ORIGINAL RESEARCH
published: 20 April 2017
doi: 10.3389/fnmol.2017.00101
Uncoupling the Trade-Off between
Somatic Proteostasis and
Reproduction in Caenorhabditis
elegans Models of Polyglutamine
Diseases
Netta Shemesh , Nadav Shai , Lana Meshnik, Rotem Katalan and Anat Ben-Zvi*
Department of Life Sciences, The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev,
Beer Sheva, Israel
Edited by:
Serena Carra,
University of Modena and Reggio
Emilia, Italy
Reviewed by:
Martin Lothar Duennwald,
University of Western Ontario,
Canada
Patricija Van Oosten-Hawle,
University of Leeds, UK
*Correspondence:
Anat Ben-Zvi
anatbz@bgu.ac.il
Received: 05 February 2017
Accepted: 24 March 2017
Published: 20 April 2017
Citation:
Shemesh N, Shai N, Meshnik L,
Katalan R and Ben-Zvi A
(2017) Uncoupling the Trade-Off
between Somatic Proteostasis and
Reproduction in Caenorhabditis
elegans Models of Polyglutamine
Diseases.
Front. Mol. Neurosci. 10:101.
doi: 10.3389/fnmol.2017.00101
Caenorhabditis elegans somatic protein homeostasis (proteostasis) is actively
remodeled at the onset of reproduction. This proteostatic collapse is regulated
cell-nonautonomously by signals from the reproductive system that transmit the
commitment to reproduction to somatic cells. Here, we asked whether the link
between the reproductive system and somatic proteostasis could be uncoupled by
activating downstream effectors in the gonadal longevity cascade. Specifically, we
examined whether over-expression of lipl-4 (lipl-4(oe)), a target gene of the gonadal
longevity pathway, or increase in arachidonic acid (AA) levels, associated with lipl4(oe), modulated proteostasis and reproduction. We found that lipl-4(oe) rescued
somatic proteostasis and postponed the onset of aggregation and toxicity in C. elegans
models of polyglutamine (polyQ) diseases. However, lipl-4(oe) also disrupted fatty acid
transport into developing oocytes and reduced reproductive success. In contrast, diet
supplementation of AA recapitulated lipl-4(oe)-mediated proteostasis enhancement in
wild type animals but did not affect the reproductive system. Thus, the gonadal longevity
pathway mediates a trade-off between somatic maintenance and reproduction, in part
by regulating the expression of genes, such as lipl-4, with inverse effects on somatic
maintenance and reproduction. We propose that AA could uncouple such germline to
soma crosstalk, with beneficial implications protein misfolding diseases.
Keywords: aging, arachidonic acid (AA), Caenorhabditis elegans, lipl-4, neurodegenerative diseases,
proteostasis, polyglutamine (polyQ) diseases, reproduction
INTRODUCTION
Aggregates or aggregation intermediates are strongly associated with the etiology of many
late-onset neurodegenerative diseases, including Huntington’s disease, amyotrophic lateral
sclerosis, Alzheimer’s disease and Parkinson’s disease (Davies et al., 1997; Nussbaum and
Polymeropoulos, 1997; Johnston et al., 2000; Glabe and Kayed, 2006; Labbadia and Morimoto,
2015a). In Huntington’s disease, for example, the expansion of polyglutamine (polyQ) repeats
is suggested to be the underlying cause of protein misfolding and gain-of-function toxicity (Orr
and Zoghbi, 2007). Expression of mutant Huntingtin containing expanded polyQ or even the
expanded glutamine tract alone is sufficient to cause cellular dysfunction in various animal models
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Uncoupling the Trade-Off between Somatic Proteostasis and Reproduction
improve proteostasis without impacting fecundity. We reasoned
that over-expression of genes down-regulated by the gonadal
longevity pathway would alleviate the protein damage associated
with age-dependent neurodegenerative diseases without affecting
reproduction.
Inhibition of germline proliferation activates DAF-16 that,
in turn, induces the expression of a large set of genes. One
of the genes up-regulated by DAF-16 is the lysosomal acid
lipase-encoding lipl-4 (Wang et al., 2008; Lapierre et al., 2011;
McCormick et al., 2012; Folick et al., 2015; Figure 1A). LIPL-4
itself modulates C. elegans lifespan, with its function resulting
in the activation of the nuclear hormone receptors NHR-49 and
NHR-80 and in the induced expression of autophagy/lipolysisrelated genes that modulate the fatty acid metabolism required
for lipl-4-dependent lifespan extension (Goudeau et al., 2011;
Lapierre et al., 2011; Ratnappan et al., 2014; Folick et al., 2015).
Moreover, LIPL-4 over-expression results in the enrichment of
long-chain fatty acids, including oleoylethanolamide (OEA), ω6 arachidonic acid (AA) and dihomo-γ-linolenic acid (DGLA)
and the ω-3 fatty acid eicosapentaenoic acid (EPA). Diet
supplementation of OEA activates NHR-49 and NHR-80, while
both DGLA and AA activate autophagy, extending lifespan
(O’Rourke et al., 2013; Folick et al., 2015; Figure 1A). Given that
lipl-4 is sufficient and required for lifespan extension (Wang
et al., 2008), we asked whether over-expression of LIPL-4 could
uncouple proteostasis from reproduction and delay the onset
of protein aggregation and toxicity. We found that LIPL-4
modulated the proteostatic switch upon transition to adulthood,
resulting in a delay in the onset of aggregation and toxicity in
C. elegans models of polyQ diseases. However, over-expression
of LIPL-4 negatively impacted fatty acid mobilization to the
developing oocytes and disrupted reproduction. Surprisingly,
diet supplementation of AA improved proteostasis without
disrupting reproduction. AA supplementation could, therefore,
uncouple somatic maintenance from reproduction, thereby
mimicking the beneficial effects of inhibiting germline
proliferation on somatic proteostasis without imposing a
cost on reproduction.
