Anders Olsen, Matthew S. Gill Eds. Ageing Lessons From C. Elegans PDF
Anders Olsen, Matthew S. Gill Eds. Ageing Lessons From C. Elegans PDF
Anders Olsen, Matthew S. Gill Eds. Ageing Lessons From C. Elegans PDF
Anders Olsen
Matthew S. Gill Editors
Ageing:
Lessons from
C. elegans
Healthy Ageing and Longevity
Volume 5
Series editor
Suresh I.S. Rattan, Aarhus, Denmark
More information about this series at http://www.springer.com/series/13277
Anders Olsen • Matthew S. Gill
Editors
1 Introduction.............................................................................................. 1
Anders Olsen and Matthew S. Gill
2 Effects of Ageing on the Basic Biology and Anatomy
of C. elegans.............................................................................................. 9
Laura A. Herndon, Catherine A. Wolkow, Monica Driscoll,
and David H. Hall
3 Dauer Formation and Ageing................................................................. 41
Pedro Reis-Rodrigues, Kailiang Jia, and Matthew S. Gill
4 Longevity Regulation by Insulin/IGF-1 Signalling............................... 63
Seon Woo A. An, Murat Artan, Sangsoon Park, Ozlem Altintas,
and Seung-Jae V. Lee
5 Mitochondrial Longevity Pathways....................................................... 83
Alfonso Schiavi and Natascia Ventura
6 Influences of Germline Cells on Organismal Lifespan
and Healthspan........................................................................................ 109
Francis R.G. Amrit and Arjumand Ghazi
7 Reproductive Ageing................................................................................ 137
Cheng Shi and Coleen T. Murphy
8 Nervous System Ageing........................................................................... 163
Claire Bénard and Maria Doitsidou
9 Stress Response Pathways....................................................................... 191
Dana L. Miller, Joseph Horsman, and Frazer I. Heinis
10 Oxidative Stress........................................................................................ 219
Bart P. Braeckman, Patricia Back, and Filip Matthijssens
11 Genome Stability and Ageing.................................................................. 245
Aditi U. Gurkar, Matthew S. Gill, and Laura J. Niedernhofer
v
vi Contents
Index.................................................................................................................. 437
Chapter 1
Introduction
Anders Olsen and Matthew S. Gill
Abstract Advances in healthcare over the last century have led to an increase in
global life expectancy. In 2015, the fraction of the world population over the age of
65 was estimated at 8.5 % and is predicted to rise to 16.7 % by 2050 [1]. Unfortunately,
with every advancing decade of life the probability of developing one or more of the
chronic debilitating conditions that we associate with ageing increases dramatically.
This in turn leads to an extended period of late life morbidity and a deteriorating
quality of life that will have huge consequences for individuals and their families.
Ageing is the primary risk factor for a number of diseases and chronic condi-
tions. Therefore, slowing the rate of ageing would be an effective approach to com-
press the period of late life morbidity and increase the healthy years of life. This
would also provide an opportunity to simultaneously prevent or delay all age-
associated chronic conditions.
The pursuit of interventions that slow the rate of ageing is not new to modern
science. However, the last 40 years have seen a revolution in the field of ageing
research and we are much closer to the goal of improving human healthspan [2]. We
now realize that ageing is not an inevitable, intractable problem but rather it is mal-
leable and the rate of ageing can be manipulated genetically, environmentally as
well as chemically. Some of the dramatic advances in our understanding of the age-
ing process stem from seminal discoveries in the nematode C. elegans
(C. elegans).
A. Olsen (*)
Department of Molecular Biology and Genetics, Aarhus University,
Gustav Wieds Vej 10C, 8000-DK, Aarhus, Denmark
e-mail: ano@mbg.au.dk
M.S. Gill (*)
Department of Metabolism & Aging, The Scripps Research Institute, Jupiter, FL, USA
e-mail: mgill@scripps.edu
It was the work of Sydney Brenner at the University of Cambridge in the late 1960s
and early 1970s that laid the groundwork for establishing C. elegans as a powerful
genetic model system. Brenner was looking for an organism which could be used to
study how genes specify organismal development, particularly development of the
nervous system. In a landmark paper in 1974, he described how genetic screens
could be used to identify mutants with visible phenotypes, and how genetic analysis
could map these traits to single genes [3]. In the years that followed, C. elegans
developed into a powerful and tractable genetic model system, alongside the well-
established fly and yeast models.
As Brenner’s former postdocs and trainees established their own independent
laboratories, the C. elegans field diversified to examine other phenotypes including
apoptosis [4], sex determination [5] and germ line biology [6, 7]. The complete
embryonic and larval cell lineage for both hermaphrodites and males provided the
blueprint for C. elegans development [8–10] and in the late 1990s C. elegans became
the first multicellular organism to have its genome sequenced [11]. Other techno-
logical advances, such as the use of green fluorescent protein (GFP) to detect gene
expression and protein localization in vivo [12] and the discovery of RNA interfer-
ence (RNAi) as a means of knocking down gene function [13], continued to add to
the utility of the worm as a model system. Indeed, in the last 20 years four Nobel
Prizes have been awarded for discoveries that stemmed from the use of C. elegans.
The short lifespan and tractable genetics of C. elegans also made it an attractive
system in which to investigate the environmental and genetic basis of lifespan. In
the late 1970s, Michael Klass demonstrated that lifespan could be manipulated by
changing the temperature of cultivation and that dietary restriction could lead to
increased longevity [14]. He was also the first person to publish a genetic screen to
identify long-lived worms that he called Age mutants [15]. This initial screen identi-
fied a number of mutants that were surmised to extend lifespan via dietary restric-
tion, based upon their inability to take up an appropriate amount of food. Another
mutant, age-1 appeared wild type in terms of development and fertility and did not
appear to be dietary restricted. In parallel, other researchers started using C. elegans
to identify genes involved in ageing. The discovery in 1993 that the dauer constitu-
tive mutant daf-2 was long-lived [16] indicated that longevity mutants could be
identified using surrogate phenotypes and that epistasis approaches could be used to
define longevity pathways. In the years that followed, a number of other long-lived
mutants were identified via a number of different approaches ([17–20]).
In the late 1990s, cloning of C. elegans longevity genes revealed that some of the
long-lived C. elegans mutants had defects in an insulin/insulin-like growth factor
signalling (IIS) pathway [21, 22]. In a short space of time it was subsequently
1 Introduction 3
discovered that mouse and fly mutants that affected the IIS pathway also showed
increased longevity, illustrating the evolutionary conservation of pathways that
affect ageing. This moved C. elegans ageing research from a niche area of nematode
biology into the broader scientific community.
The sequencing of the C. elegans genome [11] and the development of RNAi by
feeding [23] heralded a new era of reverse genetic approaches that greatly facilitated
the identification of longevity genes. It was not long after the development of the
first whole genome RNAi library by the Ahringer Lab [24, 25] that large scale
reverse genetic screens for ageing genes began to appear [26, 27], dramatically
increasing the number of genes involved with lifespan determination.
At the time of publication of this book over a 1000 C. elegans genes have been
reported to influence lifespan via loss of function or over-expression [28] and many
more have been implicated through gene expression studies. Lifespan of wild type
worms grown at 20 °C is typically 20–25 days and many of the early interventions
lead to a doubling or tripling of lifespan. Null mutations in age-1 confer the largest
increase in lifespan for single gene mutants, with maximum lifespans of more than
250 days [29]. Combinations of multiple longevity mutants can also lead to extreme
longevity [18, 20, 30, 31]. Many other interventions have much more modest effects
on lifespan, often in the region of 20–40 %. It is also important to note that despite
the fact that lifespan is measured in isogenic populations under controlled environ-
mental conditions, there is substantial variation in C. elegans lifespan both within a
population and between biological replicates. It is therefore critical that replicate
lifespan studies are performed.
Most C. elegans ageing studies have used and continue to use survival as the
primary measurement outcome. This metric simply measures the fraction of a syn-
chronized sample population that is alive on any given day. It is important to note
that increased survival does not necessarily equate to changes in the rate of ageing.
Early studies of C. elegans ageing took advantage of the ease of growing large num-
bers of worms to carry out mortality rate analyses [32]. However, in recent years
there has been a trend away from this approach. The development of automated
methods of lifespan assessment [33] provides a new opportunity to carry out such
analyses but will require widespread implementation throughout the C. elegans age-
ing research community.
In parallel, there has been a move towards developing other measures of ageing
in the worm that are not focused solely on survival. These measurements attempt to
provide a metric of the health of the animals and include movement [34, 35], pha-
ryngeal pumping [36] and autofluorescence [37]. The use of these metrics has con-
tributed to the realization that increased lifespan is not always paralleled by
increased health and, conversely, some interventions increase healthspan without
extending lifespan.
4 A. Olsen and M.S. Gill
Using the terminology of George Martin, many of these genes are likely to influence
lifespan “privately”, that is they are specific to the nematode, while others possibly
affect “public” mechanisms of ageing by affecting evolutionary conserved signal-
ling pathways [38].
The IIS pathway is perhaps the best studied pathway in C. elegans with respect
to ageing (see Chap. 4). Reduced IIS signalling in flies and mice leads to lifespan
extension and suggests that this pathway represents a public mechanism of ageing
[39]. However, there remains some controversy as to just how relevant manipula-
tions of this pathway are to human ageing [40]. Likewise, dietary restriction (DR)
remains one of the most robust means of extending lifespan in many different organ-
isms [41, 42] yet its efficacy in extending lifespan in humans remains unproven
[43]. The role of other longevity mechanisms that have been well studied in worms
and other model systems, such as autophagy (see Chap. 15) and protein translation
(see Chap. 13), still require extensive investigation to confirm human relevance.
The C. elegans ageing field has moved away from the gene discovery approach that
defined the 1990s and 2000s. We are now in more of a consolidation phase, in which
the strengths of the system for genetic analysis are being employed to understand
the mechanism of action of genes that influence ageing. A new era of discovery has
taken shape in the last 5–10 years as more laboratories have started using C. elegans
to identify drugs and chemicals that have the potential to act as therapeutic interven-
tions in the ageing process [44]. As more reports of drug-based approaches to slow-
ing ageing in mammals emerge, the utility of C. elegans in understanding the
molecular mechanisms that underpin longevity interventions is likely to be demon-
strated again. In putting together this book on C. elegans and its contribution to our
understanding of ageing, with a special emphasis on the relevance to human ageing,
we have tried to focus on the physiological, molecular and biochemical mechanisms
that underpin C. elegans longevity.
Despite the vast number of studies of ageing in C. elegans we still do not really
understand what worms die of and we have a limited appreciation of the physiologi-
cal changes that accompany ageing. Chap. 2 provides a much needed review of the
anatomical changes that take place in the ageing worm.
On the face of it, dauer formation (Chap. 3) is a specialized adaptation of the
worm to deteriorating environmental conditions but understanding the physiological
mechanisms and signalling pathways that confer extended survival in the dauer has
been instrumental in understanding the ageing process.
1 Introduction 5
The IIS pathway is perhaps the best studied longevity pathway in C. elegans and
is reviewed in Chap. 4. The role of mitochondria and the germline in lifespan deter-
mination are covered in Chaps. 5 and 6, respectively.
Much of the focus of C. elegans ageing research has been on organismal ageing,
but it is becoming clear that there are genetic determinants of tissue and organ age-
ing that have relevance to humans. Chap. 7 focuses on the emerging field of repro-
ductive ageing in C. elegans, while Chap. 8 considers the neurobiology of ageing.
In the latter half of the book, the focus shifts towards molecular mechanisms that
underpin many of the longevity interventions that have been identified in worms.
Thus the role of stress response pathways (Chap. 9), oxidative stress (Chap. 10),
DNA damage (Chap. 11), protein homeostasis (Chap. 12), protein translation (Chap.
13), lipid metabolism (Chap. 14), autophagy (Chap. 15), and dietary restriction
(Chap. 16) are discussed. Emerging areas such as integration of metabolic signals
(Chap. 17) and the relationship between the microbiome and probiotic bacteria and
lifespan determination (Chap. 18) are also covered.
Since the discoveries in the 1970s of environmental manipulations that affect C.
elegans lifespan and single gene mutations that have a profound effect on ageing
there have been dramatic advances in our understanding of the ageing process
across species. The role that C. elegans is likely to play in ageing research in the
next 25 years is discussed in Chap. 19.
We hope that this book serves to illustrate how far the C. elegans ageing field has
come in a short space of time, from the initial discovery of long-lived mutants to
forming the foundation of a new era of ageing research that has the potential to have
dramatic impacts on healthspan in human populations.
Acknowledgments We are grateful to all the authors who contributed to this book and thankful
for the support from the series editor Dr. Suresh Rattan.
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Chapter 2
Effects of Ageing on the Basic Biology
and Anatomy of C. elegans
Abstract Many aspects of the biology of the ageing process have been elucidated
using C. elegans as a model system. As they grow older, nematodes undergo signifi-
cant physical and behavioural declines that are strikingly similar to what is seen in
ageing humans. Most of the major tissue systems of C. elegans, including the cuti-
cle (skin), hypodermis, muscles, intestine, and reproductive system, undergo dra-
matic physical changes with increasing age. The ageing nervous system undergoes
more subtle changes including dendritic restructuring and synaptic deterioration.
Many of the physical changes become more apparent near the end of reproduction.
In conjunction with tissue ageing, some behaviours, such as locomotion, pumping
and defecation, decline substantially during the ageing process. Interestingly, some
aspects of physical and behavioural decline are delayed in longevity mutant back-
grounds, while other changes are not altered. This chapter provides an introduction
to the general features of C. elegans anatomy and describes what is currently known
about the physical changes that accompany the normal ageing process. It should be
noted that some descriptions summarized herein have not been previously pub-
lished, so that despite the review theme, novel aspects of the ageing anatomy are
also featured. Given the common features shared between C. elegans and humans
during ageing, a greater understanding of the anatomy of this process in C. elegans
can help illuminate the nature of ageing-related tissue decline across species.
2.1 Introduction
Fig. 2.1 Introduction to C. elegans anatomy and life cycle. (a) Schematic showing anatomy of an
adult C. elegans lying on the left lateral side (Image source: [WormAtlas]). (b) Life cycle of C.
elegans at 22 °C. Fertilization occurs at time = 0 min. Numbers along the arrows indicate the
length of time the animal spends at each stage (Image source: [WormAtlas])
the biology of C. elegans ageing through a tissue-focused lens, and can speak to the
relevance of C. elegans ageing to issues of human ageing.
adults (Fig. 2.1b). Most adult C. elegans are self-fertile hermaphrodites, although
males arise on rare occasions by non-disjunction of the sex chromosome and can
then be propagated by crossing. Each larval stage is punctuated by a moult, during
which pharynx pumping ceases, and the cuticle is shed and replaced by a newly
synthesized stage-specific cuticle.
Under harsh environmental conditions, with limited food, high temperature, or
overcrowding, early larvae may reversibly arrest development after the second lar-
val stage as dauer (“enduring”) larvae (Fig. 2.1b) [16, 17]. Dauer larvae have a dis-
tinct morphology and biology adapted for long-term survival. Recovery from dauer
arrest is triggered by food or introduction to a favourable environment. Dauers
recover into L4 larvae, which proceed on the same developmental pathway to repro-
ductive adults as larvae that bypassed dauer. For a more detailed discussion of the
dauer larva see Chap. 3.
Adult hermaphrodites are self-fertile for approximately 3–4 days and produce
about 300 progeny, limited by the number of sperm produced during spermatogen-
esis. Hermaphrodites inseminated by males receive a fresh supply of sperm and may
produce 1200–1400 progeny during an extended reproductive period [18]. After
reproduction ceases, animals enter a post-reproductive period lasting 2–3 weeks
before death [16, 19]. During the post-reproductive period, feeding and locomotory
rates decline, tissues deteriorate, and animals become more sensitive to microbial
infection [8, 12–14, 20, 21]. Post-reproductive adults lack stem cells and therefore
do not replace cells or tissues damaged by ageing. Thus, apart from the germline,
the C. elegans model features the ageing of post-mitotic tissues.
2.3.1 Cuticle
The C. elegans cuticle covers the outer surface of the body, providing protection,
maintaining body shape, and aiding motility [22, 23]. The cuticle surface is covered
by circumferential furrows and ridges called annuli. Bilateral alae, which appear as
raised ridges, run lengthwise along the body to facilitate movement (Fig. 2.2a).
The cuticle is built from collagens and noncollagenous cuticulins arranged in
layers differing in structure and composition (Fig. 2.2b). Over most of the body, the
components of the cuticle are secreted by the hypodermis and seam cells, which are
the epithelial cells covering the body. The dauer cuticle is thicker and more highly
reinforced to protect dauers from environmental threats and desiccation [24].
Cuticle also lines the major body openings, such as the anus and the excretory pore.
These “lining” cuticular domains do not appear to be composed of layers, although
they can still provide adequate structural support for function. Body openings,
including the anus, excretory pore, vulva and pharynx, are lined by interfacial cells
that produce the cuticle lining for these structures.
2 Effects of Ageing on the Basic Biology and Anatomy of C. elegans 13
a
Cuticle
Annuli Furrow
Hyp
Seam Alae
Basal
lamina
Muscle
TRANSVERSE
LONGITUDINAL
b furrow annuli
cuticle
hypodermis
muscle
sarcomeres
c annuli
furrow
cuticle
hypodermis
muscle
sarcomeres
Fig. 2.2 The C. elegans cuticle thickens and wrinkles during ageing. (a) Schematic showing cuti-
cle structure in C. elegans. A thin layer of hypodermal tissue (orange) always underlies the body
wall cuticle (grey), separating the cuticle from the four underlying quadrants of body wall muscles
(green). The basal laminae of the hypodermis and muscle fuse to make a single layer spanning the
extracellular space between the two tissues. Special features of the adult epicuticle include concen-
tric narrow annuli separated by shallow furrows, and several parallel ridges, the “alae”, that run for
most of the length of the body at the lateral line. The seam cells are a row of specialized epidermal
cells underlying the alae (Image source: [WormAtlas]). (b) TEM longitudinal section from young
adult showing cuticle, hypodermis and adjacent muscle sarcomeres. The annuli and furrows are
shown at the outer surface of the body wall (Image source: [Hall] N533 L4 Z915). Bar, 1 μm. (c)
TEM longitudinal section from 15-day-old adult. In the older adult, the cuticle layers are each
much thicker, while the furrows and annuli remain visible despite the wrinkling and thickening of
the basal and medial cuticle layers. Below the cuticle, the body wall muscle sarcomeres are thinner
and disorganized in the older adult (Image source: [Hall] N815 G0713)
14 L.A. Herndon et al.
Adult animals do not moult, so the adult cuticle must persist through the entire adult
lifespan. During ageing, the cuticle becomes progressively thicker [8], and cuticle
growth may continue until the hypodermis and seam are no longer capable of secret-
ing cuticular components. This continuous growth is likely to result from unregu-
lated biosynthesis of cuticle-related proteins as post-reproductive shut down of
overall expression does not transpire (not subject to natural selection pressures, see
discussion in [8]). The most prolific age-associated growth occurs in the basal cuti-
cle layers, which can expand in thickness by 10-fold in comparison to young adults
(Fig. 2.2c). Concomitant with this thickening, the cuticle becomes progressively
more wrinkled overall [8]. Cuticle wrinkles may arise from the combined effects of
a weaker, thinner hypodermis, loosened connections between the cuticle and hypo-
dermis, and weakening muscles. In ageing animals, the distinct cuticle linings of
body openings remain virtually intact, and are possibly reinforced (Herndon et al.
unpublished data).
While the thickened ageing cuticle generally remains intact and capable of pro-
tecting the animal from outside insults until death, the cuticle cannot provide pro-
tection from internal causes of death, such as internally-hatching embryos or vulval
muscle breakdown that allows gonad or gut extrusion. Thus, internal injuries can
ultimately induce cuticle lapses, although cuticle failure itself does not appear to be
a major cause of death.
2.3.2 Hypodermis
Fig. 2.3 (continued) right edge of the panel show debris filling the pseudocoelom (Image source:
[Hall] N801 E565). Bar, 1 μm. (d) TEM cross-section from a healthier day 15 adult showing the
lateral hypodermis filled with cellular detritus, including abundant lipid droplets. The hypodermis
cytoplasm is less electron dense than in the young adult (b) and organelles are altered or missing.
The thickened cuticle has pulled away from the hypodermis during fixation, indicating structural
weakness in cuticle attachment. In addition, acellular material has been shed into the space beneath
the thickened cuticle (Image source [Hall] N812 U3 M784). Bar, 1 μm
2 Effects of Ageing on the Basic Biology and Anatomy of C. elegans 15
Fig. 2.3 The C. elegans hypodermis becomes thinner and fragile during ageing. (a) Schematic
view at the midbody shows the hypodermis (tan) as it encloses the animal just below the cuticle
(grey). The hypodermal syncytium is quite thin where is underlies the body wall muscles (empty
green circles), but is enlarged along the lateral borders and where it provides support to the longi-
tudinal nerve cords (red). Specialized seam cells (dark orange) lie in rows just under the cuticle
alae at the lateral borders, linked to the neighbouring hypodermis by adherens junctions (aj)
(Image source: [WormAtlas]). (b) TEM cross-section of the hypodermis at the lateral line in a
young adult. In young animals, the hypodermis is filled with organelles, including abundant RER,
mitochondria (m), and stored lipids and yolk. A clear internal space, the pseudocoelom, lies
between the hypodermis and the tissues within the body cavity, such as the distal gonad (upper
right) and uterus (lower right). The excretory canal is visible (ec) at the edge of the pseudocoelom
(Image source: [Hall] N506 M700). Bar, 1 μm. (c) TEM cross-section of an older (day 15) adult
showing extremely thinned hypodermis, devoid of most cytoplasmic components, and much less
electron dense. The thickening of the cuticle is apparent in comparison to (b). Brighter areas at the
16 L.A. Herndon et al.
Growth continues for several days after the final moult, stretching the hypodermal
hyp7 syncytium [8]. As animals age, the hypodermal cylinder becomes exceedingly
thin in all regions and loses the capacity to maintain its shape. Viewed by electron
microscopy, the ageing hypodermal cytoplasm contains fewer organelles than
young hypodermis, such as smooth and rough ER and mitochondria, and those that
are present often appear damaged (Fig. 2.3c, d compared to Fig. 2.3b). The cytosol
also becomes progressively less electron dense. In very old animals, the hypodermal
cytoplasm is nearly empty and the tissue thins to the breaking point, particularly on
the basal pole facing the pseudocoelom. Ageing may disrupt “clean up” functions of
hypodermal cells, which normally clear damaged cells and other materials from the
pseudocoelom by engulfment. Old-age accumulation of debris materials in the
pseudocoelom (see below) suggests loss in efficacy of this process. Compared to
other tissues, the hypodermis may be a particularly weak link during ageing, and its
physical breakdown may have fatal consequences for old animals. Loss of hypoder-
mal cylinder integrity would allow the pseudocoelom to mix with apical contents,
which could damage anchorage of the muscles and cuticle, leaving cells or debris to
float inside the cuticle. That components of the cuticle and/or basement membranes
may be critical in healthy ageing is supported by recent findings that extracellular
matrix gene expression is enhanced in multiple long-lived mutants and modulated
expression of particular individual collagens can impact lifespan [26].
