supported by CSIR India (31/029(0251)/
2013-EMR-I and 31/34(157)/2013-EMR-I) through SRF grants.
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