(Zoghbi and Botas, 2002; Sherman and Muchowski, 2003;
Voisine and Hart, 2004). Sequestration of misfolded proteins
into aggregates is also thought to be part of the cellular defense
response against the accumulation of misfolded proteins (Cohen
et al., 2006; Tyedmers et al., 2010).
Because chronic expression of misfolded proteins could
interfere with and compete for cellular quality control
machineries, it was suggested that disruption of protein
homeostasis (proteostasis) could be a primary cause for cellular
dysfunction and death in neurodegenerative diseases (Gidalevitz
et al., 2006; Yerbury et al., 2016). Indeed, expression of
aggregation-prone proteins in various model systems was shown
to interfere with cellular proteostasis, including disruption of
the clearance and folding machineries through competition with
other proteins substrates (Suhr et al., 2001; Kim et al., 2002;
Venkatraman et al., 2004; Bennett et al., 2007; Bilen and Bonini,
2007; Kitamura et al., 2014). This, in turn, can cause instability of
the cellular proteome, affecting the folding of unrelated proteins
(Gidalevitz et al., 2006, 2009; Olzscha et al., 2011; Eremenko
et al., 2013; Yu et al., 2014; Klabonski et al., 2016). Moreover,
changes in chaperone expression levels can disrupt cellular
proteostasis and induce an accumulation of damaged protein
(Blair et al., 2013; Guisbert et al., 2013; van Oosten-Hawle et al.,
2013; Frumkin et al., 2014; Bar-Lavan et al., 2016; Lechler et al.,
2017). Thus, maintaining proteostatic capacity is critical for
protecting cells from the protein damage associated with protein
misfolding diseases.
The challenge to cellular proteostasis is exacerbated in
aged individuals as proteostasis maintenance and effective
stress response activation decline with age (Taylor and Dillin,
2011; Shai et al., 2014; Labbadia and Morimoto, 2015a). In
Caenorhabditis elegans, proteostatic capacity was shown to
decline sharply following the onset of reproduction, thereby
accelerating the accumulation of polyQ aggregation and toxicity
(Ben-Zvi et al., 2009; Liu et al., 2011; Taylor and Dillin, 2013;
Labbadia and Morimoto, 2015b; Walther et al., 2015). This
decline was, in part, linked to remodeling of the chromatin
accessibility of stress gene promoters (Labbadia and Morimoto,
2015b; Merkwirth et al., 2016; Tian et al., 2016). Proteostasis
remodeling can be negated by the actions of the gonadal longevity
pathway (Lapierre et al., 2011; Vilchez et al., 2012; Shemesh
et al., 2013; Shai et al., 2014; Labbadia and Morimoto, 2015b).
Signals from the reproductive system can regulate somatic
proteostasis in response to inhibition of germline stem cell
(GSC) proliferation by activating several transcription factors,
including DAF-16/FOXO, SKN-1/Nrf and HSF-1, that are
required for proteostasis maintenance during adulthood, as well
as for extended lifespan (Hsin and Kenyon, 1999; Libina et al.,
2003; Berman and Kenyon, 2006; Antebi, 2012; Shemesh et al.,
2013; Steinbaugh et al., 2015; Wang et al., 2017). Thus, the
gonadal longevity pathway could determine the investment in
somatic maintenance in response to reproduction competence,
making the soma available for the demands of reproduction
(Kirkwood, 2005; Antebi, 2012; Shai et al., 2014). Given that
this trade-off is a regulated switch (Shemesh et al., 2013;
Labbadia and Morimoto, 2015b), we asked whether it is possible
to uncouple somatic maintenance from reproduction and
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MATERIALS AND METHODS
Nematodes and Growth Conditions
Nematodes were grown on nematode growth medium (NGM)
plates seeded with the Escherichia coli OP50-1 strain. Unless
otherwise stated, 30–80 embryos, laid at 15◦ C, were transferred
to fresh plates and grown at 25◦ C for the duration of an
experiment. The first day of adulthood (day 1) was set at 50 h
after temperature shift, before the onset of egg-laying. Animals
were moved every 1–2 days during the reproductive period to
avoid progeny contamination. Heat shock-treated animals were
discarded after scoring.
Statistical Analysis
Experiments were repeated at least three times and >15 animals
per experimental condition were scored. Data are presented as
means ± SEM. P values were calculated using the Wilcoxon
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Motility Assay
A total of 15–30 age-synchronized animals were used for each
assay. Day 2 adults were moved into M9 buffer and thrashing
rates were measured by counting body bends for 15 s. One body
bend was defined as a change in the direction of bending at midbody. Values are presented as bends per minute.
Stiff Body Paralysis Assay
Animals expressing mutant unc-52(ts) were grown at 25◦ C until
day 1 of adulthood, when they were shifted to 15◦ C and paralysis
was scored.
Thermo-Resistance Assay
Animals were picked at the indicated ages and transferred to a
24-well plate containing heat shock buffer (100 mM Tris-HCl,
pH 7.4, 17 mM NaCl and 1% cholesterol supplemented with
bacteria). These animals were then subjected to a 37◦ C heat shock
for 6 h. Heat shock buffer was supplemented with SYTOX orange
(Invitrogene), and animal survival was scored by monitoring dye
uptake, using a Leica M165 FC fluorescent stereoscope with a
TXR filter. Fluorescent animals were scored as dead (Karady
et al., 2013).