2.3.3 Muscle
The two main types of muscle cells in C. elegans are the single sarcomere/non-
striated muscles and the multiple sarcomere/obliquely striated muscles [27]. Single
sarcomere/non-striated muscle cells include the muscles of the pharynx, the somato-
intestinal muscle, the anal sphincter and depressor, the contractile gonadal sheath,
and the sex-specific muscles of the uterus, vulva and male tail. Of these, the pharynx
muscle has been best studied in the context of age-associated structural and func-
tional changes and is discussed in greater detail in a later section.
The multiple sarcomere muscles, more commonly known as somatic or body-
wall muscles, control movement and locomotion. These 95 skeletal muscle-like
cells constitute the most abundant muscle group. The body wall muscles are
arranged as staggered pairs in four longitudinal bundles situated in four quadrants
lining the body cylinder (Fig. 2.4a–c). Evenly-distributed attachment points bind
the body-wall muscle bundles along their length to the hypodermis and cuticle. The
basic unit of the contractile apparatus is the sarcomere, and these contractile units
are repeated in body muscle, giving the cells a “striated” appearance (Fig. 2.4a).
A typical somatic muscle cell has three parts: the contractile myofilament lattice
or spindle, a noncontractile body, called the muscle belly, containing the nucleus and
the mitochondria-filled cytoplasm, and the muscle arms, slender processes extending
towards the nerve cords or the nerve ring where neuromuscular junctions (NMJ) are
2 Effects of Ageing on the Basic Biology and Anatomy of C. elegans 17
Fig. 2.4 Organization of the C. elegans body wall muscles. (a) Epifluorescence image (dorsal
view) of a body wall muscle-specific GFP reporter expressed in a young adult hermaphrodite (unc-
27:GFP reporter; Strain source: Jia, L and Emmons, SW). This view shows the full extension of
the two dorsal muscle quadrants from nose to tail (left to right). Polarized light helps to visualize
the myofilament lattice which runs virtually parallel to the body axis, separated by the narrow ridge
of dorsal hypodermis at the midline. Each quadrant consists of two parallel rows of muscle cells.
Nuclei of the muscle cells can be seen as white circles, lying near the of each spindle-like cell. Bar,
50 μm (Image source: [WormAtlas]). (b) Diagram of the midbody region. Each muscle cell (green)
along the midbody extends one to three thin “muscle arms” inward to reach the nearest nerve cord
where it receives innervation. Thus four dorsal rows in two dorsal quadrants extend arms to the
dorsal nerve cord, and four rows in two ventral quadrants extend arms to the ventral nerve cord.
Basal lamina (light orange line) separates the muscle from the nerve cords and the hypodermis.
Hypodermis, which is stylized in this diagram for illustration purposes, separates muscle from
cuticle (Image source: [WormAtlas]). (c) TEM thin section of the young adult midbody has been
false-coloured to show the layout of the muscle quadrants (green) in finer detail. Note that all
myofilament sarcomeres lie close to the cuticle, while each muscle has its cell body, the “muscle
belly”, lying more central, containing the nucleus, RER, mitochondria and other organelles.
Muscle arms extend away from the muscle belly. Bar, 1 μm (Image source: [WormAtlas])
situated (Fig. 2.4b). Somatic muscle nuclei in young adults are oblong, intermediate
in size between neuronal and hypodermal nuclei, and have spherical nucleoli.
Fig. 2.5 Body wall muscles become disorganized and deteriorate during ageing. (a) Young adult
body wall muscle cell showing five sarcomeres running side by side beneath the cuticle. The
muscle myofilaments are anchored to darkly-staining “dense bodies” (large black arrowheads)
connecting to the muscle’s plasma membrane. The plasma membrane is linked to the cuticle by
intermediate filaments extending across the thin hypodermal layer and connecting to wispy
2 Effects of Ageing on the Basic Biology and Anatomy of C. elegans 19
loss, and lipid droplets can accumulate within the muscle cells (Fig. 2.5b) [8]. Body
wall muscle nuclei also show pronounced changes with nuclei becoming misshapen
and nucleolar size increased. A few nuclei in ageing muscle have been observed to
become electron-dense and appear to undergo autophagy. The degree of nuclear
change appears to correlate with locomotory ability of the individual animal, though
there is still much variation in individual muscle cells within a single animal, sug-
gesting a stochastic component in the decline of single cells [8].
As the body wall muscles deteriorate with age, the normally sinusoidal locomotory
behaviour also declines [8, 12, 13, 20, 28–30]. Older animals move only when stimu-
lated and display increasingly irregular patterns of movement. The most decrepit ani-
mals can no longer move forward or backward, and only slightly twitch their head or
tail regions when touched. A closer look at the muscle cells in individual animals with
movement defects suggests coincident levels of sarcomere deterioration, indicating
that muscle cell deterioration may contribute to ageing-related locomotory declines
(compare Fig. 2.5b–d, which show progressive loss of myofilaments in animals with
increasingly impaired movement). Indeed, analysis of individual ageing animals
showed that decline in locomotory ability more closely predicted time of death than
did chronological age [8]. Recent studies suggest that that the earliest detectable loco-
motory declines reflect changes in neuronal signalling at the neuromuscular junction
Fig. 2.5 (continued) filaments in the basal layer of the cuticle. A prominent nucleus (large white
arrowhead) containing a large nucleolus lies beneath the sarcomeres in the muscle belly, sur-
rounded by large mitochondria. A row of mitochondria (small black arrowheads) lie in the muscle
belly, close to the sarcomere (Image source: [Hall] N513 G607). Bar, 1 μm. (b) A similar cross-
section of a body wall muscle in a relatively motile 15-day adult. In this adult, the muscle cell
retains intact sarcomeres with many myofilaments per unit volume, though reduced somewhat
compared to the young adult (a). The nucleus is present in this view and the nucleolus appears less
electron dense. The muscle belly remains fairly large with numerous mitochondria, but contains
large lipid droplets and is less electron dense. Thickening of the overlying cuticle is also apparent
in this animal (Image source: [Hall] N810 R443). Bar, 1 μm. (c) Cross-section from a slow-moving
15 day old adult shows dramatic muscle cell changes. The myofilament lattice is smaller and dis-
organized, including a dramatic decline in myosin filaments per sarcomere (long arrows). Although
mitochondria are still present (black arrowheads), the cell has shrunken and the cytoplasm is
devoid of most organelles, including RER or lipid storage. At the basal pole, wispy pieces of mem-
brane may be shedding into the pseudocoelom (short black arrows), sometimes containing small
mitochondria, and coated on the outside by basal lamina. The pseudocoelom itself has gained
volume and contains basal lamina fragments and large dark yolk granules. The basal layer of the
cuticle is now extremely thick compared to a young adult (Image source: [Hall] N813 G506). Bar,
1 μm. (d) TEM cross-section of a paralysed 15-day adult showing extreme loss of muscle integrity.
Sarcomeres have lost most myosin and actin filaments, although the remaining filaments are well
positioned between smaller dense bodies. The muscle belly is virtually absent except for a thin
projection (short arrow), indicating continuing shedding into the pseudocoelom, with a concomi-
tant loss of mitochondria and cytoplasm from the belly. Whorls of basal lamina (bl) and other
debris are floating in the huge volume of pseudocoelom. The cuticle is intact and vastly enlarged,
especially the basal layer. Arrowheads indicate the presence of intact mitochondria inside the
neighbouring hypodermis (hyp) (Image source: [Hall] N829 R157)
20 L.A. Herndon et al.
(NMJ) [12, 31, 32]. Later in life, muscle deterioration adds to this early impairment,
enhancing locomotory declines in old animals. Regardless of the important question
of initiating mechanism, substantial sarcopenia accompanies C. elegans ageing.
2.3.4 Pharynx
Pharyngeal cells deteriorate in older adults and prominent vacuoles often appear
within the organ (Fig. 2.6d). The elongated muscle cells of the isthmus become
weakened with age, as they often appear to be bent or kinked in EM images
(Fig. 2.6b) [33]. Finally, the pharynx itself becomes less efficient at crushing
bacterial cells, and intact bacteria are more likely to be observed in the pharyn-
geal lumen of older adults (Fig. 2.6e). The stress of pumping over adult life may
damage the pharynx, as mutations that limit contractions can slow functional
decline [33].
The rate of pharynx pumping decreases progressively with age, such that
pumps are rare in animals older than 8 days, which is a striking senescence fea-
ture in a ~21 day lifespan [13, 28]. Considerable heterogeneity in pump rate of
individuals has been reported [34] and this heterogeneity increases with age
[33]. Exogenous serotonin can stimulate pumping in young adults [35] and can
also stimulate pumping in old animals, although pumping rates +/− serotonin
still progressively decline over adult days 2–8 [33]. Since neurotransmitter
response is maintained, but functionality declines, the structural deterioration of
the pharynx muscle with age appears likely to limit its functional capacity.
There is a paucity of information on how the ageing of the 20 pharyngeal neu-
rons impacts organ function.
The live bacterial food upon which C. elegans nematodes are maintained in the
laboratory is at best a minor cause for decreased pharynx pumping with ageing, as
pump rates declined similarly when animals were raised on bacterial food sources
2 Effects of Ageing on the Basic Biology and Anatomy of C. elegans 21
Fig. 2.6 Deterioration of the pharynx during ageing. (a) Schematic layout of the adult pharynx
(green) with major regions labelled and showing the relative positions of the pharyngeal valve
(brown) and the intestine (pink). (c–e) indicate approximate positions of TEM thin sections shown
in lower panels (Image source: [WormAtlas]). (b) Longitudinal TEM section of the pharynx isth-
mus between the corpus and terminal bulb in a 7-day-old adult. In this middle-aged adult, the
isthmus is already weakened and kinked near the terminal bulb. Bacterial cells are seen as densely
packed plugs in the lumen of the corpus and terminal bulb. In some animals, the bacterial plugs can
be seen extending into the isthmus region (Image source: [Hall] N824 N4924). (c) Cross-section
of the corpus in a young adult. The tissue has threefold symmetry with a row of marginal cells
lying at each apex of the internal lumen, and two rows of fused pharyngeal muscles whose sarco-
meres are oriented radially on each side of the lumen. Narrow rows of pharyngeal neurons lie
between the pharyngeal muscles, named the dorsal, subventricular left and subventricular right
nerve cords (nc-d, nc-svl and nc-svr). One neuronal cell body (and its nucleus) is visible here in the
ventral left pharyngeal nerve (Image source: [MRC] N2U 411 0238-06). (d) A similar region of the
pharynx in a 15-day old adult which had maintained its locomotory behaviours in movement
assays [8]. In this animal, many pharynx myofilaments remain intact, although muscles, marginal
cells and nerve cords are vacuolated and sometimes disorganized. Intact bacteria have entered a
vacuole in one muscle cell at the lower left (V). The central lumen is filled with electron-dense
material which may be debris from partially ground up bacteria (Image source: [Hall] N812 F828).
Bar, 5 μm. (e) TEM cross section of pharyngeal isthmus region of a paralysed 15-day-old adult
showing extensive muscle deterioration and accumulation of intact bacteria in the lumen (bacterial
plug), forcing the lumen to open widely. The three marginal cells are identifiable by thick bundles
of intermediate filaments connecting radially to the lumen, but most nerve cords are difficult to
identify. The tissue is distorted in overall shape, muscle myofilaments are twisted, and the muscle
cytoplasm has become much less electron dense (Image source: [Hall] N807 G905). Bar, 5 μm
that were growth arrested due to antibiotic treatment [33]. Still, in the absence of
strong pumping the pharynx can become plugged with bacteria as animals age
(Fig. 2.6e), either as a cause or consequence of pharynx decline.
22 L.A. Herndon et al.
Fig. 2.7 Neurons (a) Epifluorescence image of panneuronal GFP reporter in an adult hermaphro-
dite showing distribution of neurons throughout the body. This is a left lateral view with anterior to
the left. NR Nerve ring, RVG retrovesicular ganglion, VG ventral ganglion, VNC ventral nerve cord,
DC dorsal cord. Motor neurons are scattered along the VNC and send processes to the DC via com-
missures (arrowheads). Several neurons are indicated by four-letter codes: ALML, CANL, NSML,
PLML, PLNL. Magnification, 400× (Image source: [WormAtlas]). (b) Epifluorescence image of the
touch receptor neurons expressing the cell-type specific GFP reporter, (mec-4:GFP) in a young
adult hermaphrodite, left lateral view. The neuron processes are straight and evenly labelled by the
reporter in the young adult (Image source: [WormAtlas]). (c, d) Ageing-related morphological
abnormalities in touch neurons. Touch neurons in older adults visualized with the same GFP marker
as in (b) appear wavy (c) or branched (d) (Image source: Toth and Driscoll). (e, f) TEM cross section
view of the ALM touch neuron from a young adult (e) and a 15-day-old adult (f). In young adults,
the touch neurons are embedded in the hypodermis just beneath the cuticle. An electron-dense
ECM, called mantle, surrounds the touch receptor processes and attaches them to the body wall.
Touch receptor processes are typically filled with 15-protofilament microtubules (MT). While the
touch neuron in f appears healthy and well structured, the cell process is filled with dozens of adven-
titious microtubules. CU Cuticle ER Endoplasmic reticulum (Image source: [Hall] N501 S3 N517;
N810 M782)
2 Effects of Ageing on the Basic Biology and Anatomy of C. elegans 23
ring in the head. Interneurons, in turn, signal to motorneurons in the dorsal and
ventral nerve cords to mediate behavioural responses, such as touch avoidance and
egg-laying. Neuromuscular junctions (NMJ) are clustered in synaptic regions where
neurons appose the muscle cell arms (Fig. 2.8a). Motor behaviours are controlled by
stimulatory cholinergic and inhibitory GABAergic inputs.
In contrast to the striking ageing-related physical decline of the body muscles, ner-
vous system changes are more subtle [8]. There is no indication that any C. elegans
neurons undergo cell death or necrosis in the course of normal ageing. Sensory
specializations, such as cilia and dendrites, remain well preserved. Touch neurons,
as analysed by electron microscopy, maintain their basic ultrastructure in aged ani-
mals (Fig. 2.7e, f). Most other aspects of the nervous system decline progressively,
with the predominant pattern of change evident from EM data involving progressive
losses of synaptic integrity and shrinkage of the neuron soma. At the cellular level,
morphological abnormalities in processes can increase with age, to a degree that
depends on individual neuron type. Further discussion of ageing in the nervous
system can be found in Chap. 8.
At the nerve cords and nerve ring, the numbers and size of intact synaptic contacts
decline substantially with age [36]. Surviving synapses are smaller and contact
zones often contain very few synaptic vesicles compared to young synapses (Fig.
2.8). The absolute cross-sectional diameter of presynaptic zones, which occur en
passant along the axons, is generally a function of the number of synaptic vesicles
and mitochondria that are locally collected near the presynaptic dense bar. In some
older synapses, the presynaptic zone shrinks to enclose only the presynaptic bar,
which itself may be flexed and shortened to fit within a tiny axonal process [36].
These findings suggest that neuronal signalling is greatly reduced in older adults,
unless electrical synapses can compensate for the loss of chemical signals.
Interestingly, 15-day-old animals that age gracefully by locomotory criteria main-
tain greater synaptic integrity than same-age, same-environment 15-day-old ani-
mals that have aged poorly (earlier onset locomotory decline) [36]. Recent
electrophysiological studies suggest that decline in presynaptic neurotransmitter
release at the C. elegans neuromuscular junction is the earliest detectable decline
in locomotory ageing, preceding detectable muscle deficits [32].
EM data support that most axons shrink in diameter during ageing. The nerve cords
remain intact and the axons appear unbroken, despite diameter shrinkage. In touch
neurons, microtubule networks appear important for adult structural maintenance [37]
24 L.A. Herndon et al.
Fig. 2.8 Synapses (a) TEM of the neuromuscular junction (NMJ) between a ventral motor neuron
and several muscle arms, transverse section. (Inset) The same synaptic region, magnified. At the
point of contact with the post-synaptic elements, the presynaptic process enlarges into a varicosity
with a specialized darkly staining bar at the active zone (thick arrow) and contains many synaptic
vesicles (thin arrows) close to microtubules (arrowheads). Bar, 1 μm (Image source: [WormAtlas]).
(b–e) Comparison of synapses in young (b, c) and 15-day-old (e, f) adult animals. Older adults
were divided into three classes based on mobility. Class A animals were highly mobile while Class
C animals moved only when prodded and primarily moved just their head and tail regions. (b) A
young adult animal exhibits a prominent presynaptic bar along the plasma membrane and the pro-
cess is swollen with synaptic vesicles. Vesicles lying close to the bar are somewhat smaller in
diameter than vesicles away from the release zone. Bar, 0.25 μm (for b and c). (c) A depleted
synapse (double arrows) in the same young adult displays a normal presynaptic bar, but a paucity
of synaptic vesicles close to the bar or at a distance. (d) In a Class A (mobile) adult at 15 days,
chemical synapses (arrows) remain well organized but have fewer vesicles near the presynaptic bar
and the presynaptic process is therefore smaller in diameter. Note that many nearby axons (away
from the synapse) remain almost the same diameter as in a young adult. Many axons still contain
clusters of synaptic vesicles and small bundles of microtubules. Bar, 0.5 μm. (e) Closeup of a
depleted synapse (double arrows) in a Class A animal at 15 days of age. A fuzzy electron dense
inclusion (white asterisks) lies close to the depleted synapse. This may represent pathological
deposition of cytoplasmic proteins. Bar, 0.25 μm. (f) Quantitation of synaptic features in ageing C.
elegans. YA, young adult; 15d A, 15-day-old class A animal that is relatively vigorous for its same-
age counterparts and considered to have aged gracefully; 15d C, 15-day-old class C animal that is
decrepit, barely mobile, and considered to have aged poorly. Data include measurements of 51
synapses from six young adults; 52 synapses from three Class A animals; 28 synapses from three
Class C animals. Synapses were from the nerve ring and lateral ganglia. “Number of vesicles”
indicates counts of all vesicles within 300 nm from the synaptic density. Asterisks indicate p < 0.02
as compared to young adult values; repeated measures analysis of variance test (SAS programme)
(Data and images in (b–f) from [36])
2 Effects of Ageing on the Basic Biology and Anatomy of C. elegans 25
and can become disorganized with age, at least near the soma [38]. Mitochondria travel
within touch neuron processes at progressively slower speeds in both anterograde and
retrograde directions [39], consistent with a declining cytoskeletal transit network.
Fluorescent reporters that allow visualization of neuronal processes have revealed
dramatic structural changes in neurons that increase in frequency with age. Neurite
branching, axon beading, axon swelling, axon defasiculation, “bubble” formation,
waviness, and new growth from the soma have been reported for touch receptor
neurons, PVD sensory neurons, PDE dopaminergic neurons, GABAergic neurons
and others (Fig. 2.7c, d) [36, 38, 40–42]. Longitudinal observations suggest these
structures are dynamic—appearing, disappearing, and progressing to different
structures [36, 38]. The age-dependent occurrence of some of these features can be
modulated by insulin-like signalling, MAP kinase and heat shock stress response
signalling and neuronal attachment. The functional significance of morphological
changes in ageing C. elegans neurons remains to be experimentally defined.
2.3.6 Glia
The nematode has a relatively small number of glial cells (56), most of which are
specialized to create special environments for protecting the ciliated endings of sen-
sory neurons (Fig. 2.9a) [43, 44]. These glial cells are known as the socket and
sheath cells, and in the adult male, the structural cells of the ray neurons in the tail.
Nematode axons are not myelinated. Only the CEP sheath cells and the GLR cells
form larger wrapping processes to enclose portions of the nerve ring neuropil, a role
similar to those of human astrocytes or microglia.
Glial cells appear to remain viable into old age in the nematode, still enclosing sen-
sory endings (Fig. 2.9b, c) ([8] and unpublished data) and are not normally required
for neuronal viability [45]. Much like that of neurons, the glial cell cytoplasm
becomes progressively more electron dense as the cells shrink in volume, and
26 L.A. Herndon et al.
Fig. 2.9 Anatomy and decline of the amphid sensillum. (a) Structure of the amphid opening in a
young adult, seen longitudinally, anterior to the top. The amphid channel (Ch) is lined by the lip
cuticle in the distal (socket) part and an electron-dense lining supported by a scaffold of cytoskel-
etal filaments (Fs) in the anterior sheath. The socket cell is connected to the hypodermis and the
sheath cell by adherens junctions (aj). Circular adherens junctions are also seen to tightly seal the
dendrites to the sheath cell (neuron-sheath junction) proximally to the level where the dendrites
enter the channel. A large Golgi apparatus located at the base of the sheath-cell process (left) gives
rise to matrix-filled vesicles bound towards the channel. Several specialized neuron dendrites
embed into the sheath cell with little or no exposure to the amphic channel (AWA, AWB, AWC,
AFD). Mitochondria (not shown) are also present in this region (Image source: [WormAtlas] modi-
fied, with permission, from Perkins et al. [59]). Bar, 1 μm. (b, c) TEM of amphid channel cilia and
AFD in young and old adults. Transverse sections through middle segments of cilia (area from
boxed region in a). In a young adult (b), the distal portions of the “channel cilia”, characterized by
nine doublet microtubules, sit inside the amphid channel lumen (black arrowheads), while the
AFD villi and its thick dendrite are encased inside individual thin channels of the amphid sheath,
away from the channel. By comparison, the 15-day adult animal (c) shows the AFD villi are
unsheathed and some have entered the main amphid channel to mix between the channel cilia.