Heat Shock Treatment
A total of 30–60 age-synchronized animals grown at 25◦ C were
used for each assay. Plates were sealed and placed in a 37◦ C bath
for 90 min. Animals were frozen or fixed immediately following
stress.
FIGURE 1 | Over-expression of lipl-4 postpones the onset of
polyglutamine (polyQ) aggregation and toxicity. (A) Schematic drawing of
the gonadal longevity cascade regulating lipl-4 when germline stem cells
(GSCs) are arrested in C. elegans. (B) The number of bright foci scored on day
2 of adulthood in age-synchronized Q35m;lipl-4(oe) animals and their siblings
(n > 70). (C) Representative images of age-synchronized
Q35m;lipl-4(oe) animals and their siblings on day 2 of adulthood. Arrows
indicate foci. (D) Motility was scored in age-synchronized
Q35m;lipl-4(oe) animals and their siblings by determining the percentage of
paralyzed animals. (E) Motility was scored in age-synchronized Q40n;lipl-4(oe)
animals and their siblings by counting the number of body bends per minute
on day 2 of adulthood. Data was compared to age-matched sibling animals
examined under the same condition. ∗ Denotes P < 0.05, ∗∗ denotes P < 0.01.
RNA Levels
Twenty animals were collected per condition. RNA was
extracted using the TRIzol reagent (Invitrogene). For cDNA
synthesis, mRNA was reverse-transcribed using the iScript cDNA
Synthesis Kit (Bio-Rad). Quantitative PCR was performed on
a C1000 Thermal Cycler (Bio-Rad) with KAPA SYBER FAST
(KAPA BIOSYSTEMS; Shemesh et al., 2013).
Progeny Quantification
Individual age-synchronized animals of all tested condition (in
parallel) were allowed to lay eggs on fresh plates at 24–25◦ C.
Animals were moved every 24 h during the first 5 days of
adulthood (i.e., past the reproduction span) and the number of
offspring was scored 48–72 h later. The progeny of >25 animals
per genotype were scored.
Mann-Whitney rank sum test to compare two independent
populations. ∗ Denotes P < 0.05, ∗∗ denotes P < 0.01.
Foci Quantification
Age-synchronized animals expressing punc-54::Q35::yellow
fluorescent protein (YFP) (Q35m) were examined using a Leica
M165 FC fluorescent stereoscope with a YFP filter and the
number of bright foci was counted.
Oil-Red-O Staining
Animals were fixed and stained as previously described
(O’Rourke et al., 2009) and subsequently mounted and imaged
using a Leica DMIL microscope with a 10× 1.0 objective.
Paralysis Assay
DAPI Staining
A total of 15–30 age-synchronized animals were used for each
assay. Animals were grown at 25◦ C for the duration of the
experiment. Animals were moved every day, and paralyzed
animals were scored by monitoring their movement 5 min after
being transferred to a new plate. Animals that did not move were
scored as paralyzed (Karady et al., 2013).
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Gonad were dissected as in Colaiácovo et al. (2003). Gonads were
fixed and stained as previously described (Karady et al., 2013) and
subsequently mounted and imaged using an Olympus Fluoview
FV1000 confocal microscope through a 60× 1.0 numerical
aperture objective with a 405-nm line for excitation.
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Yolk Levels
and motility (Ben-Zvi et al., 2009; Shemesh et al., 2013;
Feldman et al., 2014). We, therefore, crossed unc-52(ts) mutant
animals with animals expressing lipl-4(oe) and compared the
motility of unc-52(ts);lipl-4(oe) with that of their siblings.
We found that 79 ± 3% of the unc-52(ts);lipl-4(oe) mutant
animals were motile on day 4 of adulthood, as opposing
to only 12 ± 5% of the unc-52(ts) siblings, suggesting
that lipl-4(oe) can improve proteostasis in adulthood
(Figure 2A).
Cellular stress responses, such as the heat shock response, are
diminished following the onset of reproduction in C. elegans.
This is reflected in the sharp decline in the ability of animals
to survive various stresses early in adulthood (Ben-Zvi et al.,
2009; Shemesh et al., 2013; Labbadia and Morimoto, 2015b).
While non-transgenic young adults survived well (81 ± 5%)
when challenged by heat shock on day 1 of adulthood,
only 9 ± 2.5% survived when first challenged on day 2 of
adulthood. In contrast, the survival rates of lipl-4(oe) animals
remained high, namely 78 ± 9.5% and 56.5 ± 4.7% on
days 1 and 2 of adulthood, respectively (Figure 2B). Improved
survival rates were also maintained later in life (Supplementary
Figure S2A). To determine whether the increased thermoresistance observed for lipl-4(oe) animals is associated with
the ability to activate the heat shock response, we compared
the stress-dependent induction of heat shock genes, whose
expression depends on HSF-1 (F44E5.4 and hsp-70) or on
HSF-1 and DAF-16 (hsp-16.11 and hsp-16.2). When animals
were challenged by heat shock on day 1 of adulthood,
F44E5.4, hsp-70, hsp-16.11 and hsp-16.2 mRNA expression
levels were strongly induced in both lipl-4(oe) animals and
their sibling. In contrast, when animals were heat shocked
on day 2 of adulthood, the induced mRNA levels of sibling
were 40%–60% lower than in lipl-4(oe) animals (Figures 2C,D,
Supplementary Figures S2B,C). A transcriptional reporter of
hsp-16.2, a reporter that regulates green fluorescent protein
(GFP) expression in a stress-dependent manner, showed similar
behavior. When animals were challenged by heat shock on
day 1 of adulthood, strong GFP fluorescence was detected
in intestinal cells of both lipl-4(oe) animals and their sibling
(90 ± 6% and 85 ± 2%, respectively). While the percentage
of animals showing high-induction levels of GFP following a
heat shock on day 3 of adulthood was maintained for lipl-4(oe),
this was not the case for sibling animals (82 ± 3% and
45 ± 5%, respectively; Figures 2E,F). These data suggest that
lipl-4 over-expression is sufficient to modulate stress survival and
stress response activation in somatic tissues after the onset of
reproduction.