While there doesn’t appear to be neuronal or glial cell loss, a shrinkage of glial sheath cytoplasm
has led to a wider and more open amphid channel inside the sheath cell. All cilia and dendritic
segments in the 15-day animal are more electron dense than in the young adult, and the AFD villi
appear to have shrunken in diameter (Image source: [Hall] b SW8; c N813 537). Bar, 1 μm
known about the viability of the wrapping processes of the CEP sheath cells and
GLR cells during ageing. Much remains to be learned about contributions of C.
elegans glia to ageing and healthspan.
2.3.7 Intestine
After food is pumped and pulverized by the pharynx, it enters the intestine where it
is digested and nutrients are absorbed. Additionally, the intestine functions to synthe-
size and store macromolecules, initiate immune responses, and nurture germ cells by
producing and secreting yolk [46–49]. The intestine is comprised of 20 large epithe-
lial cells that are mostly positioned as bilaterally symmetric pairs to form a long tube
around a lumen (Fig. 2.10). The intestine is not directly innervated and has only one
associated muscle (the stomatointestinal muscle) at its posterior extreme. Intestinal
cells are large and cuboidal with distinct apical, lateral and basal regions (Fig. 2.10b).
Intestinal cells contain one or often two large nuclei with prominent nucleoli, many
mitochondria, extensive rough endoplasmic reticulum, many ribosomes and an
extensive collection of membrane-bound vesicles and vacuoles. Adherens junctions
seal each intestinal cell to its neighbours on the apical side and gap junctions and
septate-like junctions connect them on the lateral sides (Fig. 2.10b, c). Microvilli
extend from the apical face into the lumen forming a brush border (Fig. 2.10b, c).
The terminal web, a network of intermediate filaments, anchors the microvilli.
b c
Basolateral Apical membrane
membrane
Lumen
Glycocalyx
Microvilli
Adherens
junction Intermediate
Intestinal cell Actin filaments
Basal lamina
Pseudocoelom
Fig. 2.10 Anatomy of the adult C. elegans intestine. (a) The intestine is positioned on the left side
of the body anterior to the vulva and on the right side of the body posterior to it. At its anterior end,
the intestine is connected to the pharynx via the pharyngeal valve. The most posterior portion is
squeezed by the stomatointestinal muscle (not shown), near where the intestine connects to the
rectum and anus. Arrowhead indicates the position of the TEM cross-section shown in (c) (Adapted
with permission from [58]). (b) Key structural elements of the healthy intestinal cytoskeleton. At
its basal pole the intestine is covered by a basal lamina (orange), separating it from the pseudocoe-
lom. Pairs of intestinal cells meet to form a lumen between them, with the two cells firmly linked
by adherens junctions at their apical borders. Gap junctions and septate-like junctions form a
complex junction just beneath the adherens junctions on the basolateral membranes where the two
intestinal cells meet. Intermediate filaments help to anchor a terminal web of fibres running just
beneath the microvilli that face the lumen itself. An actin-based cytoskeleton fills each villus; the
actin fibrils anchor into the terminal web at one end, and to an electron dense cap at the tip of the
villus. A thick glycocalyx covers the outer surface of the microvilli. At adulthood, most intestinal
cells contain two very large nuclei (black circle). The lumen of the young adult intestine usually is
filled by debris from partially digested bacteria, but few if any intact bacteria (Image source:
[WormAtlas]). Graphic adapted from Wood et al. [60]. (c) Electron micrograph showing the key
features of the young adult intestine. The intestinal cytoplasm is filled with a complex mixture of
organelles, including mitochondria, Golgi apparatus, RER, yolk-filled granules, and occasional
large autophagosomes. Inset shows a complex gap junction (white arrow) next to an adherens junc-
tion (arrowhead) which seal the two intestinal cells to each other. (Image source: [WormAtlas])
deleterious to lifespan. The intestine produces yolk that nourishes embryos, but in
the old-age absence of oocytes, yolk is still produced and accumulates throughout
animal [8, 21, 50]. Inappropriate yolk accumulation appears to be detrimental and
limits lifespan [51].
In ageing animals, large clumps of undigested bacteria are often found in the intes-
tinal lumen (Fig. 2.11c), likely the result of reduced pumping and grinding effi-
ciency in these older animals [10, 50]. In rare cases, bacteria invade the lumen of the
2 Effects of Ageing on the Basic Biology and Anatomy of C. elegans 29
Fig. 2.11 Anatomical decline of the intestine during ageing (a–c) Low power transverse TEM
views of aged intestine show large changes in the cytoplasm and lumen (compare to Fig. 2.10c).
These declines are stochastic, as individual animals show markedly different rates of change. (a)
In a day 7 adult intestinal cell, the nucleus (N) has lost heterochromatin and contains an enlarged,
vacuolated nucleolus. The cytoplasm is highly vacuolated, with a profound reduction in ground
substance, or RER, although many mitochondria remain intact. The apical zones are studded with
microvilli but the lumen (L) is almost empty (Image source: [Hall] N826 5353). Bar, 5 μm. (b) A
day 15 adult intestinal cell in which the cytoplasm is choked by lipid storage droplets. L lumen, N
nucleus (Image source: [Hall] N812 F815). Bar, 5 μm. (c) A day 15 adult intestinal cell in which
the cytoplasmic contents have become highly eccentric, with substantial degradation of all remain-
ing organelles, and no intact ground substance. The lumen has intact microvilli but is swollen with
intact bacteria. The intestine may not be competent for digestion, but provides a barrier against
further bacterial invasion (Image source: [Hall] N807 G583). Bar, 5 μm. (d–h) Higher power trans-
verse TEM views displaying major defects in the adult day 7 intestinal microvilli. Progressive loss
of several barriers to bacterial invasion of the cytoplasm are evident. (d) Healthy microvilli facing
a lumen filled mostly with soluble items or a few bacterial fragments. Arrow indicates a region
where the terminal web may be separating from the base of the microvilli (Image source: [Hall]
N826 4239). Bar, 1 μm. (e) Microvilli are no longer uniform in length, and intact bacteria can be
seen in the lumen, some of which are attached to individual villi, possibly beginning to degrade
them. Arrow indicates the terminal web (Image source: [Hall] N821 4872). Bar, 1 μm. (f) In this
region, most microvilli are gone, although the terminal web (arrow) remains thickened and elec-
tron dense. The lumen contains many intact bacteria (Image source: [Hall] N821 4855). Bar, 1 μm.
(g) The microvillar border and the terminal web (arrow) separating the lumen from the intestinal
cytoplasm are less electron dense and possibly incomplete, allowing bacteria to invade the intesti-
nal cell cytoplasm (Image source: [Hall] N831 W006). Bar, 1 μm. (h) The bacteria -filled lumen
(L) (right) and the intestinal cytoplasm (left) seem to be in direct contact along an ill-defined inter-
face, with no obvious structure to divide them (Image source: [Hall] N833 W090). Bar, 1 μm
uterus or spermatheca or cross into cell cytoplasm along the alimentary canal,
infecting the marginal cells of the pharynx [50]. Enlargement of the bacterial clumps
over time suggests bacteria are able to divide inside the C. elegans body. Bacterial
cells are occasionally found within the microvilli bed and may contribute to their
destruction in patches (Fig. 2.11e–h). Studies showed that while the most decrepit
30 L.A. Herndon et al.
of the ageing animals tended to show more severe villar degeneration, sometimes
healthy microvilli were found in areas with significant intestinal distortion [50].
Conversely, some relatively healthy animals show early shortened villi phenotypes
in their intestinal cells. After much searching by TEM, we have still not found cases
where bacteria have succeeded in penetrating into the intestinal cytoplasm in aged
adults, although they must occur eventually. Even where all villi have been degraded,
the terminal web still represents a barrier to entry (Fig. 2.11f–h).
The C. elegans excretory system carries out several functions, including concentrat-
ing and expelling metabolic waste, regulating internal osmolarity, and expulsion of
exsheathment fluid after moults and hormone secretion [52]. The four cell types that
make up the excretory system are: (1) a large, H-shaped excretory canal cell extend-
ing canals bilaterally along the length of the animal, (2) a pulsatile excretory duct
cell, (3) a pore cell, and (4) two fused gland cells [52]. The normal organization and
appearance of this system in the young adult have been well illustrated in WormAtlas.
The excretory system cells are heterogeneously affected during ageing. Most com-
monly, the canal cells appear swollen or become cystic in appearance (Fig. 2.12).
Side branches may form in the canal cell lumen. The excretory gland cells may
become enlarged in older animals (not shown). The duct and pore cells must remain
intact and somewhat functional or the animal should quickly die from a fluid imbal-
ance [53]. The sudden death of some ageing animals, often typified by a “straight-
rod” death posture, may be attributed to failure of the excretory system.
The C. elegans body lacks specialized vasculature and blood cells, but nutrients and
debris can move throughout the body via the pseudocoelom, the contents of which
are distributed by internal pressure changes during locomotion. The pseudocoelom
occupies the interstitial spaces of the main body cavity, between the apical intestinal
borders and the cells lining the cuticle. Since the pseudocoelom is a fluid-filled
space, it lacks structural elements, except for the mesh-like basal lamina. The only
cells that specifically occupy the pseudocoelom are the coelomocytes. These six
cells move in limited fashion within the body cavity, removing detritus and foreign
materials from the pseudocoelom by phagocytosis (cf. [54]).
2 Effects of Ageing on the Basic Biology and Anatomy of C. elegans 31
As the cells bordering the pseudocoelomic cavity change with age, they infringe
into the pseudocoelomic space or withdraw from it. This causes the pseudocoelomic
cavity to become progressively distorted as the result of changes at its borders.
Shrinkage causes some cells to shed their basal laminae, which fold into loops and
whorls that can be found floating within the pseudocoelom (Fig. 2.5c, d). The
a b
Hy
po
de
rm
is
am
Se
m
elo
co
do
eu
Ps
Gap junction
Basal lamina
c d
Fig. 2.12 Excretory canal cell structural changes during ageing. (a) Schematic cross-sectional view
of one excretory canal cell arm. Canal cells extend lengthwise bilaterally in the body wall from head
to tail, closely apposed to the hypodermis and in register with cuticle alae over most of its length.
Abundant large gap junctions link the canal arms to the hypodermis, presumably to allow exchange
of small molecules and perhaps fluid. The canal cell has a single central lumen and many smaller
canaliculi that connect to the lumen. Any other cytoplasmic organelles tend to be excluded by the
lumen and canaliculi, coming to rest at the periphery of the excretory canal. The basal lamina of the
hypodermis is shared with that of the excretory canal cell where they face the pseudocoelom (Image
source: [WormAtlas]). (b) TEM cross-section of a canal cell in a young adult showing uniformly-
sized canaliculi surrounding a central lumen. Here the canaliculi seem disconnected from their
neighbours. In other sections, the canaliculi may appear as short chains of pearls, linking to each
other and to the lumen (Image source: [Hall] N506 Z805). Bar, 5 μm. (c, d) Canal cells in two dif-
ferent 15-day old adults, showing development of multiple lumens (c), and/or a smaller lumen and
large vacuoles (d), which might be enlarged canaliculi or endosomes. The canal has not shrunken in
size so much as the hypodermis, and is sometimes left to float on its own within the pseudocoelom
due to recession of the hypodermis (c). White arrowheads indicate gap junctions; black arrow indi-
cates basal lamina (Image sources: [Hall] (c) N813 G501; (d) N805 G490). Bars, 1 μm
32 L.A. Herndon et al.
2.3.10 Germline
The hermaphrodite reproductive system produces mature gametes and also provides
the structure and environment for fertilization, early embryonic development and
egg-laying. The C. elegans reproductive system consists of three major regions: (1)
the somatic gonad, including the distal tip cell (DTC), gonadal sheath, spermatheca
(sp), spermathecal-uterine (sp-ut) valve, and uterus; (2) the germline with mitotic
and undifferentiated cells in the distal region that become meiotic and specialized as
they progress through the proximal arm; and (3) the egg-laying apparatus, consist-
ing of the vulva, uterine and vulval muscles and specialized neurons (Fig. 2.13a, b).
In hermaphrodites, sperm production occurs in larval stages only, since at the adult
moult, germline precursors switch to forming oocytes. However, males produce
sperm continuously throughout adulthood.
Hermaphrodites produce viable embryos for about 1 week following the L4-to-
adult moult. As hermaphrodites grow older, progeny production declines sharply
due primarily to sperm depletion. Unfertilized oocytes accumulate in the uterus and
cause noticeable swelling in the hermaphrodite’s midbody (Fig. 2.13c). The oocytes
undergo nuclear endoreduplication and produce large masses of chromatin sur-
rounded by complex cytoplasm. In some regions, the borders between oocytes dis-
appear as they merge into syncytial masses. Within these syncytial zones, enlarged
nuclei aggregate and form chromatin-filled nuclear masses, which may be separated
from one another by intact nuclear membranes (Fig. 2.14c.d) [55, 56]. Cellular
debris from degrading oocytes eventually blocks the vulval opening to the exterior
and also impairs the egg-laying muscles (Figs. 2.13c and 2.14b). In very old her-
maphrodites, germline tumours begin to comprise separate sectors containing endo-
reduplicating oocytes, degenerating cells and nuclei, masses of chromatin, and a
few trapped embryos in various stages of morphogenesis (Hall, unpublished data).
2 Effects of Ageing on the Basic Biology and Anatomy of C. elegans 33
b Embryos
Uterus Vulva
DG germ line
(bare region)
op
DTC
lo
PG oocytes
uterus
vulva
c young adult
7 days
15 days
Fig. 2.13 Ageing-related changes in the germline and gonad. (a) Adult hermaphrodite, lateral
view, left side, showing the location of the reproductive system within an intact animal. The repro-
ductive system has twofold symmetry and consists of two U-shaped gonad arms joined to a com-
mon uterus. The reproductive system opens to the environment via the vulva, located in the ventral
midbody. The distal portion of each gonad arm lies dorsally, with a cluster of immature germ cells
surrounding a central rachis, to which each germ cell is linked via an open syncytial connection.
The proximal portion of each gonad arm lies ventrally, where single large oocytes are surrounded
by thin somatic sheath cells (Image source: [WormAtlas]). (b) One half of the reproductive system,
enlarged and separated from other body parts (see rectangle in a). DTC Distal tip cell, DG distal
gonad, PG proximal gonad, sp spermatheca, sp-ut spermathecal-uterine valve. Germline tissues
are shown in dark blue, somatic gonad in purple, uterine muscles in green, spermatheca in blue,
uterus in pale blue (Image source: [WormAtlas]). (c) Illustration showing progressive changes in
the germline with age. In young adults, eggs are fertilized as they pass through the spermatheca. In
middle-aged adults, egg-laying declines and fertilized and unfertilized embryos can collect in the
uterus. In older adults, complex germline masses can eventually expand to fill much of the body
cavity of the animal
34 L.A. Herndon et al.
Fig. 2.14 Ageing adults develop germline masses of electron-dense acellular material. (a) TEM,
transverse section, of a young adult hermaphrodite at low magnification. The distal portion of the
gonad arm (dorsal) consists of thin gonadal sheath cells surrounding a syncytium of germ cells that
are attached to a central cytoplasmic core (the rachis). The proximal region of the gonad (ventral)
consists of a thicker gonadal sheath surrounding the oocyte. BWM body wall muscle, hyp hypoder-
mis (Image source: N533 [Hall] F560). (b) A cross-section of the midbody in a 15-day-old in
which germline tissue occupies more than 90 % of the total volume, with intestine, body wall
muscle (BWM) and hypodermis (hyp) pushed to thin slivers at the periphery. Few normal oocytes
remain, separated from the spermatheca by a large, complex germline tumour. The tumour includes
a massive overgrowth of tightly compacted nuclear material that may be rigid enough to impair
locomotion (Image source: [Hall] N816 H027). Bar, 10 μm. (c) Low power TEM image shows a
portion of a germline tumour in a 15-day adult enclosed by a thin gonadal sheath cell (black
arrows). Nearby pseudocoelom is filled with excess lipid (L) and yolk (Y). Within the tumour there
are regions jammed with many nuclei and regions of complex cytoplasm, but no obvious maturing
oocytes (Image source: [Hall] N801 E565). (d) Boxed region in (c) is shown at higher magnifica-
tion. Each asterisk indicates a nucleus separated from other nuclei by membranes. Bar, 1 μm
The expanding germline tumour can eventually fill up to 90 % of the animal, com-
pacting the intestine and other body tissues.
In fertile young adults, yolk is produced in the intestine and transported through
the pseudocoelom to the germline, where it is absorbed by oocytes [46, 57]. As
fertility declines during ageing, defective oocytes no longer absorb the yolk, which
progressively accumulates as extracellular deposits in the pseudocoelom [8, 21, 50,
57]. There is apparently no negative feedback to intestinal yolk production, which
2 Effects of Ageing on the Basic Biology and Anatomy of C. elegans 35
continues throughout adulthood. Virtually none of this yolk lies within the germline
tumour itself, as the yolk cannot be transported into the gonad except by endocyto-
sis into a viable primary oocyte [57]. Further discussion of the germline in the con-
text of reproductive ageing can be found in Chap. 7.
2.6 Conclusions
Grounded in its simple, reproducible body plan and small size, working knowledge
of tissue origin and maintenance of C. elegans anatomy is unparalleled in the animal
world. Still, it is clear that our understanding of age-associated changes in this facile
animal model remains primitive. Systematic, high-resolution, detailed studies of
tissue changes over time could anchor critical investigations of tissue-specific age-
ing, while also providing information on stochastic occurrences of specific changes.
A striking gap is our limited understanding of how physical changes within indi-
viduals relate to overall ageing of the animal or its behaviour. Current research has
barely scratched the surface on the effort towards linking genetic or environmental
influences, thought to change ageing quality, with features of tissue-specific decline.
Acknowledgments We are grateful to the help of Zeynep Altun and Chris Crocker in designing
the schematic cartoons in this work. We gratefully acknowledge funding from NIH OD 010943 (to
DHH) and 1R01AG046358 (to MD).
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Chapter 3
Dauer Formation and Ageing
3.1 Introduction
In the natural environment, the dauer larval stage of C. elegans allows the animal to
survive adverse conditions, while seeking out new food sources [1]. Dauer larvae
are extremely long-lived, surviving for up to 70 days [2], compared with 10–15 days
for the adult animal [3]. However, the adult lifespan of animals that have recovered
from dauer is essentially the same, irrespective of the amount of time spent in dauer
arrest [3]. These observations led to the conclusion that “dauer larvae seem to be
able to reduce or suspend the rate of ageing” and that analysis of the dauer larva
“may offer valuable insights into the controlling mechanisms of ageing” [3].
Due to the limited availability of energy resources in the wild, organisms must
decide to direct their efforts toward reproduction or somatic maintenance [9]. In the
event that environmental conditions are not conducive to survival of the offspring,
animals may suspend reproduction and focus on survival, in order to produce off-
spring at a later stage when the environment is favourable. To facilitate this, several
organisms have developed specialized diapause states in which somatic mainte-
nance is favoured over reproduction [10].
Under conditions of plentiful food, low temperature and low population density,
C. elegans will develop from egg to adult through four larval stages that are punctu-
ated by moults. However, if the developing L1 larva encounters an environment
with diminishing food availability, high temperature and/or high population density
it can divert development towards formation of the dauer larva. Dauer development
involves a longer second larval stage, termed L2d or predauer, followed by moulting
into the dauer larva itself. Once the dauer larva encounters a more favourable envi-
ronment, i.e. one that can support reproductive growth, it will exit the dauer stage
directly into the L4 stage and subsequently develop into a fertile adult.
The decision to commit to dauer entry is critical to the reproductive success of
the animal. Failure to form dauers in an adverse environment will leave progeny at
a developmental disadvantage. In contrast, if dauer development occurs under con-
ditions that support reproductive growth, reproductive potential will be jeopardized.
In this respect, the ability of the animal to exit the L2d stage prematurely and enter
the L3 stage provides a means of escaping dauer commitment. In fact, it has been
proposed that in uncertain environments the L2d stage is favoured as it provides a
greater degree of developmental flexibility [11]. The importance of making the
3 Dauer Formation and Ageing 43
During the dauer stage worms are stress resistant [1] and live roughly four times
longer than a reproductive adult [3]. Given that this diapause is specifically intended
to promote survival for long periods in unfavourable conditions, it is perhaps not
surprising that it is accompanied by a general upregulation of stress resistance
mechanisms that range from anatomical changes to altered signalling pathways
44 P. Reis-Rodrigues et al.
The primary environmental inputs that govern the decision to proceed with repro-
ductive growth or to enter the dauer stage are food availability, population density
and temperature. Some of the first Daf-d mutants showed chemotaxis defects and
their characterization indicated morphological abnormalities in the amphid sensory
neurons [25], indicating that one or more of the sensory neurons were involved in
detecting dauer pheromone. Subsequent analysis of other mutants defective in che-
mosensation revealed additional mutants with altered sensory sensilla that were also
Daf-d [26]. Interestingly, daf-19 mutants, which have defects in a transcription fac-
tor that is responsible for formation of all cilia, are Daf-c [27], suggesting that food
signals are also sensed by ciliated sensory neurons. The specific chemosensory neu-
rons involved in dauer formation were identified through an elegant series of experi-
ments using laser ablation of neurons, either singly or in combination [28]. Thus,
ADF, ASI, ASJ and to a lesser extent ASG are important for dauer entry, while in
contrast, dauer recovery is primarily determined by the presence of an intact ASJ
neuron [28].