Age-synchronized animals expressing pwIs98(YP170::tdimer2)
were fixed as in Karady et al. (2013) and imaged using a Leica
M165 FC fluorescent stereoscope with a TXR filter. Pictures were
analyzed using imageJ software (NIH).
Diet Supplementation of Fatty Acids
AA (50 µM dissolved in NP40) and control plates (containing
NP40) were prepared as previously described (Deline et al.,
2013). A total of 30–80 embryos were transferred to fresh plates
and grown at 25◦ C for the duration of an experiment.
RESULTS
lipl-4 Over-Expression Postponed the
Onset of PolyQ-Associated Toxicity
Extended polyQ stretches (>35Q) fused to fluorescent proteins
have been used in C. elegans as models for polyQ-associated
toxicity (Morley et al., 2002; Brignull et al., 2006). To ask
whether lipl-4 over-expression can modulate polyQ aggregation
and toxicity, we first crossed animals expressing YFP fused to
35 repeats of glutamine in body wall muscle (Q35m; Morley et al.,
2002) with animals expressing lipl-4 under the regulation of its
own promoter as an extra chromosomal array, lipl-4(oe). We
then monitored protein aggregation and toxicity and compared
the same properties with non-transgenic siblings of lipl-4(oe)
animals (siblings). By day 2 of adulthood, animals expressing
Q35m in the lipl-4(oe) background showed 40% less visible foci
than did their siblings expressing Q35m alone (Figures 1B,C
and Supplementary Figure S1). When we examined motility
as a measure of Q35m toxicity, we found that the onset of
Q35m-mediated paralysis was delayed in lipl-4(oe) animals.
By day 8 of adulthood, only 35 ± 11% of lipl-4(oe) animals
were paralyzed, as compared to 85 ± 4% of their siblings
(Figure 1D). Similar results were observed when we crossed
animals expressing cyan fluorescent protein fused to 40 repeats
of glutamine in neurons (Q40n; Brignull et al., 2006) with
animals over-expressing lipl-4. The motility of Q40n:lipl-4(oe)
animals, as measured by thrashing rate (body bends per min),
was ∼1.6-fold faster than that of their siblings (Figure 1E). Thus,
lipl-4 over-expression modulated the onset and progression of
protein aggregation and toxicity in C. elegans polyQ disease
models.
lipl-4 Over-Expression Was Sufficient to
Maintain Somatic Proteostasis
To extend our observations to other aspects of proteostatic
function, we next analyzed the impact of lipl-4(oe) on the
folding of metastable proteins. The ability of C. elegans to
maintain metastable proteins is dependent on the cellular
folding capacity. This ability becomes highly restricted early
in adulthood, resulting in age-dependent misfolding (BenZvi et al., 2009). A well-established protein folding reporter
is the product of a temperature-sensitive mutation in the
gene encoding perlecan, unc-52(e669, su250; unc-52(ts)), that
causes an age-dependent disruption of muscle organization
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lipl-4-Dependent Rescue of Proteostasis
Required the Reproductive System
In wild type animals, lipl-4 mRNA levels decline upon
transition to adulthood (Supplementary Figure S3A). lipl-4 is
regulated by DAF-16 as part of the gonadal longevity pathway,
one of several transcription factors that are activated upon
inhibition of GSCs proliferation (Hsin and Kenyon, 1999;
Wang et al., 2008; Antebi, 2012; McCormick et al., 2012).
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FIGURE 2 | Over-expression of lipl-4 maintains proteostasis in adulthood. (A) Stiff-body paralysis was scored for age-synchronized lipl-4(oe) animals and their
siblings on day 4 of adulthood. (B) Thermo-resistance was examined in age-synchronized lipl-4(oe) animals and their siblings. Animals were subjected to heat shock
(6 h at 37◦ C) at the indicated times and survival was assayed. (C,D) Quantification of F44E5.4 (C) and hsp-16.11 (D) mRNA levels from age-synchronized lipl-4(oe)
animals and their siblings following heat shock (90 min at 37◦ C). The data presented are normalized to treated siblings on day 1 of adulthood. (E) Representative
images of age-synchronized lipl-4(oe) animals and their siblings expressing phsp-16.2::green fluorescent protein (GFP) that were subjected to heat shock (90 min at
37◦ C) on day 3 of adulthood. (F) Heat shock gene induction was examined in age-synchronized lipl-4(oe) animals and their siblings expressing phsp-16.2::GFP.
Animals were subjected to heat shock (90 min at 37◦ C) and the percentage of animals expressing GFP was scored. Data was compared to age-matched sibling
animals examined under the same condition. ∗ Denotes P < 0.05, ∗∗ denotes P < 0.01.