After the initial description of a dauer-inducing pheromone extract [6], it was
another 20 years before the first chemical structure of the dauer pheromone was
determined [29]. It then became apparent that the dauer pheromone was not a single
chemical entity, but rather a mixture of small molecules that shared a similar chemi-
cal backbone of an ascarylose sugar unit coupled to a short chain fatty acid deriva-
tive [30, 31]. Following the identification of these ascarosides (ascr) as the bioactive
components of dauer pheromone a number of candidate pheromone receptors have
been proposed that are expressed in sensory neurons. Two GPCRs, DAF-37 and
DAF-38, have been shown to bind ascr#2 to promote dauer formation by repressing
TGF-β signalling [32], while two other GPCRs, SRBC-64 and SRBC-66, have been
shown to bind both ascr#2 and ascr#3 to induce dauer formation and likely function
upstream of TGF-β and insulin-like signalling [33]. Likewise, srg-36 and srg-37
encode GPCRs that are expressed in the sensory cilia of ASI neurons and mutations
3 Dauer Formation and Ageing 45
in these genes confer resistance to ascr#5, suggesting that they might bind this mol-
ecule to induce dauer formation [34].
In contrast to the progress in identifying the dauer pheromone, the chemical
identity of the food signal that promotes reproductive growth and dauer recovery [7,
35] still remains elusive. Although partial characterization suggested that a yeast
derived food signal was a nucleoside [7], the exact identity has yet to be determined.
Others have identified bacterially derived fatty acids as weak food signals and pro-
posed that, like pheromone, there is not one universal food signal but rather that
multiple food signals are likely to exist [36]. It has also been proposed that the food
signals that suppress dauer entry may be different from the food signals that pro-
mote recovery from the dauer larva [37].
Temperature has long been recognized as being a signal that promotes dauer
formation [35]. In wild type animals a small percentage of dauers can be observed
at 27 °C, even in the presence of ample food and the absence of pheromone [38].
The relationship between temperature and dauer formation was crucial in the isola-
tion of temperature dauer formation mutants [39]. Many of the original Daf-c
mutants were identified at 25 °C and found to recover and develop normally at lower
temperatures. Subsequently a number of other mutants were identified that grow
well at 25 °C but form dauers at 27 °C [38, 40]. It has been suggested that the pres-
ence of temperature-sensitive mutants was a reflection of dauer formation being a
temperature regulated process [39]. In C. elegans, temperature is sensed by the AFD
neuron and transduced by the AIY interneuron [41]. A mutation that affects the
function of the AIY neuron (ttx-3), and therefore disrupts the transduction of the
temperature signal, suppresses dauer formation in daf-7 mutants at high tempera-
tures and enhances dauer formation at low temperatures [42]. These data suggest
that the temperature sensing neurons can influence the activity of the endocrine
pathways involved in dauer formation. This may be mediated by changes in the
activity of heat-shock factor-1 [43].
Several endocrine signals have been identified that regulate dauer formation
and most of them emanate from the sensory neurons. The guanylyl cyclase
encoded by daf-11 is expressed in ASI and other chemosensory neurons and is
thought to be involved in second messenger signalling downstream of chemosen-
sory signalling that leads to secretion of insulin and TGF-β neuropeptides [44,
45]. daf-11 mutants are strongly Daf-c [44] and are suppressed by Daf-d mutants
with defective chemosensory cilia [46]. Further evidence for a neuroendocrine
component to dauer formation comes from studies of the calcium activated pro-
tein for secretion (CAPS) ortholog in C. elegans, encoded by unc-31 [47]. UNC-
31 is required for docking of dense core vesicles (DCVs), which contain
neuropeptides, at the cell surface and is expressed in neurons [48]. unc-31 mutants
are Daf-c at 27 °C indicating that neuropeptide secretion from DCVs is important
for reproductive growth [49]. Consistent with this, unc-31 mutants have been
shown to be defective in insulin release [50].
46 P. Reis-Rodrigues et al.
The insulin signalling pathway is defined by the insulin receptor ortholog DAF-2
[51] and its role in ageing is covered in detail in Chap. 4. Many components of the
signal transduction pathway downstream of daf-2, such as pdk-1 [52], akt-1 [53,
54], daf-18 [55] and daf-16 [56, 57] were defined through the identification of dauer
formation mutants. In contrast, age-1, which encodes a subunit of a PI3 kinase [54],
was originally identified in a mutant screen for longevity, and was subsequently
shown to be identical to daf-23 [58]. Many mutations that affect the insulin signal-
ling pathway confer a Daf-c phenotype (e.g. daf-2, pdk-1, age-1) while others result
in a Daf-d phenotype (e.g. daf-16, daf-18). daf-16 encodes an ortholog of the
FOXO3A transcription factor and is fully required for dauer formation in daf-2
mutants [56, 57]. Under dauer-inducing conditions DAF-16 translocates from the
cytosol to the nucleus and initiates programmes of gene expression required for
dauer entry.
Microarray studies have shown that reduced insulin signalling, and the conse-
quent activation of DAF-16, is associated with a set of genes that are upregulated
(Class I genes) and a set of genes that are down-regulated (Class II genes). While
the Class I genes mostly consist of stress response genes, the Class II genes are
mostly involved in regulation of development [59]. DAF-16 regulates transcrip-
tion in cooperation with another transcription factor, PQM-1 [60]. While DAF-16
promotes transcription of Class I genes involved in the stress resistance and lon-
gevity of dauers, PQM-1 regulates the Class II genes that are involved in develop-
ment [60]. Consistent with its role in promoting development, pqm-1 mutants
have been shown to have a delayed dauer recovery phenotype. DAF-16 and
PQM-1 nuclear localization and activity appears mutually exclusive and it is
unclear if the dauer recovery phenotype of pqm-1 mutants is due to DAF-16 activity
or loss of PQM-1 [60].
In contrast to the relative simplicity of insulin and insulin-like signalling in mam-
mals, in which insulin, IGF-I and IGF-II can bind to the insulin receptor, the role of
ligands for DAF-2 is complicated by the presence of 40 candidate insulin like pep-
tides (ILP) in the C. elegans genome [58, 61]. Systematic analysis of deletion
mutants, as well as RNAi studies, have indicated that a number of insulin peptides
regulate dauer entry and dauer recovery [59, 62, 63]. Several chemosensory neurons
express different complements of insulin peptides [64]. Of the chemosensory
neurons that are known to be important for promoting reproductive growth, the ASI
neuron expresses ins-4 [65], ins-6 [62] and daf-28 [66], and the ADF neuron
expresses ins-7 [67]. Simultaneous knock-down of ins-4, ins-6, and daf-28 generates
a fully penetrant Daf-c phenotype suggesting that these insulin peptides are the
principle regulators of reproductive growth [68], and these ligands are hypothesized
to act as agonist ligands for DAF-2. Human insulin, when expressed transgenically
in C. elegans, appears to act as a DAF-2 antagonist and enhances dauer formation
[61]. INS-1 and INS-18 are structurally most similar to human insulin and also
appear to act as antagonists [61, 68]. However, a direct interaction between a nema-
tode insulin peptide and DAF-2 either in vivo or in vitro is yet to be demonstrated.
3 Dauer Formation and Ageing 47
Epistasis studies defined a second pathway for dauer formation that acts in parallel
to the DAF-2/DAF-16 pathway. Subsequent cloning of these genes indicated that it
defines a TGF-β-like signalling pathway. daf-7 encodes a TGF-β-like neuropeptide
that is expressed exclusively in the ASI sensory neuron and DAF-7 levels are
responsive to food levels and downregulated by pheromone [69]. DAF-7 acts via a
heteromeric TGF-β receptor comprised of DAF-1 and DAF-4 which in turn influ-
ences the activity of the SMAD transcription factors DAF-8 and DAF-14 [70].
Hypomorphic and loss of function mutations in daf-7, daf-1, daf-4, daf-8 and daf-14
are Daf-c, and are suppressed by mutations in the co-SMAD daf-3 and the SNO/
SKI daf-5 which are Daf-d. Although daf-7 is primarily expressed in the ASI neuron
[69], the components of its signal transduction pathway are expressed throughout
the animal [70], supporting an endocrine role for TGF-β.
Evidence for the TGF-β and insulin signalling pathways acting in parallel came
from the observations that the Daf-c phenotype of daf-2 mutants could be sup-
pressed by Daf-d mutations in daf-16 but not by mutations in daf-3 or daf-5 [71].
Conversely, daf-3 and daf-5 mutations, but not daf-16 mutations, were able to sup-
press Daf-c mutants in the TGF-β pathway. However, we now know that two major
endocrine signalling mechanisms are initiated from TGF-β signalling through DAF-
3; the regulation of DAF-12 ligands and the expression of insulin peptides [20, 72].
Insulin signalling activity is also influenced by the TGF-β pathway at the level of
signal transduction cross talk [73].
3.3.5 HEN-1/SCD-2
Another, less well-defined, pathway that acts upstream of DAF-3 involves the tyro-
sine kinase receptor SCD-2 and its putative ligand HEN-1 [74]. scd-1, scd-2 and
scd-3 mutations were previously identified as suppressors of the TGF-β pathway
[75]. None of these gene mutations suppress the Daf-c phenotype of daf-2 mutants,
indicating that they function in parallel to insulin/IGF signalling. Mutations in the
scd-2 gene, which encodes a receptor tyrosine kinase orthologous to anaplastic lym-
phoma kinase, lead to dauer formation in response to pheromone at 27 °C, but not
25 °C [74]. The epistatic pathway includes hen-1 (a putative ligand), soc-1 (a recep-
tor tyrosine kinase adapter) and sma-5 (MAP kinase), as well as daf-3. Mutations in
this pathway are thereby Daf-d and are able to suppress the Daf-c phenotype of
daf-7 and daf-8 mutations. Furthermore, the hen-1/scd-2 pathway regulates DAF-3
transcriptional activity [74]. Taken together, these data place hen-1/scd-2 in a new
pathway that influences dauer formation by modulating TGF-β signalling at the
level of DAF-3.
48 P. Reis-Rodrigues et al.
The terminal portion of the dauer formation pathway is defined by the cytochrome
P450 DAF-9 and the nuclear hormone receptor DAF-12. Mutations in daf-9 are
Daf-c [76], while the majority of daf-12 loss-of-function mutations are Daf-d [77].
DAF-9 is involved in the synthesis of a steroid hormone that acts as a ligand for
DAF-12 [78, 79]. Other dauer genes that influence the production of this ligand
have been placed in this arm of the dauer formation pathway [80–82].
daf-9 mutations are fully suppressed by Daf-d daf-12 mutations, but not by daf-
16 or by daf-3 and daf-5 mutations [76, 83]. Additionally, daf-12 mutations fully
suppress the Daf-c mutations of the TGF-β pathway but only partially suppress
Daf-c mutations in the insulin signalling pathway, indicating that daf-12 acts down-
stream of daf-7 but in parallel to daf-2 [76, 83]. Taken together, these studies sug-
gest that daf-9 acts upstream of daf-12 and downstream of the TGF-β signalling
pathways.
During development DAF-9 is expressed exclusively in the XXX cells [76, 84],
a pair of neuron-like cells derived from the hypodermal lineage that have a thin,
flattened projection adjacent to the pseudocoelomic space. The spatial arrangement
of chemosensory neurons and the XXX cell has led to the current model that exter-
nal signals are transduced by chemosensory neurons to ultimately influence the
secretion of DAF-7/TGF-β and insulin peptides. Insulin peptides have been hypoth-
esized to act on DAF-2 receptors that are expressed on the XXX cells. Indirect evi-
dence for this comes from daf-2 transcriptional reporters that indicate that daf-2 is
expressed in XXX cells [85] as well as from studies of the sdf-9 gene. sdf-9 encodes
as phosphatase that is expressed exclusively in the XXX cells and sdf-9 mutants
form partial dauers that are suppressed by daf-16 mutations [86]. SDF-9 has been
proposed to interact directly with DAF-2 to stabilize the active state of the receptor
or to act as an adapter protein [87]. It is yet to be determined whether the XXX cells
are a direct target of TGF-β signalling [88].
The sequence homology of daf-9 and daf-12 with cytochrome P450s and nuclear
hormone receptors respectively indicated that DAF-9 synthesizes a steroid-like hor-
mone that acts as a ligand for the DAF-12 nuclear receptor. Since the identity of
such a ligand could not be determined by genetic means alone, there was a great
deal of effort directed towards identifying this ligand using chemical genetic
approaches [89, 90]. Two 3-ketocholestenoic acids, termed dafachronic acids (DA),
isolated from C. elegans sterol extracts that could transactivate DAF-12 in a cell
culture based assay were proposed as DAF-12 ligands [78]. Shortly afterwards Held
et al., using a candidate approach, determined that 3-hydroxy cholestenoic acid
could also act as a DAF-12 ligand [79]. While the ∆7ketocholestenoic acid is likely
to be an endogenous ligand for DAF-12 [78], subsequent work has indicated that
multiple DAF-12 ligands can be detected in the worm, including
3-hydroxycholestenoic acids [91].
Although dafachronic acids are proposed to act as endocrine signals, it appears
that they may act more like a paracrine signal in vivo. daf-9 expression propagates
3 Dauer Formation and Ageing 49
An integrated model of how the sensory inputs and signalling pathways are inte-
grated to promote reproductive growth or promote dauer formation is shown in Fig.
3.1. Under conditions of high food, low pheromone and low temperature, DAF-7
and insulin peptides are secreted from ASI and ADF (Fig. 3.1a). Activation of the
TGF-β signalling pathway in target tissues leads to phosphorylation of the SMAD
transcription factors DAF-8 and DAF-14. A transcriptional complex of DAF-8,
DAF-14 and DAF-3 directs programmes of gene expression that promotes repro-
ductive growth. Activation of DAF-2 by insulin peptides results in a signalling cas-
cade that ultimately leads to phosphorylation of DAF-16 and its retention in the
cytoplasm, where it is transcriptionally inactive. Dafachronic acids produced by
DAF-9 are thought to bind to DAF-12 and DAF-12 activity upregulates daf-9
expression in a positive feedback loop [92].
Under dauer inducing conditions, DAF-7 expression in ASI is reduced (Fig.
3.1b). Expression of some insulin peptides are reduced, but others, that presumably
act as DAF-2 antagonists, are increased [20]. Reduced signalling through the TGF-β
signalling pathway results in dephosphorylation of DAF-8 and DAF-14 and the
DAF-3/DAF-5 complex directs gene expression programmes that promote dauer
formation. Reduced signalling through DAF-2 results in dephosphorylated DAF-16
entering the nucleus. Activated DAF-16 downregulates daf-9 mRNA levels in some
models of dauer formation, indicating that it might regulate its activity [95]. The
consequent decrease in DA allows unliganded DAF-12 to recruit the transcriptional
co-repressor DIN-1 [93], leading to down regulation of gene expression of genes
involved in promoting reproductive growth [96]. DAF-12–DIN-1 silences genes
required for reproductive growth [96], while DAF-16 activation upregulates genes
involved in stress resistance and longevity [59].
When the dauer larva encounters conditions that are conducive for reproductive
growth it will exit the dauer stage. In contrast to reproductive growth from L1 to L2,
TGF-β does not appear to be important for dauer recovery, as ablation of the ASI
50 P. Reis-Rodrigues et al.
XXX XXX
DAF-9 DAF-9
INS-7 DAF-7
DA
DAF-28
DAF-12 DAF-12
P
DA DIN-1
DAF-8 DAF-3
X DAF-14 DAF-12 DAF-16 DAF-5 X DAF-12 X
STRESS GROWTH GROWTH STRESS GROWTH GROWTH
GENES GENES GENES GENES GENES GENES
Fig. 3.1 (a) Environmental and genetic interactions that lead to reproductive growth. (b)
Environmental and genetic interactions that lead to dauer entry
neurons does not affect dauer exit [28]. However, the ASJ neurons become more
important, due to the requirement for INS-6 in promoting dauer recovery [28, 62].
Importantly for this chapter, genes involved in stress response (e.g. daf-21, hsp-20,
sod-3 and ctl-1) seem to be upregulated in dauer larvae and their expression is
reduced upon exit [17]. Lastly, epigenetic modifications take place in dauer larvae
that are evident in adult animals that have been through dauer but not present in
animals that have never been in dauer [97].
One of the key discoveries that linked dauer formation to adult longevity was the
observation that temperature-sensitive daf-2 mutants that developed into adult ani-
mals at the permissive temperature were long-lived [8]. Furthermore, the longevity
of daf-2 mutants is fully dependent on the presence of functional DAF-16, since
daf-2;daf-16 mutants are not long-lived [8]. Subsequent work demonstrated that
3 Dauer Formation and Ageing 51
other Daf-c mutants in the insulin signalling pathway also exhibit increased lifespan
[71]. In parallel, studies of the age-1 gerontogene [98] indicated that age-1 and daf-
23 act in the same pathway [99] and are in fact the same gene [58]. Thus, the first
forward genetic screen for longevity, performed by Klass [100], had identified a
component of the dauer formation pathway. Since then many genes and processes
involved in dauer formation have been implicated in ageing (Fig. 3.2).
Daf-d mutants that have defects in the structure of ciliated neurons are also long-
lived, indicating that C. elegans lifespan is regulated by sensory perception of sig-
nals from the environment [101]. In most of these mutants the lifespan extension is
partially daf-16 dependent [101], suggesting that the sensory defects that mediate
ASG ASI X
ASG X
ASI
DAF-7
INS?
P
DAF-16 DA
DA DIN-1
X DAF-12 DAF-16 DAF-12 X
STRESS NORMAL STRESS NORMAL
LONGEVITY LIFESPAN LONGEVITY LIFESPAN
GENES GENES GENES GENES
Fig. 3.2 (a) Dauer genes involved in normal lifespan. (b) Dauer genes involved in longevity
52 P. Reis-Rodrigues et al.
lifespan extension not only act via insulin signalling but also via an additional
mechanism. Laser ablation of specific neurons shown to be important for dauer
formation [28] also leads to extended adult lifespan in a daf-16 dependent manner
[102]. Interestingly, while ADF and ASI play a major role and ASG plays a minor
role in dauer formation [28], ablation of ASI and ASG neurons alone extend lifes-
pan, while loss of ADF has no effect [102]. In addition to the gustatory neurons ASI
and ASG, ablation of the AWA olfactory neuron extends lifespan but via a mecha-
nism that involves the somatic gonad [102].
Alcedo and Kenyon also considered whether the dauer pheromone influenced
adult lifespan, since it had been proposed to repress the activities of the ASI, ADF
and ASG. However, they found no effect of crude dauer pheromone extracts [102].
In contrast, treatment of worms with different combinations of purified ascarosides
isolated from dauer pheromone has been shown to extend adult lifespan in a sir-2.1-
dependent manner [103].
Nutrient availability affects both dauer formation and longevity. During develop-
ment, low food availability induces dauer formation, while in adults dietary restric-
tion (DR) induces longevity (see Chap. 16). Although different DR methods have
different dependencies [104], these likely arise from different sensory inputs that
remain largely undefined. Some methods of DR require daf-16, clearly indicating a
commonality with insulin-like signalling [104]. However, it has also been shown
that dafachronic acid production is required for dietary restriction-induced longev-
ity through a mechanism that is independent of DAF-12, but dependent on the
nuclear hormone receptor NHR-8 [105].
In addition to external signals of nutrient availability that act through sensory
neurons, dauer formation and longevity are likely influenced by internal signals of
nutrient status. However, the identity of these endogenous signals and their mecha-
nism of action remain enigmatic. A class of lipid-derived signalling molecules
called N-acylethanolamines may provide a link between internal nutrient sensing,
dauer formation and longevity [106]. Eicosapentaenoyl ethanolamine (EPEA), an
N-acylethanolamine produced in worms, not only suppresses dauer formation in
both insulin signalling and TGF-β mutants, but also counteracts dietary restriction-
induced longevity [106].
Following the discovery that daf-2 mutants are long-lived, other Daf-c mutants from
the insulin signalling pathway that are epistatic to daf-16 were found to be long-
lived [52, 71, 107]. A more detailed discussion of the role of insulin signalling in
ageing can be found in Chap. 4. A key finding that is relevant to this chapter is that
reduction of insulin signalling only in adult animals is sufficient to extend adult
lifespan [108]. Moreover, if the activity of the insulin signalling pathway is reduced
only during development and then restored in adulthood, lifespan is not extended,
thus separating developmental effects from ageing effects.
3 Dauer Formation and Ageing 53
The early longevity studies with Daf mutants suggested that lifespan extension is
restricted to the Daf-c mutants from the insulin signalling pathway. However, more
than a decade later, Murphy et al. compared the gene expression profiles of mutants
from the TGF-β pathway with long-lived insulin receptor mutants in order to sepa-
rate out those genes that are dauer specific [72]. It turns out that there is very little
correlation between those genes regulated by the TGF-β pathway during dauer for-
mation and those regulated in adult animals. In fact, there is a greater degree of
correlation between long-lived IR mutant adults and adult animals carrying muta-
tions in TGF-β signalling components. The previous failure to observe a longevity
phenotype was attributed to the fact that daf-7, and other TGF-β mutants, tend to
retain their eggs, which in turn leads to internal hatching and subsequent matricide.
When these mutants were grown on 5-fluorodeoxyuridine (FUDR) to inhibit prog-
eny production, the longevity phenotype emerged. Interestingly, the lifespan exten-
sion of TGF-β pathway is dependent on daf-3 and daf-16, suggesting that daf-3
activity regulates insulin secretion which, in turn, affects activity of the insulin/IGF
receptor and consequently, daf-16 [72].
12 [113]. Taken together, these studies suggest a component of the effect of steroid
signalling on lifespan may be related to adaptation to different temperatures.
Another robust mechanism of lifespan extension in C. elegans occurs when the
germline precursor cells are ablated [114]. A more detailed discussion of this topic
is provided in Chap. 6. This mechanism of lifespan extension is dependent on daf-
16, daf-9 and daf-12 activity, suggesting the existence of a steroid hormone that
mediates this longevity [114]. In adults, daf-9 expression appears in the sperma-
theca but it is not known if DA is the exact product of DAF-9 in this situation or
whether an alternative product of DAF-9 is responsible for lifespan extension.