LIPL-4 is expressed in the intestine, where it regulates
DAF-16 function (Wang et al., 2008; Lapierre et al., 2011;
Antebi, 2012; Folick et al., 2015). To determine whether the
effects of lipl-4 on somatic proteostasis require the reproductive
system, gonadogenesis-defective gon-2(q388), (gon-2) mutant
animals were crossed with lipl-4(oe)-expressing animals and
their ability to survive stress in adulthood was examined.
Although lipl-4(oe) animals maintained high heat shock survival
rates (Figure 2B), lipl-4(oe);gon-2 animals lost the ability to
mount an effective heat shock response by day 2 of adulthood
and their survival rates sharply declined from 82 ± 7% to
25.5 ± 6% (Figure 3D), similar to what was seen with
the wild type (Figure 2B) and non-transgenic gon-2 animals
(70 ± 7% to 24 ± 7%, Figure 3D). These data indicate that
lipl-4-dependent rescue of somatic proteostasis required the
gonad.
Accordingly, lipl-4 mRNA levels were strongly induced (7-fold)
in glp-1(e2141) (glp-1) germline proliferation mutant animals
(Supplementary Figure S3B; Wang et al., 2008; McCormick
et al., 2012). We, therefore, asked if down-regulation of
lipl-4 is sufficient to impact germline-dependent rescue of
proteostasis. For this, we examined whether lipl-4(RNAi) can
affect Q35m-associated toxicity and heat shock activation of
glp-1 mutant animals during adulthood. Q35m;glp-1 animals
show reduced aggregation and reduced paralysis during
adulthood (Shemesh et al., 2013). However, Q35m;glp-1 paralysis
was induced 3.5-fold when treated with lipl-4(RNAi), as
compared to Q35m;glp-1 animals treated with an empty
vector (EV) control (Figure 3A). Likewise, glp-1 animals
that were treated with lipl-4(RNAi) lost their ability to
induce an effective heat shock response. Induction of hsp16.2-dependent GFP on day 3 of adulthood was reduced
3-fold in lipl-4(RNAi)-treated glp-1 animals, as compared
to glp-1 animals treated with an EV control (Figure 3B).
We next ask whether over-expression of lipl-4 could further
enhance proteostasis in glp-1 animals. glp-1 mutant animals
were crossed with lipl-4(oe) animals and subjected to heat
shock. Heat shock survival rates of glp-1;lipl-4(oe) animals
and their siblings were unaffected by lipl-4(oe) (87.5 ± 4.8%
vs. 89.7 ± 4%, Figure 3C). Thus, LIPL-4 is required
for proteostatic remodeling downstream of the longevity
reproductive pathway.
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Over-Expression of lipl-4 Disrupted
Reproduction
We established that lipl-4(oe) could modulate proteostasis and
alleviate the protein damage associated with age-dependent
misfolding, and that such rescue required the gonad. We
next asked whether there is lipl-4(oe)-dependent impact on
reproduction. lipl-4(oe) animals were smaller than their siblings
but were not developmentally delayed. Therefore, we first
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FIGURE 3 | lipl-4-dependent rescue of somatic proteostasis requires the somatic gonad. (A) Motility was scored on day 6 of adulthood. Age-synchronized
glp-1 animals expressing Q35m were treated with lipl-4 or empty vector (EV) control RNAi and the percentage of paralyzed animals was determined. (B) Heat shock
gene induction was examined. Age-synchronized glp-1 animals expressing phsp-16.2::GFP were treated with lipl-4 or EV control RNAi and subjected to heat shock
(90 min at 37◦ C) on day 1 and day 3 of adulthood. The percentage of animals showing a fluorescent signal in the gut was scored. (C) Thermo-resistance was
examined in age-synchronized glp-1;lipl-4(oe) animals and their siblings. Animals were subjected to heat shock (6 h at 37◦ C) on day 2 of adulthood and survival was
assayed. (D) Thermo-resistance was examined in age-synchronized gon-2;lipl-4(oe) animals and their siblings. Animals were subjected to heat shock (6 h at 37◦ C)
on day 1 and day 2 of adulthoods and survival was assayed. For (A,B) data was compared to EV control. For (C,D) data was compared to age-matched sibling
animals examined under the same condition. (n.s.) no statistical significance, ∗∗ denotes P < 0.01.
examined the effect of lipl-4(oe) on fecundity by determining
the number of progeny lipl-4(oe) and their siblings produced.
Notably, lipl-4(oe) had ∼40% less offspring than did their
siblings (127 ± 16 and 211 ± 12 offspring, respectively;
p < 0.0001; Figure 4A). This strong reduction in progeny
number was not due to arrest of germline proliferation, since
lipl-4(oe) and their siblings contained proliferating germ cells
(Supplementary Figure S4).
The gonadal longevity cascade modulates fatty acids
metabolism and affects the levels of various fatty acids and
their distribution in the nematode body (Wang et al., 2008;
Goudeau et al., 2011; Lapierre et al., 2011; McCormick et al.,
2012; Lapierre et al., 2013; Ratnappan et al., 2014; Steinbaugh
et al., 2015). For example, when germline proliferation is
inhibited, fatty acids accumulate in the body cavity, where
they activate the transcription of genes required for fatty acid
mobilization (Lynn et al., 2015; Steinbaugh et al., 2015). To ask
how lipl-4(oe) could negatively impact reproduction, we first
monitored total fat stores in lipl-4(oe) animals and their siblings
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using Oil-Red-O staining. Most of the lipl-4(oe) animals (83%)
showed a strong reduction in lipid levels, with lipids being mostly
located in the developing oocytes (Figure 4B and Supplementary
Figure S5).