However, DA supplementation to germline ablated and somatic gonad ablated
worms does extend lifespan in a manner dependent on daf-12, with no effect
observed in animals where the somatic gonad is intact, suggesting that this tissue
may suppress the germline effects of DA on longevity [115].
daf-12 mutations have differing effects on longevity of daf-2 mutants, according to
the nature of the daf-2 defect. Class I daf-2 mutants are Daf-c and long-lived, with
little larval arrest [116] and their molecular lesions tend to cluster in the extracellular
ligand binding domain of the nematode insulin receptor [117]. Class II daf-2 mutants
share class I defects but also exhibit other pleiotropic phenotypes, including reduced
adult motility, high levels of embryonic and L1 arrest, reduced brood size and late life
progeny [116]. The increased severity of the phenotypes of the Class II mutants gener-
ally correlates with their molecular lesions being more severe, with many affecting the
tyrosine kinase signalling domain [117]. Interestingly, certain daf-12 alleles are able
to suppress the longevity of Class I daf-2 mutants but confer a synergistic increase in
lifespan when combined with Class II daf-2 mutants [71, 116].
Others have found that longevity of Class I daf-2 mutants is suppressed when DA
biosynthesis is compromised or when DAF-12 activity is completely abolished,
suggesting that liganded DAF-12 promotes longevity [118]. If daf-2 activity is
reduced using RNAi, which results in a milder reduction in function, both liganded
and unliganded DAF-12 promote longevity. However, in Class II daf-2 mutants,
both liganded and unliganded DAF-12 act in opposition to control lifespan [118].
Taken together these data suggest that the relationship between steroid hormone
signalling and lifespan extension is complex and context specific. This is perhaps in
part due to our lack of knowledge regarding the small molecule landscape in C.
elegans. Based on gene expression studies, and the molecular identities of some of
the target genes of DAF-16 [59] and DAF-12 [96], it is likely that other small mol-
ecules, including steroids, play a role in conferring lifespan extension. Future
approaches that combine molecular genetics, analytical chemistry and comparative
metabolomics will be required to comprehensively identify these small molecules.
The discovery that post-dauer adult lifespan is not affected by the amount of time that
the animal spends in dauer led to the early suggestion that the dauer stage is essen-
tially non-ageing [3]. Thus, when it was discovered that Daf-c mutations confer
3 Dauer Formation and Ageing 55
lifespan extension in animals that have never been arrested as dauers [8], a reasonable
conclusion was that the longevity was due to a mis-expression of dauer programmes
in adult animals. To some extent this hypothesis is supported by the observations that
daf-2 dauers and long-lived daf-2 adults have largely similar transcriptional profiles
[18, 19, 119]. However, recent data suggests that it may not be as simple as this. When
IIS is reduced at temperatures of 20–25 °C a number of dauer-like traits are induced
and lifespan extension primarily requires daf-16, but not on the Nrf-like transcription
factor, skn-1. However, when IIS is reduced at 15 °C, dauer-like traits are not induced
and lifespan becomes fully dependent on skn-1 [120]. This suggests that the down-
stream processes that confer lifespan extension are highly context specific. The study
of dauers at colder temperatures is impractical due to their propensity to exit the stage
at lower temperatures. Nevertheless, genes that confer stress resistance and longevity
in the dauer stage, may inform on condition specific mechanisms of longevity.
In the case of temperature-dependent survival, daf-2 mutation has also been
shown to improve resistance to low temperatures [121], although the specific factors
that differentially contribute to each case are largely unexplored. One exception is
trpa-1, the ortholog of TRPA in humans, which downregulates insulin signalling
through sgk-1 to activate DAF-16, specifically at colder temperatures, leading to
lifespan extension [122]. In this way, long-lived dauer mutants reveal genetic path-
ways and stress defence mechanisms that inform on adult longevity even in condi-
tions where the animals are not predisposed to dauer formation. It is likely that some
adult dauer mutants may or may not share traits with dauers, but the mechanisms
that are employed during the dauer stage to increase stress resistance and lifespan
extension are important tools to modulate adult ageing in diverse contexts.
The identification of single gene mutations that extend lifespan in C. elegans raised
the possibility that the ageing process could be dissected using genetic analysis [8,
98, 100]. However, it was the discovery that daf-2 encodes an insulin/IGF-like
receptor [51] that really revolutionized the field of gerontology, as it provided the
foundation for the idea that genetic analysis of lifespan in model systems could lead
to the identification of evolutionarily conserved pathways that could be targeted
therapeutically to improve human healthspan and ageing [123].
Of the several pathways involved in dauer formation, the activation of DAF-16
and subsequent expression of stress-resistance genes is perhaps the most-well stud-
ied in the context of ageing research. Although activation of DAF-16 was originally
identified as a requirement for longevity under low IIS conditions, several other
longevity inducing conditions seem to require this transcription factor. Notably,
DAF-16’s mammalian ortholog, FOXO3A, also seems to play an important role in
longevity and healthspan . In mammals, reduction of insulin signalling specifically
in fat tissue confers longevity [124]. Moreover, genetic association studies have
implicated specific single nucleotide polymorphisms of FOXO3A in human longev-
ity [125, 126].
56 P. Reis-Rodrigues et al.
Fig. 3.3 Many processes related to ageing are impacted by genes involved in dauer formation in
C. elegans
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Chapter 4
Longevity Regulation by Insulin/IGF-1
Signalling
Abstract For the past three decades, many ageing-regulatory pathways have been
identified using C. elegans as a model organism. The insulin/insulin-like growth
factor (IGF)-1 signalling (IIS) pathway is one of the most evolutionarily well-con-
served ageing-regulatory pathways ranging from worms to mammals. Here, we
review the molecular mechanism and the functional significance of IIS in C. elegans
ageing. Specifically, we describe the roles of key components of IIS in ageing, sys-
temic ageing regulation by IIS, and other known physiological functions of IIS that
contribute to longevity. We also discuss possible implications of IIS in mammalian
health and ageing.
4.1 Introduction
The IIS pathway is composed of various signal-transducing factors, and the role of
each component in lifespan regulation is relatively well-characterized in C. elegans
(Fig. 4.1). age-1 mutants were the first long-lived IIS mutants identified through a
genetic screen [3, 4]. Subsequently, daf-2 mutants, which have been known to dis-
play phenotypes in the development of dauer (an alternative diapause larva, dis-
cussed in Chap. 3), were shown to live twice as long as wild-type C. elegans [5].
age-1 and daf-2 were eventually shown to encode a phosphoinositide-3 kinase
(PI3K) and an insulin/IGF-1 receptor, respectively [6, 7]; these are the key upstream
components of IIS. Since then, many more factors that act downstream of the IIS
pathway have been identified in C. elegans.
Inhibition of IIS promotes long lifespan in C. elegans. Specifically, the reduced
function of DAF-2 results in the inactivation of the downstream kinase cascade,
starting from AGE-1/PI3K [[8]; reviewed in [9]]. Down-regulation of AGE-1 then
leads to the inactivation of 3-phosphoinositide-dependent kinase 1 (PDK-1) [10],
likely through a decrease in the PI(3, 4, 5)P3/PI(4, 5)P2 ratio [11]. This, in turn,
Fig. 4.1 Reduced IIS increases lifespan in C. elegans. Inhibition of DAF-2/insulin/IGF-1 recep-
tor decreases the PI(3,4,5)P3/PI(4,5)P2 ratio through down-regulation of AGE-1/PI3 kinase, whose
function is antagonized by the activation of DAF-18/PTEN. This decrease leads to the inactivation
of PDK-1 and AKT-1/2, which subsequently promotes the nuclear translocation and activation of
DAF-16/FOXO, and SKN-1/NRF2 transcription factors. HSF-1/heat shock factor 1 also collabo-
rates with DAF-16 in the nucleus. These transcription factors regulate the expression of various
genes that contribute to longevity in C. elegans
4 Longevity Regulation by Insulin/IGF-1 Signalling 65
down-regulates the Akt/protein kinase B (PKB) family members, AKT-1 and AKT-2
[10, 12]. The PI(3, 4, 5)P3/PI(4, 5)P2 ratio can also be decreased by the activation of
DAF-18/phosphatase and tensin (PTEN) phosphatase, which mediates dephosphor-
ylation of PI(3, 4, 5)P3 and increases lifespan [8, 13–17]. Down-regulation of IIS
also leads to the activation of transcription factors, which up-regulate the expression
of various target genes that contribute to longevity, including chaperones, antioxi-
dants, and antimicrobials. The representative longevity transcription factors down-
stream of IIS are DAF-16/Forkhead box O (FOXO), heat-shock transcription
factor-1 (HSF-1), and skinhead-1 (SKN-1)/Nuclear factor-erythroid-related factor
(Nrf).
DAF-16 DAF-16 is a FOXO transcription factor homologue [18, 19] that mediates
a diverse array of cellular processes by regulating the expression of numerous genes,
including those involved in ageing [20–25]. A variety of post-transcriptional regula-
tors of this protein, including protein kinases and phosphatases, have been identi-
fied. Both AKT-1 and AKT-2 phosphorylate and inactivate DAF-16 by preventing
nuclear translocation [26–30]. Phosphorylation of DAF-16 by serum/glucocorticoid-
inducible kinase 1 (SGK-1)/SGK was also shown to obstruct the translocation into
the nucleus [30]. However, subsequent studies using a sgk-1 gain-of-function
mutant or overexpression of sgk-1 indicate that SGK-1 may activate DAF-16 [31,
32]. AMP (5′ adenosine monophosphate)-activated protein kinase (AAK-2) can
also activate DAF-16 by phosphorylation and increases lifespan [33–36]. Similarly,
CST-1/MST kinase and JNK-1/c-Jun N-terminal kinase phosphorylate and up-
regulate DAF-16 to extend lifespan [37, 38]. Protein phosphatases also appear to
regulate the activity of DAF-16 directly or indirectly. For example, SMK-1/suppres-
sor of MEK null (SMEK), a homologue of the protein phosphatase 4 regulatory
subunit, is required for the long lifespan of daf-2 mutants in a daf-16-dependent
manner [39]. PPTR-1/protein phosphatase 2A regulatory subunit (PP2A) decreases
the phosphorylation of AKT-1 and leads to both activation of DAF-16 and increased
longevity in daf-2 mutants [40].
Other regulatory modes for DAF-16 include protein acetylation, protein stability
control, protein-protein interactions, and transcriptional control of its isoforms.
CBP-1/CREB-binding protein (CBP), which is an acetyl-transferase, contributes to
the longevity of daf-2 mutants [41], likely via acetylating and activating DAF-16
[42]. DAF-16 is also required for the long lifespan conferred by the overexpression
of sir-2.1/NAD-dependent protein deacetylases [[43–45] but see also [46]].
Components of the ubiquitin proteasome system regulate the stability and activity
of DAF-16. Specifically, an E3 ligase, RLE-1/RC3H1, ubiquitinates DAF-16, and
consequently, rle-1 mutants live long due to increased stability of DAF-16 [47].
MATH-33/deubiquitylase counteracts the RLE-1-dependent degradation of DAF-
16 and extends lifespan [48]. In addition, components of the Skp1-Cul1-F-Box E3
ligase complex contribute to the longevity of daf-2 mutants, perhaps by indirectly
up-regulating DAF-16 [49]. Additionally, proteasome activation promotes long
lifespan by increasing DAF-16 activity [50]. Scaffold proteins are also important for
DAF-16 regulation. Genetic inhibition of the 14-3-3 scaffold protein, PAR-5 or
66 S.W.A. An et al.
C. elegans has a simple nervous system, comprised of 302 neurons, which have
been mapped in detail [87] (see also Chaps. 2 and 8). Well-known functions of sen-
sory neurons include the perception of environmental stimuli and the transmission
of signals for proper physiological responses. Interestingly, sensory neurons in C.
elegans also contribute to lifespan regulation [reviewed in [88]]. Chemosensory
neurons appear to affect lifespan mostly by acting through IIS [89], whereas ther-
mosensory neurons regulate lifespan via steroid signalling at high temperature [90].
Impairment of general chemosensory neuronal functions leads to the activation of
DAF-16 and longevity via modulating the expression of insulin-like peptides (ILPs);
chemosensory mutations also do not further extend the longevity of daf-2 mutants
[27, 89, 91–93]. Thus, it is likely that the inhibition of chemosensory neurons down-
regulates IIS activity, and this may in turn activate DAF-16 to promote longevity
(Fig. 4.2).
68 S.W.A. An et al.
Fig. 4.2 Neuroendocrine regulation of IIS and longevity. Inhibition of sensory neural functions
leads to down-regulation of IIS. This inhibition modulates the expression of hormonal insulin-like
peptides that are secreted from sensory neurons, triggering the activation of DAF-16 in non-
neuronal tissues, such as the intestine. Activated DAF-16 then translocates into the nucleus, where
it induces the expression of target genes that confer organismal longevity
complexity [93, 104–106, 108–116]. ILPs are known to modulate the activity of
DAF-2 by acting as either agonists (e.g., INS-6 and DAF-28) or antagonists (e.g.,
INS-1) [21, 93, 105, 106, 110, 116–119]. However, some ILPs, such as INS-18 and
INS-7, can serve as both agonists and antagonists of DAF-2 in a context-dependent
manner [104, 105, 108, 111, 115, 120]. Recent studies have characterized the
expression patterns and functions of all ILPs systematically [120, 121]. In contrast
to the previous notion that ILPs function redundantly [[116, 121] also reviewed in
[122]], these studies have suggested that ILPs can constitute combinatorial codes
for the regulation of development and physiology in C. elegans [120]. Thus, ILPs
appear to have distinct roles as individuals and to regulate various physiological
outputs as members of an intricate ILP-regulatory network.
Various tissues in C. elegans express ILPs and appear to regulate IIS in an endo-
crine manner. ILPs are mainly expressed in neurons, although a few have also been
shown to be expressed in other tissues, such as the intestine and the hypodermis [93,
105, 106, 108, 110, 114–116, 118, 119, 121, 123]. These expression patterns of
ILPs imply that the nervous system of C. elegans may be a key regulatory centre for
endocrine IIS. Consistent with this idea, neuronal IIS has a large impact on organis-
mal physiology. For example, DAF-2, AGE-1, and DAF-18 regulate lifespan cell
non-autonomously in the nervous system [124–126]. In addition, disruption of sen-
sory neurons increases lifespan and up-regulates DAF-16 in the intestine and the
hypodermis by decreasing the expression of INS-6 and DAF-28 [93]. Neuronal daf-
16 contributes to the long lifespan of daf-2 mutants [127], again pointing to the
important role of the nervous system in endocrine regulation of IIS-induced
longevity.
Tissues other than neurons also play substantial roles in the endocrine IIS-
regulated lifespan in C. elegans. The intestine of C. elegans is the major digestive
organ [128] and serves as a signalling centre for nutritional status. Thus, IIS in the
intestine may transmit signals regarding nutritional status to regulate organismal
physiology. In fact, intestine-specific expression of daf-16 substantially restores the
longevity of daf-2 mutants [127]. The intestine also regulates the expression of
ILPs, in particular ins-7, to modulate IIS in distant tissues via a positive feedback
loop [108]. In addition, intestinal daf-16 prevents age-dependent deterioration of
muscle [60]. Overall, this endocrine IIS system appears to coordinate the rates of
ageing among different C. elegans tissues.
In addition to lifespan, the C. elegans IIS pathway regulates various other physio-
logical processes. For example, reduced IIS enhances resistance to a number of
stresses, including heat [129, 130], oxidative stress [131–133], and osmotic stress
[134], as well as hypoxia [135, 136]. Reduced IIS also allows C. elegans to
70 S.W.A. An et al.
successfully cope with heavy metal toxicity [137], ultraviolet (UV) radiation [138],
endoplasmic reticulum (ER) stress [61], and cytosolic proteotoxicity [67, 68, 139].
This signifies the importance of IIS pathway-regulated mechanisms for healthy
ageing.
Stress resistance resulting from reduced IIS is mediated by a variety of factors,
including longevity-promoting transcription factors DAF-16, HSF-1, and SKN-1
(see Sect. 4.2). For example, DAF-16 contributes to enhanced thermotolerance and
resistance to hypertonicity, UV, heavy metals, and hypoxia conferred by reduced IIS
[67, 129, 130, 134–138, 140–142]. Reduced IIS also protects against oxidative
stress by triggering the activation of DAF-16 and SKN-1 [26–28, 39, 79, 80, 86,
131–133, 143]. The SMK-1 and EGL-27/GATA transcription factor promote UV
resistance in daf-2 mutants [39, 141, 144]. XBP-1, a key mediator of the ER
unfolded protein response (UPRER), collaborates with DAF-16 to enhance UPRER in
daf-2 mutants [61]. Additionally, HSF-1, together with DAF-16, contributes to
enhanced cytosolic protein homeostasis conferred by reduced IIS [67, 68]. The
decreased levels of IIS also protect somatic cells from various stresses by equipping
these cells with many characteristics of germline stem cells [145]. Overall, IIS-
mediated stress resistance contributes to the proper management of stresses through
a variety of factors, which are also essential for longevity.
Innate immunity ensures survival in the presence of pathogenic threats. C. ele-
gans has an innate immune system that is regulated by evolutionarily conserved
signalling pathways, one of which is the IIS pathway. Reduced IIS activity increases
resistance to various fungal and bacterial pathogens via DAF-16 [146, 147], in par-
allel to the well-known immune regulator, p38 MAP kinase [146–150]. The tran-
scription factors SKN-1 and HSF-1 also mediate the enhanced pathogen resistance
under conditions of reduced IIS [151, 152]. daf-2 mutants display mitigated internal
bacterial colonization, enhanced bacterial clearance, and increased expression of
antimicrobial genes [21, 150]. Moreover, daf-2 mutants display enhanced efficiency
in RNA interference (RNAi) [153], which is important for antiviral defence in
C. elegans [154–156]. Thus, it will be interesting to test whether daf-2 mutants are
resistant to viral infections as well.
Importantly, reduced IIS has been shown to alleviate the pathological features of
various disease models in C. elegans, including Huntington’s disease [139, 157],
Alzheimer’s disease [158, 159], Parkinson’s disease [160], and amyotrophic lateral
sclerosis (ALS) [161] (Fig. 4.3). In a Huntington’s disease model, reduced IIS ame-
liorates the polyglutamine (polyQ) aggregation mediated by CAG repeats in a DAF-
16- and HSF-1-dependent manner [67, 139, 162, 163]. In a model for Alzheimer’s
disease, reduced IIS protects C. elegans from the toxicity caused by Aβ1−42 expres-
sion via DAF-16, HSF-1, and autophagy [164, 165]. In a Parkinson’s disease (PD)
model, C. elegans expressing human α-synuclein in neurons displays both a motor
deficit and progressive degeneration of dopaminergic neurons [160]; however, daf-2
mutations result in complete retention of these dopaminergic neurons [163]. ALS
originates from mutations in various genes, including superoxide dismutase 1
(SOD1) [166]. In a C. elegans model, daf-2 mutations protect against the toxic
mutant SOD1-induced motor neuron dysfunction by decreasing protein aggregation
4 Longevity Regulation by Insulin/IGF-1 Signalling 71
Fig. 4.3 The role of IIS in stress resistance and human disease models. Reduced IIS confers
enhanced resistance against a variety of stresses, including heat, hypoxia, high osmolarity, heavy
metals, UV radiation, proteotoxicity, and pathogens. Reduced IIS also ameliorates the impact of
age-related human disease models in C. elegans, including those for Huntington’s disease, amyloid
lateral sclerosis (ALS), Alzheimer’s disease, and Parkinson’s disease. These features correlate with
healthy ageing and longevity.
4.6 Conclusions
In this chapter, we reviewed the functions of IIS and the mechanisms by which it
influences C. elegans longevity. The entire IIS pathway appears to play a central
role in linking environmental signals, such as food availability and stresses, to vari-
ous physiological outputs, including ageing, reproduction, and development.
Therefore, one possible reason why the IIS pathway has a huge impact on ageing is
72 S.W.A. An et al.
Acknowledgments We thank the members of Lee laboratory for critical comments on the manu-
script. This work was supported by the National Research Foundation of Korea (NRF) Grant
funded by the Korean Government (MSIP) (NRF-2013R1A1A2014754) to S.-J.V.L.
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Chapter 5
Mitochondrial Longevity Pathways
Several genes in C. elegans were named Gerontogenes [7], based on their ability to
confer extended lifespan when mutated. One fascinating class of Gerontogenes, due
to the paradoxical nature of its initiating event, is ascribed to interventions that
5 Mitochondrial Longevity Pathways 85
directly or indirectly affect the functionality of the ETC, now known as Mitochondrial
(Mit) mutants [8–12]. Mitochondrial functional and structural alterations are typical
hallmarks of ageing [13, 14] and the mitochondrial free radical theory of ageing,
adapted from Harman’s free radical theory of ageing [15], has been for many
decades one of the prevailing and indisputable theories to explain the degenerative
processes occurring during the ageing process. Moreover, severe mitochondrial
dysfunction is the common denominator of a variety of genetic disorders ranging
from early onset syndromes (e.g. Leigh syndrome, Friedreich’s ataxia) to age-
associated diseases (e.g. Parkinson, diabetes) [16–18]. The discovery that mito-
chondrial dysfunction can lead to lifespan extension was therefore initially very
surprising, but only a few years after the initial findings [19, 20] the first plausible
explanations on this paradoxical effect were proposed and experimentally proved
[8, 9, 21, 22].
In this chapter we will first describe the three major categories into which the
long-lived C. elegans Mit mutants have been previously divided, depending on how
lifespan extension is achieved [9]. The first category is the largest one, and derives
from gene inactivation by RNA interference (RNAi). The second category is
ascribed to classical genetic mutations, while the third one involves external inter-
ventions (mainly drugs), which extend lifespan by directly targeting ETC com-
plexes. As might be expected, several reports indicate that genetic and
pharmacological interventions targeting mitochondria affect their structure, reduce
ATP, oxygen consumption rate and respiratory capacity [9 and references therein].