Levels of vitellogenic proteins are also affected by
reproductive signals (DePina et al., 2011). Moreover,
over-expression of vitellogenic proteins was previously shown
to reduce lipl-4 levels (Seah et al., 2016). To further elucidate
the effect of LIPL-4 on reproduction, we next examined
transport of lipids from the intestine to the germline by
monitoring the levels and localization of the vitellogenic protein
VIT-2 tagged with tdimer2 (VIT-2::tdimer2). Coinciding with
diminished fat stores, VIT-2 levels were reduced by ∼60% in
lipl-4(oe) animals, as compared to their siblings, on the day 2
of adulthood (Figures 4C,D). Fatty acid uptake by oocytes
was, therefore, disrupted in lipl-4(oe) animals, which could, in
turn, impact progeny production. Thus, while enhanced lipl-4
levels improved somatic proteostasis, they were detrimental to
reproduction.
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FIGURE 4 | Over-expression of lipl-4 disrupts reproduction. (A) Progeny numbers were scored in age-synchronized lipl-4(oe) animals and their siblings.
(B) Representative images of total fat stores. Age-synchronized lipl-4(oe) animals and their siblings were collected on day 2 of adulthood and total fat stores were
analyzed using Oil-Red-O staining. Values indicate the percentage of animals showing the presented staining pattern (n > 18). (C) Representative images of
age-synchronized lipl-4(oe) animals and their siblings carrying vit-2::tdimer2 on day 2 of adulthood. (D) VIT-2::tdimer2 levels were quantified from images using the
ImageJ software and normalized to siblings levels. Data was compared to age-matched sibling animals examined under the same condition. ∗∗ Denotes P < 0.01.
AA Uncouples Somatic Proteostasis from
the Reproductive System
63 ± 4%, respectively). Likewise, the percentage of sibling
animals showing induction of hsp-16.2-dependent GFP upon
heat shock was increased in animals supplemented with AA,
as compared to controls. However, diet supplementation of
AA to lipl-4(oe) animals did not further increase hsp-16.2dependent GFP expression following heat shock (Supplementary
Figure S6B). This was not due to AA-dependent increases in
lipl-4 levels, as diet supplementation of AA did not affect lipl4 mRNA levels (Supplementary Figure S7). These data thus
support a role for AA in proteostasis remodeling.
We then tested whether diet supplementation of AA
could affect Q35m aggregation. A ∼30% reduction in foci
number was observed in Q35m siblings supplemented with AA.
Whereas only a slight reduction in foci formation was observed
in Q35m;lipl-4(oe) animals supplemented with AA and this
reduction was not significantly different from Q35m siblings
supplemented with AA (Figure 5B). Similar behavior was
observed for Q35m- and Q40n-associated toxicity. Q35m;lipl4(oe), Q40n;lipl-4(oe) animals and their siblings were grown on
control or AA-supplemented plates and motility was monitored.
Diet supplementation of AA to siblings expressing Q35m or
Q40n improved their thrashing rates by 1.4- and 1.8-fold,
respectively, compared to animals grown on control plates. In
contrast, diet supplementation of AA did not further improve
the motility of Q35m;lipl-4(oe) or Q40n;lipl-4(oe) animals
Over-expression of lipl-4 did not uncouple the trade-off
between somatic maintenance and reproduction. Metabolite and
lipid profiling of lipl-4(oe) identified accumulation of several
long-chain fatty acids, including OEA, EPA, DGLA and AA
(O’Rourke et al., 2013; Folick et al., 2015). While DGLA
causes GSC death and complete sterility, EPA does not affect
fertility (Watts and Browse, 2006). We, therefore, asked whether
modulating lipl-4(oe) downstream products could potentially
improve proteostasis without disrupting reproduction. We
first examined whether diet supplementation of OEA, EPA
or AA could regulate heat shock survival, as a simple
readout of proteostasis. Embryos were transferred to fatty
acid-supplemented or control plates (containing NP40) and
their survival was examined on day 2 of adulthood. Diet
supplementation of OEA or EPA had no significant effect on
animal survival following heat shock on day 2 of adulthood
(Supplementary Figure S6A). In contrast, when non-transgenic
siblings were supplemented with AA, heat shock survival rates
were significantly improved on day 2 of adulthood, as compared
to siblings grown on control plates (56 + 5% and 25 + 3%,
respectively; Figure 5A). Diet supplementation of AA to lipl4(oe) animals did not further improve survival, as compared
to lipl-4(oe) animals grown on control plates (65 + 4% and
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Uncoupling the Trade-Off between Somatic Proteostasis and Reproduction
FIGURE 5 | Diet supplementation of arachidonic acid (AA) could mimic the effects of lipl-4(oe) on proteostasis. (A) Thermo-resistance was examined in
age-synchronized lipl-4(oe) animals and their siblings. Animals were grown on control (NP40) or AA-supplemented nematode growth medium (NGM) plates,
subjected to heat shock (6 h at 37◦ C) and survival was assayed on day 2 of adulthood. (B) The number of bright foci was scored in age-synchronized
Q35m;lipl-4(oe) animals and their siblings (n > 40). Animals were grown on control (NP40) or AA-supplemented NGM plates and the number of foci was counted on
day 2 of adulthood. (C,D) Motility was scored in age-synchronized Q35m;lipl-4(oe), Q40n;lipl-4(oe) animals and their siblings. Animals were grown on control (NP40)
or AA-supplemented NGM plates and the number of body bends per minute was scored on day 2 of adulthood. Data was compared to control plates (NP40). (n.s.)
no statistical significance, ∗ denotes P < 0.05, ∗∗ denotes P < 0.01.
indicate that diet supplementation of AA can uncouple somatic
proteostasis from the reproductive system.
Any benefits derived from AA supplementation in the soma
came with no apparent cost to reproduction. We, therefore,
asked whether AA-dependent rescue of proteostasis required
the reproductive system. To test this directly, we examined
the effect of AA supplementation on gonad-less animals by
monitoring heat shock survival of gon-2 animals grown on
control or AA-supplemented plates. While the survival of gon-2
animals grown on control plates was low on day 2 of adulthood
(43 ± 4%; Shemesh et al., 2013), survival rates of gon-2
animals grown on AA-supplemented plates were surprisingly
high (63.7 ± 4.4%, Figure 6C), similar to AA-supplemented
siblings (Figure 5A). Thus, as opposed to lipl-4, the impact of
AA on somatic proteostasis does not require the reproductive
system. Rather, a small increase in AA levels was sufficient to
improved proteostasis in adulthood with no apparent cost to
reproduction.
(Figures 5C,D). Thus, diet supplementation with AA mimics
the effects of lipl-4(oe) on proteostasis, suggesting that the
increased AA level, observed in lipl-4(oe) animals is sufficient to
remodel proteostasis in adulthood, likely via agents downstream
of LIPL-4.
Given that AA mimics the effects of lipl-4(oe) on somatic
proteostasis, we next asked whether AA supplementation has
a negative impact on the reproductive system, similar to lipl4(oe). For this, we examined the effect of AA on progeny
production by lipl-4(oe) animals and their siblings. A previous
study showed that only accumulation of high levels (>20%
of their lipids) of AA affected fertility (Watts and Browse,
2006). We, therefore, asked whether diet supplementation of
AA at a concentration that improved proteostasis (0.05 mM
AA) affected reproduction. AA diet supplementation had no
significant effect on the number of progeny produced by lipl4(oe) siblings. Specifically, animals grown on control plates
produced 210 ± 14 offspring, while AA-supplemented animals
produced 196 ± 13 offspring. Likewise, lipl-4(oe) animals grown
on control or AA produced similar numbers of offspring
(160 ± 12 and 145 ± 10, respectively; Figure 6A). Moreover,
diet supplementation of AA to lipl-4(oe) animals or their siblings
did not affect fatty acid levels or localization, as monitored by
Oil-Red-O staining or VIT-2 levels. Most siblings grown on
either AA-supplemented or control plates (88%) showed normal
fatty acid stores, while lipl-4(oe) animals showed low fatty acid
levels localized in oocytes, regardless of diet (Figures 4B, 6B).
Moreover, AA supplementation to wild type animals did not
affect VIT-2 levels (Supplementary Figure S8). These data
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DISCUSSION
Complex signals control the proteostatic switch employed at the
onset of reproduction to connect somatic maintenance to the
reproductive system (Antebi, 2012). In wild type animals, the
onset of egg laying is linked to a sharp decline in the ability
to activate stress responses and maintain proteostasis mediated
by signals from the gonad (Ben-Zvi et al., 2009; Liu et al.,
2011; Taylor and Dillin, 2013; Labbadia and Morimoto, 2015b;
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Shemesh et al.
Uncoupling the Trade-Off between Somatic Proteostasis and Reproduction
FIGURE 6 | Reproduction is unaffected by diet supplementation of AA. (A) Progeny numbers were scored in age-synchronized lipl-4(oe) animals and their
siblings grown on control (NP40) or AA-supplemented NGM plates. (B) Representative images of total fat stores. Age-synchronized lipl-4(oe) animals and their
siblings were grown on control (NP40) or AA-supplemented NGM plates and collected on day 2 of adulthood. Total fat stores were analyzed using Oil-Red-O
staining. Values indicate the percentage of animals showing the presented staining pattern (n > 14). (C) Thermo-resistance was examined in age-synchronized
gon-2 animals. Animals grown on control (NP40) or AA-supplemented NGM plates were subjected to heat shock (6 h at 37◦ C) on day 2 of adulthood and survival
was assayed. Data was compared to control plates (NP40). (n.s.) no statistical significance, ∗ denotes P < 0.05.
LIPL-4 has Inverse Effects on
Reproduction and Somatic Maintenance
Walther et al., 2015). The inhibition of germline proliferation,
however, activates the gonadal longevity pathway, resulting in
enhanced somatic proteostasis (Shemesh et al., 2013). Here, we
showed that this proteostatic switch can be activated upon overexpression of LIPL-4 or AA enrichment, both of which act
downstream of the gonadal longevity cascade (Wang et al., 2008;
Lapierre et al., 2011; O’Rourke et al., 2013). We propose that
the observed decline in lipl-4 levels upon transition to adulthood
is associated with reduced proteostatic capacity but with
improved progeny production (Figure 7A). Over-expression of
lipl-4 thus restores somatic proteostasis, yet disrupts fatty acid
accumulation in growing oocytes and thus, progeny production
(Figure 7B). Because AA diet supplementation resulted in
improved somatic proteostasis but did not affect or require
the reproductive system, we suggest that the adverse effects of
LIPL-4 function on reproduction can be bypassed and that its
beneficial effects on somatic proteostasis can be retained by
increased AA levels (Figure 7C). The somatic proteostasis rescue
mediated by AA could thus delay the onset of toxicity and
aggregation in C. elegans models of polyQ diseases without cost
to reproduction.