Moreover, they not only extend lifespan but also concurrently affect additional ani-
mal phenotypes and behaviours, such as fertility, development, body size, neuro-
muscular activities and resistance to stress. We will then summarize evidence
indicating that the ability of the initiating events to extend lifespan is dose, time and
tissue dependent, and describe the main molecular mechanisms thought to be caus-
ally involved in Mit mutants longevity (Fig. 5.1). Finally, we will briefly discuss the
pros and cons of reducing mitochondrial function to improve healthspan/lifespan
from a translational point of view.
Although mitochondrial proteins other than those directly or indirectly involved
in ETC functionality (e.g. mtDNA translation or mitochondrial antioxidants) have
been shown to modulate C. elegans lifespan, in this book chapter we will mainly
focus on those belonging to the different ETC complexes and only briefly mention
other mitochondrial proteins. Moreover, mutations leading to pathological pheno-
types such as lifespan shortening (mev-1, gas-1) [23, 24] or arrest animal develop-
ment (nuo-1, atp-2, frh-1, phb-1/2, atad-3) [22, 25–28] have also been identified,
but will not be comprehensively described in this chapter.
86 A. Schiavi and N. Ventura
Fig. 5.1 A moderate reduction of mitochondrial activity either through gene silencing, genetic
mutations or pharmacological targeting of different mitochondrial proteins (mainly involved in
ETC functionality) (a) affects intrinsic mitochondrial physiological functions (b), which in turn
triggers cellular genetic and metabolic reprogramming (c), ultimately leading to extension of ani-
mals’ healthy lifespan (d)
whose suppression extends C. elegans lifespan [29–32]. Besides the long list of
genes identified through screening, other genes that fall into this category were
found to extend lifespan through targeted investigations, such as frh-1 [28], atp-3
[21], mics-1 [33], nuo-6 [34]. Many of these genes identified by gene silencing
belong to complexes of the ETC (except complex II), their regulatory subunits or
assembly factors, including the ATPase [10 and references therein]. This “RNAi-
mediated” category (Mit RNAi) is the largest one of the mitochondrial interventions
that extend lifespan, and demonstrates the advantage of addressing partial, rather
than complete suppression of mitochondrial proteins through silencing (dose depen-
dent effect – see 5.3.2.1). The different silencing potency of individual RNAi con-
structs, along with additional technical variables, contributes to the differences in
the genes identified in the different RNAi screens [35]. Compared to wild-type ani-
mals, lifespan extension in this RNAi-mediated category is in most cases associated
with other phenotypes and behaviours, suggesting the induction of protective,
healthy ageing promoting responses: prolonged fertility period with minor altera-
tion of the total brood size, slightly reduced adult body size without dramatic con-
sequences on animal development, increased resistance to various types of stress,
mildly reduced ATP and ROS production, altered mitochondrial structure and
increased mitophagy, slightly reduced movement and chemosensory function early
in life which nonetheless decline much slower during ageing as compared to wild-
type animals [21, 28, 31, 32, 34, 36–38].
Most of the studies investigating molecular mechanisms underlying mitochon-
drial stress control of longevity have been so far conducted using genetic-derived
Mit mutants (see below), thus, little is known about how Mit RNAi extend lifespan.
Very few downstream genes and pathways have been shown to be modulated by Mit
RNAi, and include cep-1, hif-1, UPRmt, autophagy/mitophagy, detoxification and
antioxidant genes [22, 36, 37, 39–42], but only in some instances are they causally
involved in animals’ longevity (e.g. cep-1, hif-1, autophagy and mitophagy regula-
tory genes). Although evidence is accumulating to indicate that genetic- and RNAi-
mediated Mit mutants may act through partially independent signalling [34, 37],
lots remains to be done to understand how silencing of nuclear-encoded mitochon-
drial proteins actually extends lifespan and concurrently affects other phenotypes
associated with healthy ageing.
The initial discovery that mutations in genes coding for mitochondrial proteins may
prolong lifespan in C. elegans came from the identification of a long-lived clk-1 [43,
44] mutant. Since these original findings, few additional genetic mutants have been
identified that fall into this second category, clearly reflecting the importance of the
ETC in survival and the difficulty of obtaining hypomorphic mutations [11 and
references therein]. clk-1 encodes a demethoxyubuiquinone (DMQ) monoxygenase
necessary for the synthesis of ubiquinone and its mutation leads to DMQ9 accumu-
lation. Three different clk-1 alleles have been described (e2519, qm30 and qm51)
88 A. Schiavi and N. Ventura
each displaying a different degree of phenotypic severity, but all accumulating the
same amount of DMQ9, thus ruling out a role for this quinone intermediate in regu-
lating lifespan [45]. Similar to the RNAi-mediated category, clk-1 mutations
decrease ETC functionality and ROS production and do not dramatically affect
developmental. However, like other genetic-mediated Mit mutants, clk-1 mutants
display significantly reduce brood size, movement and pharyngeal pumping
[44, 46, 47].
isp-1(qm150) is another extensively studied genetically-defined Mit mutant that
was identified in a screen for mutants displaying a Clk phenotype [20]. isp-1 encodes
the Rieske Fe-S protein subunit of complex III and the qm150 allele contains a mis-
sense point mutation that most likely affects its redox potential. This long-lived
mutant is characterized by low oxygen consumption, decreased sensitivity to ROS,
and a dramatic reduction in brood size and both embryonic and post-embryonic
development [20]. A mutation in the mitochondrial encoded gene ctb-1 suppresses
most isp-1 phenotypes but not its longevity [20], indicating that lifespan and devel-
opmental changes are not necessarily coupled. The transcriptomic and metabolomic
profile of the clk-1(qm30) and isp-1(qm150) mutants have been analysed revealing
both common and unique signatures and changes in the expression of genes which
in some cases are responsible for the phenotypes [39, 48–50].
Other much less well-characterized genetic-mediated Mit mutants are gro-1, lrs-
2, tpk-1, and nuo-6. gro-1 encodes for isopentenylphosphate:tRNA transferase, an
enzyme that modifies a subset of mitochondrial tRNAs and is necessary for the
efficient translation of mtDNA genes. gro-1(e2400) mutants have a phenotype simi-
lar to clk-1 with prolonged lifespan, reduced brood size, delayed development and
slowed behavioural rates [51]. The lrs-2(mg312) mutant was instead identified in a
screen for genetic alterations extending C. elegans lifespan in a daf-16-independent
manner [32]. lrs-2 encodes a mitochondrial leucine tRNA synthetase and the mg312
allele is predicted to form a truncated, non-functional version of the protein, thus
affecting the expression of all mtDNA encoded proteins and therefore is very likely
to severely impact mitochondrial ETC functionality. As a result, lrs-2 mutants have
a very slow development and become sterile adults with arrested gonad develop-
ment. Based on its human ortholog, the protein encoded by the C. elegans tpk-1
gene is predicted to have thiamine (vitamin B1) diphosphokinase activity. Dietary
thiamine consists mainly of thiamine pyrophosphate (TPP), which is then trans-
formed into thiamine in the intestine before absorption. A partial loss-of-function
mutant, tpk-1(qm162), displays altered cellular thiamine levels and slow behav-
ioural rates that are partially rescued by TPP, but not thiamine, supplementation
[52]. Thiamine is necessary for the appropriate functionality of the OXPHOS and
the pentose phosphate pathways [53] since it acts as a cofactor for α-ketoacid dehy-
drogenases (pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, branched-
chain α-ketoacid dehydrogenase, and transketolase), whose inhibition has been
proposed as a common underlying mechanism for Mit mutants longevity [54]. The
nuo-6(qm200) mutant was recently identified [34] in a genetic screening looking for
mutants displaying phenotypes similar to isp-1(qm150). Interestingly, the two
genetic mutants have very similar phenotypes and mitochondrial metabolic changes
5 Mitochondrial Longevity Pathways 89
but differ substantially in many respects (i.e. development, fertility, oxygen con-
sumption, ATP and autophagy levels) from isp-1 and nuo-6 RNAi. Moreover, while
the lifespan of the double mutant is not additive, isp-1 RNAi longevity is fully addi-
tive to nuo-6 genetic and vice versa [34]. This last finding might simply reflect the
fact that the genetic mutants are not fully activating the pro-longevity adaptive
response, which is fully activated by the RNAi. However, along with other studies,
it most likely reflects the differential modality of lifespan extension between RNAi-
and genetic-mediated Mit mutants (see 5.4.3). Additional systematic studies are
required to fully understand common and unique molecular mechanisms activated
by RNAi and genetic modification of the mitochondria to extend lifespan and to
concurrently modulate their phenotypic features.
The third and perhaps most interesting, but less explored, category of interventions
comprise various chemicals and drugs targeting mitochondria that in the past decade
have been shown to extend lifespan [55 and references therein]. Because of the
endosymbiotic origin of mitochondria, different antibiotics, which inhibit bacterial
growth by targeting its DNA synthesis, transcription or translation, such as ethidium
bromide (EtBr), doxycycline and chloramphenicol, also affect mtDNA, and were
shown to extend C. elegans lifespan [27, 40]. Antimycin A is another antibiotic that
instead prevents bacterial growth by inhibiting the functionality of ETC complex III
and it was shown to extend lifespan [31].
Other drugs have been shown to extend C. elegans lifespan by directly targeting
mitochondrial ETC complexes. Metformin is a biguanide drug largely prescribed in
patients with type 2 diabetes and was originally shown to promote C. elegans
healthspan through a dietary restriction-like mechanism and oxidative stress
response [56]. However, more recently, a mechanism has been proposed in which
metformin targets complex I, and in turn induces ROS-mediated PRDX-2 activation
[57]. Rotenone is another inhibitor of complex I of the ETC that extends C. elegans
lifespan through generation of ROS [58]. Paraquat also generates superoxide inside
mitochondria, by a redox cycling reaction, and can significantly extend C. elegans
lifespan when used at low concentrations [59]. On the other hand, mitochondrial-
targeting antioxidants such as MITOQ, ubiquinone, vitamin E, and superoxide dis-
mutase and catalase mimetics can also extend lifespan [60–62].
Some drugs also promote worm longevity by targeting the mitochondrial ATPase.
Oligomycin is a potent inhibitor of the mitochondrial ATPase whose treatment
beginning from adult (40 μM) or embryo (2 μM) extends worms lifespan [55, 63].
LYC-30904, an allosteric modulator of the mitochondrial F1F0-ATPase with thera-
peutic properties in murine models of autoimmune disease [64], and targeting the
same ATPase subunit encoded by the atp-3 C. elegans homologue, was recently
identified as a new complex V inhibitor with lifespan extending effect in C. elegans
[55]. Of note, the ketone, Krebs cycle intermediate, α-ketoglutarate extends lifespan
by inhibiting complex V activity [63]. Additional products of mitochondrial
90 A. Schiavi and N. Ventura
which leads to slow growth and extended lifespan [20], the isp-1(gk267) allele is a
large knock-out mutation that results in early larval arrest. Finally and perhaps more
intuitively, mitochondrial targeting drugs also act through a bimodal dose response:
low doses of most of the assessed pro-longevity drugs induce pathological effects
when used at higher concentrations [55].
The trade-off between beneficial vs detrimental biological effects (here lifespan
extension or shortening) regulated by a threshold (here mitochondrial stress) clearly
resemble the concept of hormesis: a protective adaptive response to a low dose of a
particular type of stressor, which is instead toxic or detrimental at higher doses.
According to this theory, the term mitohormesis has been proposed for a particular
form of hormesis by which mild mitochondrial stress promotes a beneficial response
that may decrease the susceptibility to diseases and delay ageing [72–74]. The con-
cept of mitochondrial hormesis has mainly been associated with the production of
low doses of ROS inducing positive pro-longevity effects while shortening lifespan
at higher doses [75, 76]. However, neither ROS nor ATP levels correlate with lifes-
pan extension in the RNAi-mediated Mit mutants [21, 31, 37]. It is therefore con-
ceivable that other molecular parameters possibly affected by mitochondrial
targeting interventions (e.g. NAD/NADH, iron content, mitochondrial membrane
potential [36, 69, 77]) lead to opposite biological effects by activating different
pathways (or the same pathways to a different extent) in a dose-dependent manner
[9]. As such, while ROS-mediated mitohormesis can explain the life-extending
effect of caloric restriction and of reducing the IGF/insulin signalling, when the
stress is directly applied to mitochondria other factors may alternatively or concur-
rently play a role.
Mild changes in mitochondrial parameters may thus activate mitochondrial qual-
ity control pathways and protective metabolic/genetic cellular reprogramming (see
Sect. 5.4) that help coping with a low degree of mitochondrial stress. On the con-
trary, when the threshold for healthy mitochondrial function is surpassed due to
complete mitochondrial protein deficit or more robust RNAi effects, the overt mito-
chondrial damage may either fail to induce or hyper-activate protective pathways,
which in turn leads to the observed detrimental effects on organismal health and
ageing (developmental arrest, sterility, short life span and lethality) [9, 78].
Another important and unique aspect of this class of long-lived mutants that distin-
guishes it from most other longevity interventions is that, especially when it comes
to the RNAi-mediated category, mitochondrial reprogramming has to occur during
animal development in order to trigger the pro-longevity effect [21, 31]. This was
originally described showing that if Mit RNAi is applied only during larval develop-
ment and then interrupted (but not if it is only applied during adulthood), the life
extending effect is observed [31]. The relevant timing window was further refined
by showing that RNAi only during L3/L4 larval stage is enough to extend lifespan
[21]. This stage-specific requirement is very interesting considering that on the
92 A. Schiavi and N. Ventura
other hand, reducing caloric intake or insulin signalling can extend lifespan also if
applied during adulthood. Indeed, it implies that specific developmental signals
exist or programmes must be reprogrammed to extend lifespan upon mitochondrial
stress. The nature of this signal and/or programme is still unknown and awaits fur-
ther investigation but epigenetic changes, metabolic reprogramming, endocrine-like
mechanisms, as well as germline or neuronal specific developmental processes can
all be envisioned.
Interestingly, severe mitochondrial dysfunction leading to development arrest
has also been associated with lifespan extension [22, 27]. These findings suggest
that although detrimental effects may arise upon severe suppression of mitochon-
drial proteins, likely due to the inability of cells to repair or cope with the over-
whelming mitochondrial stress, pro-longevity pathways can still be triggered. This
notion clearly indicates that mechanisms regulating development and lifespan are
somewhat uncoupled. The early life requirement for the beneficial effects of mito-
chondrial reprogramming brought about by appropriate degree of protein expres-
sion is reminiscent of the antagonistic pleiotropic theory of ageing originally
proposed by George Christopher Williams in 1957 [79]. This theory poses that a
gene (e.g. p53, TOR) or one of its cellular regulated processes (e.g. cellular senes-
cence, protein synthesis), which controls both a beneficial and a detrimental pheno-
typic trait of an organism, can be evolutionarily selected if it has beneficial effects
early in life while having its negative effects later in life [80–83]. Consistent with
this theory, it might be hypothesized that “normal”, rather than reduced expression
of specific mitochondrial proteins (or mitochondrial activity) early in life, is subject
to selection to protect against developmental problems or the occurrence of
mitochondrial-associated disorders [9, 84], but would accelerate the ageing process
later in life. A fine-tuned reduction of mitochondrial function during development
could then be a strategy (similar to what has been shown for appropriate levels of
p53 expression [85]) to promote healthy lifespan without inducing deleterious con-
sequence during development. It is interesting to note that the only gene so far
shown to have clear double-edged effect in mediating the opposite longevity out-
comes in response to different degrees of mitochondrial stress is indeed the C. ele-
gans p53 homologue cep-1 [78, 86].
Tissue specific control of longevity in C. elegans has been observed in several stud-
ies. Specific subsets of neurons can be affected to extend lifespan or are required for
the pro-longevity effects of other interventions such as caloric restriction [58, 87,
88], and germline signalling [89, 90] (see also Chap. 6). These findings indicate that
control of longevity in C. elegans is cell non-autonomous and mediated by secreted,
diffusible molecules, which promote systemic beneficial effects via endocrine-like
mechanisms [91]. Interestingly, in humans, a peculiar aspect of mitochondrial-
associated disorders is their tissue specificity. Although these are multi-system
5 Mitochondrial Longevity Pathways 93
As mentioned above, hormesis is the bimodal dose response by which a low dose of
a particular stressor promotes a protective response to a high dose of the same or to
different stressors, while being detrimental at higher doses. Accordingly, the term
mitohormesis has been coined to define a particular form of hormesis by which mild
mitochondrial stress promotes a beneficial response that may decrease the sensitiv-
ity for diseases and delay ageing [72–74]. In line with this concept, different C.
elegans studies have demonstrated that ROS, provided extrinsically or possibly pro-
duced by mitochondria, have a causal role in the beneficial responses that lead to
lifespan extension [75, 76] (for details on stress response and oxidative stress con-
trol of C. elegans ageing see Chaps. 9 and 10 respectively). One of the first studies
demonstrating the hormetic response to ROS showed that reducing glucose avail-
ability in C. elegans induces ROS formation, which in turn are required to promote
stress resistance and extend lifespan [99]. In another example the same group dem-
onstrated that a very low (not lethal) exposure to arsenite promotes longevity by
transiently increasing ROS levels, while a higher concentration reduces longevity
by blocking mitochondrial respiration [100]. Furthermore increasing levels of
superoxide by treatment with low concentration of paraquat extends the lifespan in
C. elegans [101]. In most of the mentioned studies, reducing ROS levels using anti-
oxidants prevented the lifespan extension indicating that their common denomina-
tor is ROS’ role as signalling molecule to most likely activate beneficial signalling
pathways in turn extending lifespan. One report identified a C. elegans peroxire-
doxin, PRDX-2, as a mediator of ROS-production extension of lifespan upon met-
formin treatment [57]. Another study showed that HIF-1, the hypoxia inducible
factor-1, which is required to extend longevity in both genetic- and RNAi-derived
Mit mutants [102], does so in genetic Mit mutants by ROS dependent mechanisms
[102].
In agreement with the mitohormesis concept, as described above (Sect. 5.3.2.1),
RNAi-mediated suppression of different ETC subunits (namely atp-3, isp-1, cco-1,
nuo-2, and frh-1) promote C. elegans longevity only within a specific range of sup-
pression, outside which they show no or negative effects [21, 55]. However, these
studies showed no correlation between oxidative stress and lifespan and failed to
reveal a role for ROS and/or antioxidants [9, 21, 22] indicating that ROS induction
is not a common pro-longevity denominator in all Mit mutant categories. Consistent
with the different ways to activate downstream pro-longevity responses, HIF-1-
mediated extension of lifespan is most likely independent from ROS in the RNAi-
derived Mit mutants, which in fact display reduced levels of ROS [36].
5 Mitochondrial Longevity Pathways 95
The unfolded protein response (UPR) is the main cellular reaction to the accumula-
tion of misfolded proteins in the endoplasmatic reticulum (ER) [112]. Environmental
changes and ageing can promote accumulation of misfolded proteins, which in turn
may lead to pathological conditions without an appropriate functionally UPR.
Mitochondrial unfolded protein response (UPRmt) specifically helped in maintaining
functional mitochondria from the accumulation of misfolded proteins inside mito-
chondria, and it is known to be involved in delaying ageing in C. elegans [40, 113].
96 A. Schiavi and N. Ventura
induced in isp-1 genetic mutants, knocking down bec-1 and vps-34 by RNAi reduces
the lifespan in the isp-1 and clk-1 genetic mutants but not in the wild type [123,
124]. Even though there are some controversial results regarding the role of autoph-
agy as a compensatory pathway in response to mitochondrial dysfunction, it is clear
that autophagy plays an important role in the ageing process. Besides classical
autophagy regulatory genes, cep-1 and hif-1 are also causally involved in autophagy
induction and lifespan extension RNAi-mediated Mit mutants [36, 37].
Elevated ROS levels could induce autophagy possibly through p53 and HIF-1
activation [125–127]. However, this is unlikely to be the main mechanism inducing
autophagy in this category of long-lived Mit mutants, which, as mentioned above,
showed reduced ROS levels. Mild ETC perturbation might nevertheless affect both
oxygen and iron metabolism, which can control both p53 and HIF1 activation [128].
Under physiological oxygen and iron levels, in C. elegans as in mammals, HIF-1 is
continuously expressed but is degraded by its negative regulators EGL-9 and VHL-1
[129]. EGL-9 is a proline hydroxylase which promotes the hydroxylation of proline
residues in HIF-1 that can then be ubiquitinated by E3 ubiquitin ligase VHL-1, lead-
ing to the consequent degradation of HIF-1 in the proteasome. Down regulation of
egl-9 or vhl-1 as well hif-1 overexpression or induction by hypoxia, extend longev-
ity in C. elegans. HIF-1 as well as VHL-1 and EGL-9 are required to properly
extend the lifespan in different Mit mutants [36, 102, 130, 131], which also appear
to be more resistant than wild-type animals to hypoxia or iron deprivation indicating
the induction of a protective hypoxia-like response in response to mitochondrial
stress [36, 49]. Notably mitophagy, a specific form of autophagy dedicated to the
selective removal of damaged mitochondria [132], is induced in different RNAi-
mediated Mit mutants most likely through iron deprivation [36, 133]. Major mitoph-
agy regulatory genes (e.g. pink-1/PINK, pdr-1/PARIN, sqst-1/p62 and dct-1/BNIP3)
were indeed shown to be required for induction of mitophagy and lifespan extension
in RNAi-mediated Mit mutants or upon iron depletion [36].
As mentioned above, the main role of mitochondria is to produce ATP through oxi-
dative phosphorylation coupled to the Krebs cycle. Mitochondrial stress may there-
fore lead to an altered ratio of ATP/ADP and NADH/NAD+, which can both affect
C. elegans longevity [77, 134]. Increased levels of AMP may activate AMP-
regulated protein kinase (AMPK) and aak-1 and aak-2, the C. elegans homologues
of the catalytic alpha subunit of AMPK, are required to extend lifespan in isp-1 and
clk-1 genetic mutants [134]. However, neither aak-1 or aak-2, nor C. elegans homo-
logues of other AMPK subunits, are required to specify Mit RNAi (frh-1, isp-1,
nuo-2, cco-1) longevity [37] and ATP levels do not directly correlate with lifespan
outcomes upon different levels of mitochondrial stress [31, 135, 136]. These obser-
vations underscore the differential mode of action between genetic- and RNAi-
mediated Mit mutants.