The antagonistic pleiotropy theory suggests that genes that
have beneficial effects early in life could be selected for, even
if they have detrimental effects later in life (Williams, 1957).
Given how LIPL-4 has inverse effects on progeny production
and proteostasis, we propose that lipl-4 represents such a
gene, as elevated levels of lipl-4, although beneficial to the
organism later in life, reduce reproductive success. The actions
of LIPL-4 resulted in a trade-off between somatic maintenance
and reproduction (Kirkwood, 2005). However, LIPL-4 function
activates several different pathways that differ in terms of their
effects on somatic proteostasis and reproduction. For example,
lipl-4(oe) results in elevated levels of OEA that, in turn, activated
NHR-49 and NHR-80. OEA did not affect proteostasis. However,
NHR-49 and NHR-80 inversely regulated lipl-4 and vitellogenic
gene expression (Seah et al., 2016; Figure 4), suggesting that
OEA could regulate lipid resource allocation from yolk-bound
lipoprotein to intestinal lipid droplets. In contrast, elevated levels
of AA did not affect lipid distribution (Figure 6B) but rather were
FIGURE 7 | Signals from the reproductive system regulate somatic proteostasis. (A) The onset of reproduction regulates proteostasis cell-nonautonomously
(1). This activates the gonadal longevity pathway that down regulates lipl-4 expression at the onset to reproduction (2). When embryo production begins proteostasis
sharply declines. This is associated with decreased lipl-4 and AA levels (3). (B) Over-expression of lipl-4 (1) restores somatic proteostasis and enhances AA levels but
also disrupts fatty acid mobilization to developing oocytes and thus disrupt reproduction (2). (C) Diet supplementation of AA (1) restores somatic proteostasis (2), like
lipl-4 over-expression, but did not disrupt fatty acid mobilization or reproduction.
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Uncoupling the Trade-Off between Somatic Proteostasis and Reproduction
shown to induce autophagy in C. elegans and mammalian cells
and extend C. elegans lifespan (O’Rourke et al., 2013). Moreover,
lipl-4(oe) also resulted in a significant increase in DGLA (Folick
et al., 2015). While DGLA, like AA, can induce autophagy and
increase lifespan (O’Rourke et al., 2013), diet supplementation
of DGLA resulted in a complete loss of germ cells and sterility,
mediated by DGLA-derived epoxides (Watts and Browse, 2006;
Deline et al., 2015). Finally, EPA did not affect proteostasis,
autophagy, lifespan or reproduction in C. elegans (Watts and
Browse, 2006; O’Rourke et al., 2013). Thus, the pleiotropic
effects of LIPL-4 on reproduction and somatic maintenance are
due to activation of different downstream pathways. Targeting
metabolites that affect proteostasis but that have no or only
mild effects on reproduction could artificially uncouple the two
processes.
our findings could have consequences for the treatment of
neurodegenerative diseases. The question, however, remains as
to whether this proteostatic switch is conserved. While it is clear
that proteostasis is limited in adults across species, currently
there are only indications that the proteostatic switch itself is
conserved (Shai et al., 2014; Labbadia and Morimoto, 2015b).
These include the impact of the reproductive system on aging
in flies and mice and the conservation of many of the players
in these signaling pathways, including LIPL-4 (Flatt et al., 2008;
Mason et al., 2009; Shai et al., 2014; Folick et al., 2015; Labbadia
and Morimoto, 2015b). By finding ways to uncouple proteostatic
collapse from the reproductive system, we might be able to
target multiple age-dependent protein misfolding diseases with
different etiologies but with similar underlying biology.
AUTHOR CONTRIBUTIONS
AA-Modulated Proteostasis Collapse
How does LIPL-4 impact proteostasis? Diet supplementation of
AA did not affect lipid distribution (Figure 6B) but was sufficient
to recapitulate the impact of lipl-4(oe) on proteostasis (Figure 5
and Supplementary Figure S6B). Thus, although redistribution
of lipids could activate several transcriptional programs that
affect stress response pathways (Steinbaugh et al., 2015), it is
less likely that lipid redistribution impacts somatic proteostasis.
Accumulation of polyunsaturated fatty acids, such AA and
DGLA, induce autophagy. Such induction itself could enhance
proteostasis, for example, by removal of protein aggregates
(Vilchez et al., 2014). However, AA could potentially activate
other somatic maintenance pathways. For instance, exposure
of HeLa cells to AA was shown to activate HSF1 and induce
the expression of heat shock genes (Jurivich et al., 1994;
Horikawa and Sakamoto, 2009; Balogh et al., 2013). Moreover,
AA could be further metabolized to eicosanoids, including
various prostaglandins that could also induce the heat shock
response and affect the expression of heat shock genes in
human cells (Amici et al., 1992; Balogh et al., 2013). Thus, AA
supplementation has the potential to modulate proteostasis, a
function that could also be conserved in humans. Determining
when AA is required for proteostasis remodeling might help us
elucidate the function this molecule serves.
Regulation of the proteostatic switch has a strong impact on
the maintenance of somatic tissues (Shai et al., 2014). Given
that this switch can impact the onset of protein aggregation,
NShemesh and AB-Z designed the experiments and wrote the
manuscript. NShemesh, NShai, LM and RK performed the
experiments, analyzed the data and interpreted the results.
NShemesh, NShai, LM, RK and AB-Z revised the text.
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This research was supported by a grant from the Legacy Heritage
Biomedical Science Partnership Program of the Israel Science
Foundation (grant No. 804/13; https://www.isf.org.il/#/) and
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Conflict of Interest Statement: The authors declare that the research was
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