98 A. Schiavi and N. Ventura
Different studies also revealed that C. elegans Mit mutants exhibit altered energy
metabolism in response to mitochondrial stress, suggesting that extended longevity
could derive from metabolic reconfiguration. Indeed, RNAi (frh-1, nuo-5 and nduf-
7) or genetic-derived (isp-1, cco-1, clk-1) Mit mutants, modulate metabolic path-
ways involved in carbohydrate, amino acid, and fatty acid metabolism, as well as
genes regulating OXPHOS, glycolysis, TCA cycle, and lipid metabolism [37, 48–
50, 111, 135, 137]. Moreover, long-lived Mit mutants accumulate a set of com-
pounds, enriched in α-ketoacids and α-hydroxyacids [48, 50] and have reduced lipid
content [37, 138]. Interestingly, different products of intermediate metabolism
(especially ketone bodies) [63, 65, 66], as well as different types of lipids [90, 139,
140], have been shown to modulate C. elegans longevity.
As described in the previous section, ROS, ATP, and UPRmt are intrinsic mitochon-
drial parameters clearly affected in different Mit mutants, which to a certain extent
influence animal longevity by triggering protective compensatory mechanisms to
cope with mitochondrial stress. However, most mechanistic studies on mitochon-
drial stress control of longevity have been carried out, with a few exceptions, with
genetic-mediated Mit mutants and changes in ROS, ATP, and UPRmt, as explained
above, do not directly correlate with lifespan extension upon silencing of different
ETC subunits. Therefore, additional, non-mutually exclusive, mitochondrial param-
eters, as well as downstream processes that are causally involved in specifying Mit
mutant longevity remain to be discovered. It is envisioned that between the three
different categories, as well as between the different targeted mitochondrial pro-
teins, both unique and common mechanisms exist. Examples of under-investigated
mitochondrial parameters which might modulate longevity in the Mit mutants are
mtDNA damage, transcription and translation [40, 141–144], iron and iron-sulphur
cluster proteins homeostasis [36, 86, 145], NAD levels [77, 146], mitomiRNAs
[147], mitochondrial membrane potential and mitochondrial protein translocation
systems [69]. Downstream signalling molecules, which may specify longevity in
Mit mutants (as well as other Mit phenotypes), include byproducts of metabolism
[48, 63, 66], additional transcription factors [54, 95, 123], lipids and nuclear-
hormone-receptors [148–152], DNA-damage responses and apoptosis [103, 111].
As noted in the previous sections, several lines of evidence support the notion that
genetic- and RNAi-derived Mit mutants promote healthy ageing through different
mechanisms. (i) The most direct evidence comes from the additive effects on
5 Mitochondrial Longevity Pathways 99
lifespan observed when isp-1 and nuo-6 genetic and RNAi interventions are com-
bined [34]. (ii) Additional evidence is provided by the more generic observations
that the phenotypes of genetic- and RNAi-mediated mutants differ substantially in
many respects (i.e. development, fertility, oxygen consumption, ATP and autophagy
levels) [34]. (iii) Although isp-1(qm150) mutant and cyc-1 RNAi affect the same
ETC complex they display a different gene expression profile [39]. (iv) AMP kinase
is not involved in Mit RNAi lifespan extension [37] but does mediate the longevity
of at least two different genetic-mediated Mit mutants (isp-1 and clk-1) [134]. (v)
ROS have repeatedly been shown to be involved in lifespan extension of different
genetic-mediated Mit mutants [100, 102, 153], all RNAi-mediated Mit mutants
tested so far display reduced ROS levels and their longevity is not affected by the
use of antioxidants [21, 36, 37, 40, 42] (and our unpublished observations).
However, a transient increase in ROS during development following Mit RNAi that
activates compensatory pathways ultimately reducing ROS and extending lifespan
cannot be ruled out. (vi) Apoptosis regulatory genes were shown to mediate lifespan
of genetic- but not RNAi-mediated isp-1 and nuo-6 [103]. (vii) In contrast, although
autophagy regulatory genes were shown to mediate both genetic- and RNAi-
mediated longevity [37, 124], the autophagic process was only induced in the RNAi
[37] but not genetic [34] Mit mutants. The observed differences could reflect the
different type of alteration of the targeted protein (reduced expression vs reduced
function) and/or the amount of protein suppression. More work is clearly required
to define common and unique mechanisms of lifespan extension elicited by the dif-
ferent pro-longevity categories and even by the single interventions. It will be par-
ticularly interesting to understand whether pharmacological interventions act via
the same mechanism as genetic or RNAi Mit mutants, or in another unique way.
It is evident from this book chapter that, although mutations in some of the same
genes that promote healthy ageing in C. elegans result in devastating diseases in
humans, there might be clear advantages in partially reducing mitochondrial func-
tion. This notion was originally discovered in yeast and C. elegans [19, 154], and
subsequently observed in Drosophila [155] and even more strikingly in different
mice models, where either knockout of the cytochrome c assembly factor (Surf1),
or hemizygous knockout of mouse clk1 (Mclk1), or reduced expression of a mito-
chondrial ribosomal subunit (MrsS5), have been associated with extended and
healthier life [40, 156, 157]. The evolutionarily conserved nature of this pro-longev-
ity effect from yeast to mammals, opens the door to the possibility of eventually
translating this effect to humans. This appears especially true when coupled to the
possibility of modulating mitochondrial activity through pharmacological or nutri-
tional interventions with proven beneficial effects in human diseases states [55, 57,
158]. The challenging aspects of the translational potential of these interventions
will be defining a discrete window of intervention as well as identifying which
100 A. Schiavi and N. Ventura
specific tissues should be targeted to sense and translate the effect of the initial
mitochondrial-targeting compound. However, the exploitation of a food supplement
or skin lotion with anti-ageing effects is clearly not far from possible applications.
Many food supplements, such as resveratrol or vitamins, as well as protective anti-
ageing skin lotions, are indeed already being used to possibly improve health and
prevent diseases development. Another concern is if, like in the nematode, mito-
chondrial reprogramming has to occur before the pre-fertile age in order to extend
healthy ageing. To this end, a more reasonable use of mitochondrial targeting inter-
ventions would be to prevent or delay the onset and progression of known genetic
disorders, or in the presence of risk factors or familiar predispositions for age asso-
ciated disorders (such as Parkinson’s or Alzheimer’s diseases) where the activation
of specific mitochondrial stress response pathways would be proven to be useful.
Last but not least, the notion of timing requirements should clearly make us very
conscious of exposure to environmental and nutritional factors before the pre-fertile
age which might negatively impact on our health span.
In conclusion, the fundamental role of mitochondria in the ageing process and
their growing role in the development of age-associated diseases make it undoubt-
edly worthwhile to keep investigating this paradoxical class of longevity mutants in
C. elegans. It is increasingly clear that the ageing process and associated changes in
the nematode closely resembles mammalian ageing in many respects (e.g. progres-
sive degeneration of different tissues and decline of different biological functions),
and can be easily manipulated and studied following genetic, nutritional and phar-
macological interventions.
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Chapter 6
Influences of Germline Cells on Organismal
Lifespan and Healthspan
6.1 Introduction
As animals grow older, their somatic tissues undergo deterioration that is mani-
fested as symptoms of ageing. However, the germline is passed on to the next gen-
eration in an immaculate condition for the maintenance of the species. This
dichotomy between the preservation of the two tissues underscores the complex
association between reproduction and ageing. This relationship is fascinating
because both are such central, intimately linked aspects of an animal’s life history.
Traditionally, research has focused on the lamentable, but well-documented, reduc-
tion in fertility associated with increasing maternal age. But, in recent years, there
has been emerging evidence that the germline and soma exchange signals that help
coordinate the health of both tissues. In this article, we provide an overview of the
existing knowledge on how signals emanating from the proliferating germline influ-
ence the rate of somatic ageing in C. elegans. Studies on ageing of the reproductive
system are addressed in Chap. 7.
The relationship between reproduction and ageing has proven to be intractable
partly because it is rife with paradoxical observations. The mating process is known
to have a negative impact on the lifespan and healthspan of females, not only due to
the physical damage caused by copulation but also by the presence of mortality-
inducing chemicals in the males’ seminal fluid. In Drosophila, males transfer pep-
tides along with sperm that reduce female lifespan [1], whereas, in many
Caenorhabditis species, mating causes hermaphrodites and females to undergo
shrinkage [2] and reduces stress resistance and lifespan [3]. Similar observations
have been made in other organisms [4, 5]. However, in numerous other species,
especially arthropods, males are known to donate edible or glandular products dur-
ing copulation that provide direct benefits to the female. These ‘nuptial gifts’ can
range from simple food and energy supplies to seminal secretions that are immuno-
protective to the recipient and its progeny, and even extend the females’ lifespan [6,
7]. Even in C. elegans, the deleterious impact of male pheromones on lifespan is
accompanied with enhanced thermotolerance in the hermaphrodite [8]. Such para-
doxes illustrate the complexity of the mutual interactions between germline and
somatic health and have made it difficult to arrive at simplistic principles about the
relationship between reproduction and ageing. This is particularly applicable to the
effect that sterility has on the lifespan and healthspan of animals.
Correlative studies in many animal and some plant species have found sterility to
be associated with increased lifespan [9–11], and these have informed the ‘Somatic
Maintenance’ or ‘Disposable Soma’ theory of ageing [12]. It is founded on the con-
sideration that reproduction is an energy intensive process that consumes cellular
resources that could otherwise be devoted towards somatic repair and maintenance.
This ‘trade off’ is postulated to be fundamental for the survival of the species but
detrimental to the individual by increasing post-reproductive mortality. However,
recent data negate this simplistic interpretation. Field studies on thirty mammalian
and bird species showed either no correlation or even a positive correlation between
fertility and longevity [13]. While some evaluations of human genealogical data
have supported a ‘trade off’ phenomenon [14], there have been many more that have
either found no association or detected positive correlations between fertility and
lifespan [15]. In fact, both contemporary and historical data from European popula-
tions have revealed significant positive correlation in this relationship in both men
and women [16–18]. Overall, observational human and animal studies provide a
growing body of evidence that fails to support the theoretical notion of a cost of
reproduction in fertile animals. Instead, recent experimental approaches in labora-
tory animals have suggested a complex, nuanced relationship between reproduction
and ageing [9, 19]. In particular, studies in C. elegans have revealed a wealth of
6 Influences of Germline Cells on Organismal Lifespan and Healthspan 111
The discovery, that reproductive signals reflective of the procreative status of the
animal modulate lifespan, led to the inevitable search for genes involved in this
soma-germline dialogue. Early observations suggested that GSC removal produces
112 F.R.G. Amrit and A. Ghazi
widespread transcriptional changes in the animal because two of the genes essential
for longevity, daf-16 and daf-12, encode transcription factors [20]. DAF-16 is the
worm homologue of FOXO3A and is the main pro-longevity factor repressed by
insulin/IGF1 signalling (IIS), the conserved and most well known lifespan-
regulatory pathway [26]. A detailed discussion of the role of IIS in ageing can be
found in Chap. 4. DAF-16/FOXO3A involvement initially indicated that reproduc-
tive signals modulate IIS to alter ageing. But, DAF-16/FOXO3A relocates to the
nuclei of intestinal cells in GSC-less young adults, whereas, upon reduced IIS,
nuclear localization occurs in many tissues [27]. Similarly, GSC-less worms
expressing DAF-16/FOXO3A only in the intestine undergo lifespan extension to the
same extent as worms that have DAF-16/FOXO3A in all tissues, but the intestinal
protein is not sufficient to increase lifespan in IIS mutants [28]. Other such observa-
tions suggested that reproductive cues and IIS are independent physiological stimuli
that share DAF-16/FOXO3A as a downstream effector.
The discovery of two genes, kri-1 and tcer-1, that selectively enhance the longev-
ity of glp-1 mutants was especially instrumental in consolidating this premise. kri-1,
encoding an Ankyrin-repeat containing protein homologous to the human disease
gene KRIT1/CCM1, is expressed only in gut cells and stimulates the nuclear local-
ization of DAF-16/FOXO3A [29]. It also mediates the transcriptional upregulation
of TCER-1/TCERG1 [30] (Fig. 6.1). We identified tcer-1, encoding the worm
homologue of a human transcription elongation and splicing factor, TCERG1, in a
screen designed to isolate genes essential for lifespan extension following GSC loss
[30]. In exploring the role of TCER-1/TCERG1, it became apparent that DAF-16/
FOXO3A regulated overlapping but distinct targets in GSC-less animals and IIS
mutants, and that TCER-1/TCERG1 increased lifespan specifically following germ-
line loss by facilitating a distinct pattern of DAF-16-dependent gene expression.
TCER-1/TCERG1 is widely expressed in nuclei of somatic tissues and its expres-
sion is elevated following germline loss in intestinal cells and (unlike DAF-16/
FOXO3A) in neurons. Elevating TCER-1/TCERG1 in the soma of fertile animals
augments their lifespan without loss of fertility and is accompanied with increased
expression of DAF-16/FOXO3A target genes [30]. This is an important discovery
because it implies that TCER-1/TCERG1 serves as a switch that connects germline
signals to the activity of a broadly deployed transcription factor such as DAF-16/
FOXO3A, and because it opens up the possibility that health benefits accrued by
GSC removal can be obtained by activating this pathway without loss of fertility.
Recent experiments, including ours, with other pro-longevity genes acting in this
pathway (discussed below) have further substantiated this beguiling possibility.
Fig. 6.1 Transcription factors activated by GSC removal in C. elegans’ intestinal cells and cellular
processes modulated by them. Proteins undergoing nuclear relocation (DAF-16/FOXO3A, SKN-1/
NRF2, HLH-30/TFEB and MML-1) are shown on membrane of, and within, the nucleus; upward
arrow next to proteins indicates transcriptional upregulation upon GSC loss (TCER-1/TCERG1,
PHA-4/FOXA, NHR-80/HNF4 and NHR-49/PPARα). DAF-16/FOXO3A nuclear localization is
governed by multiple inputs including the dafachronic-acid (DA) cascade (featuring DAF-12/VDR
and its miRNA targets), a neuronal miRNA, mIR71, and KRI-1/KRIT1. KRI-1 also enhances
TCER-1/TCERG1 transcription and SKN-1/NRF2 nuclear entry. NHR-80/HNF4 upregulation is
controlled by DAF-12/VDR and, in part, by DAF-16/FOXO3A. NHR-49/PPARα upregulation is
partially triggered by DAF-16/FOXO3A and TCER-1/TCERG1. NHR-49/PPARα participates in a
positive feed-back loop, possibly in collaboration with NHR-71/HNF4, to potentiate DAF-16/
TCER-1 activity by altering the subcellular localization of KRI-1/KRIT1. The main cellular pro-
cesses modulated by these factors include lipid metabolism, autophagy and protein homeostasis.
DAF-16/FOXO3A acts with TCER-1/TCERG1 to elevate both lipid-synthetic and lipid-degradative
pathways. SKN-1/NRF2 shares the regulation of some of these processes. NHR-49/PPARα (likely
in cooperation with MDT-15) stimulates β-oxidation and fatty-acid desaturation, whereas, NHR-
80/HNF4 promotes fatty-acid desaturation alone. SKN-1/NRF2 and DAF-16/FOXO3A enhance
proteasomal activity, while autophagy is augmented by PHA-4/FOXA, HLH-30/TFEB and the
MML-1/MXL-2 complex. Improved heat- and oxidative stress resistance is mediated by HSF-1/
HSF, SKN-1/NRF2 and, partly, DAF-16/FOXO3A
114 F.R.G. Amrit and A. Ghazi
lipophilic hormones and steroids to modulate gene expression, including the expres-
sion of regulatory microRNAs (miRNAs). The Antebi lab has shown that DAF-12/
VDR influences the choice between normal growth and diapause during larval
development through binding and activation by 3-keto bile acid-like steroid ligands
called Δ4 and Δ7 dafachronic acids (DAs) [32]. In GSC-less worms, DAs activate
DAF-12/VDR which in turn promotes DAF-16/FOXO3A nuclear localization [33]
by increasing the levels of (at least) two miRNAs, mIR-84 and mIR-241 [34]. Both
were reported to be upregulated by DAF-12/VDR in intestinal and epidermal tissues
upon GSC ablation and were redundantly required for DAF-16/FOXO3A nuclear
localization and glp-1 mutants’ longevity. Genetic evidence suggests that mIR84
and mIR241 promote DAF-16/FOXO3A nuclear traffic by repressing the expres-
sion of (at least) two known anti-longevity proteins [34]. One of these, SGK-1, is a
kinase that phosphorylates DAF-16/FOXO3A to inhibit nuclear entry [35]. The sec-
ond, LIN-14, a developmental timing protein has also been previously shown to
limit lifespan by repressing DAF-16/FOXO3A [36]. In contrast, DAF-12/VDR
exerts no influence on mIR-71, another pro-longevity miRNA shown by the Horvitz
lab to act in neuronal cells to augment DAF-16/FOXO3A’s intestinal nuclear pas-
sage [37]. It remains to be seen if additional miRNAs, regulated by DAF-12/VDR
or not, have roles in this longevity pathway.
In a series of exhaustive studies, the Antebi lab also identified multiple genes of
the biosynthetic pathway responsible for DA production, including daf-9 (encodes
a cytochrome P450 enzyme) [38], daf-36 (encodes a Rieske-like oxygenase enzyme)
[39] and dhs-16 (encodes a 3-hydroxysteroid dehydrogenase) [40] (Fig. 6.1).
Expectedly, each of these genes is essential for lifespan extension upon GSC abla-
tion, and supplementation of DA in GSC-less daf-9 and daf-36 (but not daf-12)
mutants extends lifespan [33]. Interestingly, worms lacking both the germline and
the somatic gonad, that would otherwise exhibit wild-type lifespan, live signifi-
cantly longer upon DA supplementation [41]. This observation raises the possibility
that the somatic gonad may be the site of DA production following GSC removal,
and may partly explain the importance of the organ in GSC-less longevity. However,
DA levels between wild-type worms and glp-1 mutants have been reported to be
similar, although the study examined whole worms and would not have detected
localized changes in levels [42]. daf-12 mutants expressing a nucleus-restricted ver-
sion of DAF-16/FOXO3A selectively in intestinal cells do not undergo life length-
ening suggesting that, besides regulating nuclear relocation, DAF-12/VDR is also
required for DAF-16/FOXO3A’s transcriptional activity [29]. Unlike the well-
characterized role of DAF-12/VDR in modulating DAF-16/FOXO3A sub-cellular
traffic, how it influences the latter’s activity within the nucleus is unknown.
Besides DAF-12/VDR, the ‘NHR’ gene family in C. elegans includes ~284 mem-
bers. The Aguilaniu lab showed that one of these, NHR-80, an ortholog of mam-
malian hepatocyte nuclear factor 4 (HNF4), is essential for glp-1 mutants’ longevity
6 Influences of Germline Cells on Organismal Lifespan and Healthspan 115
The forkhead box, or FOX, gene family includes transcription factors belonging to
subfamilies ranging from FoxA to FoxP. While DAF-16 is a member of the FoxO
sub-family, PHA-4 represents the FoxA branch and is orthologous to genes encod-
ing mammalian FOXA1, FOXA2 and FOXA3 proteins. PHA-4/FOXA was initially
reported to be critical only for worms that are long lived due to reduced food intake
or dietary restriction (DR), a paradigm that also extends lifespan in a variety of
metazoan species [52, 53]. However, this study did not examine GSC-less mutants
and in a subsequent report from the Hansen lab, PHA-4 was shown to be essential
for GSC-less longevity as well [54]. PHA-4 mRNA levels rise upon GSC-removal,
independent of DAF-16/FOXO3A. Although the tissues where this upregulation is
orchestrated are not known, it is brought about by repression of the nutrient sensing
kinase, target of rapamycin (TOR), and results in increased expression of multiple
autophagy genes [54].
116 F.R.G. Amrit and A. Ghazi
role in embryonic development [60], has been shown to extensively influence adult
health and longevity by regulating multiple stress-response pathways, in normal
worms as well as in diverse long-lived mutants [53, 59]. SKN-1/NRF2 overexpres-
sion extends lifespan modestly as well [59]. The protein translocates to the nuclei of
intestinal cells in IIS mutants, wherein, it promotes longevity and stress resistance
in a genetically parallel pathway from DAF-16/FOXO3A [59]. However, in mutants
representing DR longevity, its function in a pair of sensory neurons is sufficient for
lifespan to be augmented [53]. skn-1 inactivation was previously reported to shorten
glp-1 mutants’ longevity [61], and subsequently, the Blackwell and Kenyon labora-
tories confirmed this observation [62, 63]. Both groups showed that SKN-1/NRF2
nuclear localization occurs in intestinal cells of glp-1 mutants. The Blackwell lab
reported that the nuclear entry was partially dependent upon tcer-1 and kri-1, and
completely under control of pmk-1 that encodes a p38 MAP Kinase known to phos-
phorylate SKN-1/NRF2 in other contexts [62]. However, by testing the induction of
a SKN-1/NRF2-target gene, gst-4, the Kenyon lab found that SKN-1/NRF2 is acti-
vated only marginally by the p38 MAP Kinase pathway, and strongly by the trans-
sulphuration pathway [63]. The transsulphuration pathway leads to the production
of sulphur-containing metabolites, including hydrogen sulphide (H2S) and previous
reports have shown that H2S extends worm lifespan via SKN-1/NRF2 activity [64].
Interestingly, KRI-1/KRIT1 was partially responsible for H2S production and gst-4
induction, whereas, both events were DAF-16/FOXO3A independent, suggesting
that KRI-1/KRIT1 has independent effects on DAF-16/FOXO3A and SKN-1/NRF2.
Using both site-specific overexpression and loss-of-action studies, the authors also
showed that SKN-1/NRF2 acts in the adult intestine to extend lifespan [63].
The worm homologue of the human heat-shock factor (HSF), HSF-1, an essen-
tial component of the heat-shock response (HSR) and proteostasis is also required
for glp-1 mutants’ longevity [65]. Similar to SKN-1/NRF2, HSF-1/HSF impacts
multiple longevity paradigms as well as normal lifespan [66]. Other transcription
regulators such as MDT-15 (component of the mediator complex that putatively
works as a co-activator of NHR-49/PPARα) [67] and SBP-1 (homologue of human
SREBP1 transcription factor) [68] have also been implicated in glp-1 longevity [69]
and stress resistance [62], respectively, but details of their regulation and molecular
function are as yet unaddressed.
As evinced from the paragraphs above, eliminating the worm germline triggers the
activation of a host of transcription regulators largely in intestinal cells (Fig. 6.1)
suggesting that GSC removal is accompanied by a major gene-expression shift. It
raises questions about the nature of these transcriptional changes, their physiologi-
cal outcomes and the relationships between the regulatory factors involved.
118 F.R.G. Amrit and A. Ghazi
Contemporary studies have provided some insights into these queries, although
there are many more questions than there are answers in the field. In a microarray-
based study, about 3440 genes were reported to be upregulated and 150 downregu-
lated (of the approximately ~18,000 genes in the worm genome) in GSC-ablated
worms as compared to whole-gonad ablated sterile worms [70]. Similar transcrip-
tional changes accompany GSC removal in Pristionchus pacificus, a related nema-
tode that exhibits GSC-less life extension too, with a significant overlap between the
transcriptomes of GSC-less worms of the two species [70]. In contrast, an RNA-
Sequencing (RNA-Seq)-based study identified a smaller group upregulated in glp-1
mutants’- 1306 and 615 genes, by more than fourfold and fivefold, respectively
[62]. The differences in numbers between these studies notwithstanding, they sup-
port broad transcriptional remodelling upon GSC removal. Experiments aimed at
identifying the targets of some of the transcription factors discussed above have
suggested the involvement of specific biochemical pathways. McCormick et al.
reported the identification of 230 and 130 genes whose expression was altered in
glp-1 mutants dependent upon DAF-16/FOXO3A and DAF-12/VDR, respectively
[69], whereas, Steinbaugh et al. identified 529 SKN-1/NRF2 targets in glp-1 mutants
[62]. In a recent study, we discovered 835 and 801 downstream genes whose expres-
sion is governed by TCER-1/TCERG1 and DAF-16/FOXO3A, respectively, in glp-
1 adults. About one-third of the targets are shared between the two factors [71]. A
similar comparison of the downstream targets of HLH-30/TFEB and the MML-1/
MXL-2 complex identified a substantial number of co-regulated genes; MML-1 and
MXL-2 shared 827 targets, whereas, 202 were common between all three factors
[58]. In all these studies, the gene lists were strongly enriched for lipid-metabolic
functions. In addition, proteasomal degradation, autophagy and stress resistance are
also consistently represented. In the following sections, we focus on each of these
cellular processes and discuss their impacts on the reproductive control of ageing.
Germline removal in C. elegans not only increases lifespan and stress resistance it
also causes elevated fat accumulation, observed using lipid-labelling dyes as well as
biochemical approaches [72]. At first glance this is astonishing as obesity is associ-
ated with increased mortality, not better health and long life. However, gonadec-
tomy precipitates enhanced fat accumulation in many organisms besides worms, in
both invertebrates (e.g., fruit flies, blow flies, locusts, grasshoppers) [73–77] and
vertebrates (e.g., mice, rats, cats and monkeys) [78–81]. In humans, deficient
gonadal hormone production results in obesity and metabolic disorders [82]. But,
all fat is not equal and all fat accumulation is not detrimental to the organism. Long-
lived IIS worm mutants manifest greater adiposity but are healthier and longer-lived
than their leaner, wild-type counterparts [72, 83]. In Drosophila, interventions that
6 Influences of Germline Cells on Organismal Lifespan and Healthspan 119
extend lifespan, such as reduced IIS and TOR inhibition, elevate fat [84, 85]. Obese
mice that exhibit healthy metabolic profiles have been described too [86, 87].
Similarly, a small but striking group of ‘metabolically healthy obese’ individuals are
notable because they retain excessive weight without developing clinical patholo-
gies [88]. Altogether, there is increasing awareness of the nuanced and multi-layered
relationship between adiposity, reproduction and lifespan (for reviews see [89, 90]).
Studies in GSC-less worms have revealed interesting answers to questions regard-
ing this trifecta.
In our RNA-Seq study [71], key conserved genes encoding enzymes responsible for
initiating de novo fatty acid synthesis were identified as being upregulated by DAF-
16/FOXO3A and/or TCER-1/TCERG1 upon GSC removal. These included pod-2
(encodes acetyl CoA carboxylase, ACC), fasn-1 (encodes fatty-acid synthase, FAS)
and mlcd-1 (malonyl CoA decarboxylase 1, MLCD). Accordingly, lipid-labelling
120 F.R.G. Amrit and A. Ghazi
6.4.2.4 Lipolysis
One of the first fat-metabolic genes found to be essential for glp-1 mutants’ longev-
ity by the Ruvkun lab encoded a lipase, LIPL-4, orthologous to a human lipase
LIPA. lipl-4 expression is intestine restricted and upregulated by DAF-16/FOXO3A
in glp-1 mutants; it’s overexpression lengthens fertile animals’ lifespan [97]. Besides
LIPL-4, at least six other lipases and lipase-like proteins encoded in the C. elegans
genome are upregulated (or predicted to be so) following GSC loss, dependent upon
DAF-16/FOXO3A, TCER-1/TCERG1 or SKN-1/NRF2 [62, 69, 71]. RNAi of many
of these reduces glp-1 longevity [71]. The involvement of multiple lipases in this
lifespan paradigm is inexplicable, especially in the light of the marked adiposity
manifested by GSC-less animals. LIPL-4/LIPA has been shown to link lipid metab-
olism to autophagic flux upon GSC removal (discussed in the next section) [54] and
the SKN-1/NRF2 target LIPL-3 is postulated to prevent excess fat accumulation
[62]. In fertile worms, OEA production is LIPL-4 dependent [48, 97] and in human
cardiac cells, the cytosolic lipase ATGL-1 is essential for PPARα ligand synthesis
[98]. Hence, it is plausible that one or more of these lipases may also facilitate the
production of signalling molecules or ligands for factors such as NHR-49/PPARα or
NHR-80/HNF4. But, overall little is understood about the function of the lipolytic
genes and is likely to be a major focus of future studies.
The current data on lipid metabolism and reproductive control of ageing raises
many important questions. Why does GSC-ablation increase fat content and what is
the nature of these adipose depots? Why are lipid catabolism and anabolism simul-
taneously augmented and how? Lipid content is increased largely in the intestine,
and to some extent, in the epidermis. However, the worm intestine is not simply a
part of the alimentary canal and the major fat-storage depot, it also subsumes func-
tions of the liver and pancreas, is the main site for induction of immunogenic
responses and the sole centre for yolk synthesis. Yolk, made up of lipids and pro-
122 F.R.G. Amrit and A. Ghazi
teins, is generated in gut cells, secreted into the body cavity and transported to the
gonad to be deposited into oocytes for nourishment of the embryo. It is a logical
supposition that the adiposity of GSC-less animals is derived from yolk lipids that
can no longer be deposited into eggs, and much of it is likely to be so. Indeed,
Steinbaugh et al. showed that GSC-less mutants continue to produce a yolk lipopro-
tein, vitellogenin 2 (VIT-2) [62]. But in this study, VIT-2 localization was predomi-
nantly in the body cavity, not the intestine. Instead, there was increased level of
GFP-labelled DHS-3 (DHS-3::GFP), a protein that almost exclusively labels TAGs,
in gut cells of GSC-less mutants [62]. Additionally, the gene-expression data and
biochemical evidences showing elevated de novo fatty-acid synthesis [71], TAG
production [44, 71, 72] and lipid desaturation [43, 44, 71] suggest that the excess fat
in GSC-less worms is derived at least in part from bona fide increase in lipid pro-
duction. But why is lipogenesis triggered when fertility is thwarted? While DGATs
can help immure fats (normally designated for oocytes in fertile animals) into lipid
droplets, what purpose is served by elevating de novo fatty-acid synthesis? The
reason for this is unknown, but vertebrate and worm evidences suggest that it may
serve signalling functions. Fatty acids have been known as lipid ligands for long.
But, recent reports have begun to emphasize the importance of the ‘source’ of lipid
signals. For instance, mice incapable of synthesizing ‘new fat’ due to FAS/FASN-1
deletion in the liver or hypothalamus cannot activate PPARα [99]. So, augmenting
de novo lipid synthesis may help synthesize ligands for factors activated upon germ-
line depletion. As mentioned above, PPARα ligand production in cardiac cells is
dependent upon the lipase ATGL-1 [98], so it is equally possible that one or more of
the lipases upregulated upon GSC removal also contributes to the synthesis of such
ligands.
Why is lipid desaturation increased upon GSC loss? The elevated MUFA:SFA
ratio observed in glp-1 mutants suggests that the metabolic shift involves not only
quantitative but also a qualitative remodelling of the lipid profile. SFAs are critical
for reproductive health as they make up >70 % of human oocyte lipids, whereas,
MUFAs make up <15 % [100]. But, SFAs are poor substrates for incorporation into
TAGs and major causes of lipotoxicity [95, 96]. Alternatively, lipids with higher
UFAs are generally associated with improved cellular maintenance [101], and with
enhanced lifespan in human centenarian studies [102]. The transformation of a
SFA-rich, reproduction-oriented lipid profile of a fertile adult into one that is
enriched in UFAs by proteins such as NHR-49/PPARα and NHR-80/HNF4 may
mitigate the deleterious effects of GSC loss and organize a lipid profile conducive
for somatic maintenance and health. Whether this transformation is simply an adap-
tation to sterility or the bedrock for longevity remains to be discovered, and the two
possibilities are not mutually exclusive either. Another important question emerging
from these studies is how these transcription factors simultaneously elevate ostensi-
bly antagonistic lipid-metabolic steps in the same animal. Future experiments will
address if, and how, this coordination is managed, and if the strategy is widely used
in the animal kingdom for retaining lipid homeostasis in the face of metabolic
challenges.
6 Influences of Germline Cells on Organismal Lifespan and Healthspan 123
One of the major quality control mechanisms that influence cellular homeostasis is
the ability to degrade proteins. Autophagy and the ubiquitin proteasome system
(UPS) are the two main proteolytic systems, the UPS being the primary pathway for
protein degradation in eukaryotic cells. The most well-known function of UPS is the
spatially and temporally controlled destruction of regulatory proteins that inform
various cellular processes. Regulatory proteasomal activity has been implicated in
the lifespan extension of C. elegans mutants, including GSC-less worms [110–112]
(see also Chap. 12). In addition, the ‘housekeeping’ function of UPS in destroying
damaged, misfolded, old and aggregation-prone proteins is critical for maintenance
of the proteostasis network. Loss of proteostasis is one of the hallmarks of ageing to
which numerous age-related pathologies are attributed [113]. In C. elegans, the abil-
ity to retain proteostasis falls dramatically once peak reproductive age is reached,
and declines over time [114]. Conversely, germline-deficient animals exhibit
enhanced resistance to conditions that tax the proteostasis machinery such as high
6 Influences of Germline Cells on Organismal Lifespan and Healthspan 125
Many interventions that prolong life also confer tolerance to environmental stress-
ors, in worms and in many other species, though exceptions exist [117]. glp-1
mutants also exhibit this positive correlation between longevity and stress resis-
tance. In C. elegans, the most commonly studied stress paradigms include oxidative
stress, heat shock, protein misfolding in the endoplasmic reticulum (ER) and mito-
chondria that evokes an unfolded protein response (UPR) in these organelles
(UPRERand UPRmt, respectively) and immuno-competence, or ability to combat
pathogen attack [118]. Arantes-Oliveira et al. first reported the enhanced oxidative-
stress resistance of glp-1 mutants [22]. Steinbaugh et al. substantiated this data and
characterized the vital role for SKN-1/NRF2 in mediating this resilience [62]. Wei
and Kenyon linked GSC removal to altered redox signalling and showed that GSC
removal causes elevation in the levels of reactive oxygen species (ROS) and H2S
cell non-autonomously [63]. These events are important because quenching ROS
with anti-oxidants or inhibiting the transsulphuration pathway responsible for H2S
production curtailed longevity. Interestingly, ROS and H2S appear to activate differ-
ent stress-response paradigms during adulthood; ROS leads to UPRmt, whereas, H2S
causes SKN-1/NRF2 activation. Using two chemical redox sensors, it was found
126 F.R.G. Amrit and A. Ghazi
that ROS production was induced in two waves. The first one, a mitochondrial sig-
nal, was detected late in larval life, just before the animal reaches adulthood. The
second cytoplasmic ROS signal appeared during early adulthood and activated
UPRmt (as evidenced by the induction of the UPRmt reporter, hsp-6) through upregu-
lation of the transcription factors DVE-1 that mediates UPRmt, and its co-activator
UBL-5 [63]. Interestingly, atfs-1, that encodes the key mediator of UPRmt, is
included in the list of genes upregulated by DAF-16/FOXO3A and its RNAi knock-
down suppresses glp-1 longevity [71], but hsp-6 induction was found to be DAF-
16/FOXO3A independent [63]. The finding that SKN-1/NRF2-dependent
detoxification systems and DVE-1-dependent UPRmt are induced by different redox
systems is highly intriguing, and may reflect the response of somatic tissues to loss
of individual aspects of germ-cell physiology. How the other transcription factors of
the network play into these stress-response initiatives remains to be described.
Studies focusing on the interaction between reproductive fitness and somatic
endurance have revealed considerable information on the ability of GSC-less
mutants to mount a chaperone-driven heat-shock response (HSR) following expo-
sure to high temperatures. In C. elegans, thermo-resistance declines with the onset
of reproduction, at least in part due to diminished expression of a histone H3 tri-
methyllysine-27 (H3K27me3) demethylase, JMJD-3.1, that antagonizes
transcription-repressive chromatin marks [119]. glp-1 mutants are exceptionally
thermotolerant, dependent upon many of the genes discussed here, including daf-
16, tcer-1, kri-1, hsf-1 and jmjd-3.1 [115, 119]. However, other mutants that exhibit
sterility due to gonadogenesis defects (not GSC loss) and have normal lifespan (e.g.
glp-4, gon-2) are also thermotolerant, although genetic evidence hints that GSC
removal (not just arresting reproduction) may trigger their HSR.
As with ageing, immuno-competence also manifests an inverse correlation with
reproduction in many organisms, including C. elegans. Expectedly, glp-1 mutants
exhibit superior resistance against gram-negative pathogens such as Salmonella
enterica [120], Pseudomonas aeruginosa [121, 122] and Serratia marcescens [122],
the gram-positive pathogen Enterococcus faecalis [120], and the fungal pathogen
Cryptococcus neoformans [120]. Of these, the response to P. aeruginosa has been
well studied. Alper et al. reported that DAF-16/FOXO3A’s requirement for glp-1
mutants’ immunoresistance was influenced by worm-culture conditions, indicating
that nutrient status may have an impact on the reproduction-immunity relationship
as well [122]. Interestingly, the DAF-16/FOXO3A-driven immuno-resistance
appears to be associated with sterility and not especially GSC status, as similar
resistance is exhibited by other gonadal mutants that are sterile but not long lived
(e.g., the feminized mutant, fog-2 and the somatic-gonad defective mutant, glp-4)
[122, 123]. DAF-16/FOXO3A also undergoes nuclear relocation in many gonadal
mutants not just those lacking GSCs. However, GSC removal seems essential to
confer broader and stronger immunity, because both glp-1 and glp-4 mutants are
resilient against P. aeruginosa, whereas, only glp-1 mutants are resistant to S.
enterica infection [120]. Altogether, these findings reiterate that GSCs may signal
to inhibit immunocompetence, and upon their removal innate immunity is enhanced.
In few cases, the genetic basis of this improved immunity has been dissected and
6 Influences of Germline Cells on Organismal Lifespan and Healthspan 127
The initial observations of Hsin and Kenyon that demonstrated the control exerted
by the germline on the lifespan of the animal [20], and challenged the simplistic
‘trade off’ interpretation, laid the groundwork for a field that has burgeoned into
great significance and mainstream science interest. The early worm studies led to a
renewed examination of the germline-soma dialogue in other model organisms and
species. The remarkable ease of molecular-genetic analysis and large-scale RNAi
screening in worms allowed the identification of innumerable genetic players with
roles in this dialogue, many of them with conserved functions in lifespan regulation.
These studies have not only revealed knowledge about ageing but have also led to
important discoveries in the fields of metabolism, autophagy and proteostasis. The
128 F.R.G. Amrit and A. Ghazi
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6 Influences of Germline Cells on Organismal Lifespan and Healthspan 135
Cheng Shi and Coleen T. Murphy
7.1 Introduction
C. Shi
LSI Genomics and Department of Molecular Biology, Princeton University,
Princeton, NJ, USA
C.T. Murphy (*)
Glenn Center for Aging Research, Princeton University, Princeton, NJ, USA
LSI Genomics and Department of Molecular Biology, Princeton University,
Princeton, NJ, USA
e-mail: ctmurphy@princeton.edu
distances (including humans and C. elegans), and for these species, it is one of the
earliest age-related phenotypes manifested, appearing in mid-adulthood. For
humans, reproductive ageing has both medical and societal implications, as the inci-
dence of infertility, maternal age-related birth defects, and miscarriage begin to rise
in the mid to late 30s. This is more than a decade before oocyte depletion and the
onset of menopause (after age 50), and reproductive decline in women is thought to
be due to declining oocyte quality. Therefore, whether C. elegans can be a model of
human reproductive ageing hinges upon whether its reproductive ageing is also due
to oocyte quality decline, or other factors.
How does reproductive ageing occur? Why doesn’t every species maximize its
reproductive period (i.e., minimize its post-reproductive lifespan)? What limits the
reproductive span of an animal? What are the similarities and differences between
reproductive ageing and somatic longevity, and what is the relationship between
them?
In this chapter, we will try to address these questions as we review the most
recent progress in the field of C. elegans reproductive ageing, which has provided
many insights into the underlying genetic pathways and molecular mechanisms that
regulate this process. We will first introduce how reproductive ageing is measured
in C. elegans. Then we will summarize the current knowledge of reproductive age-
ing from C. elegans studies, such as the known conditions, tissues, and genetic
pathways involved in reproductive ageing regulation. We will also compare and
contrast worm and human/mammalian reproductive decline and illustrate why C.
elegans is a good model for studying mammalian reproductive ageing. Finally, we
will discuss how the knowledge gained from worm studies contributes to the under-
standing of relationship between reproductive ageing and somatic longevity.
The total progeny produced (brood size) is often used as a simplified equivalent of
reproduction (although “reproduction” has broader meanings). By contrast, “repro-
ductive span” is used to reflect the rate of reproductive ageing, and reports the frac-
tion of mothers still reproductive with age. Before we focus on the relationship
between reproductive and somatic ageing, it would be helpful to discuss whether
and how reproduction (brood size) and reproductive ageing are correlated.
Hughes et al. [2] found that early reproduction does not affect reproductive age-
ing. fog-2 hermaphrodites, which do not produce self sperm [1], display similar
rates of decline in reproductive capability after being mated with wild-type males at
different time points (Day 1, 3, 5, 7, 10). This result was unexpected, differing from
theories that suggested that the C. elegans germline could continually produce
oocytes of high quality throughout life. The number of progeny produced each day
depended on the age of hermaphrodites, and was independent of the time of mating.
The total number of progeny produced ranged from 7 (mated on Day 10) to over
500 (mated on Day 1); however, the last day of reproduction for all the groups were
the same [2]. Therefore, early reproduction and brood size have no effect on the rate
7 Reproductive Ageing 139
of reproductive ageing. Likewise, a separate study reported that early progeny pro-
duction does not cause reproductive ageing [3]. Thus, delaying reproduction does
not allow reproduction to continue longer at a later age. Later work showed that
oocytes age regardless of usage [4], similar to human reproductive ageing, thus
uncoupling total early reproduction from duration of reproduction.
Reproductive Span Assays Reproductive span (RS) assays measure how long
each individual mother in a population reproduces [2, 7, 8]. Because the data are
binary (reproductive vs non-reproductive mothers), the focus of this type of assay is
the length of time that the mothers reproduce, rather than on the number of progeny
they produce, analogous to the “live vs dead” assessment in survival analyses. Late
L4 hermaphrodites are placed on individual plates and transferred every day until
reproduction ceases for at least 2 days (or are censored due to death, matricide, loss
from plates, etc.). The data from individuals with the same genotype or treatment
are then pooled and plotted as “percent reproductive” for each day, analogously to
traditional lifespan curves. Reproductive span curves can be compared using stan-
dard survival statistical tests such as the log-rank (Mantel-Cox) method [2, 7].
The self-sperm reproductive span assay is commonly used, largely because it is
easier to perform than mated reproductive spans. However, as noted above, because
self RS assays measure sperm count, which is not the limiting factor for reproduc-
tive ageing in wild-type C. elegans [4], self-reproductive span is not an accurate
reflection of C. elegans reproductive ageing. Instead, oocyte quality is the limiting
factor for normal reproductive span [4], and can only be assessed through mated
reproductive span assays. Mating with males provides an excess of sperm and sig-
nificantly extends reproductive span [2, 7], as the male sperm “use up” the hermaph-
rodite’s oocytes with age.
The mated reproductive span assay is more laborious than the self-reproductive
span assay, as it involves mating hermaphrodites with an excess of males on Day 1
of early adulthood, then transferring the mothers and monitoring the sex ratio of
progeny to ensure that there was abundant male sperm supply throughout the entire
reproductive span [2, 7]. Old (Day 10) hermaphrodites whose self-reproduction has
ceased for several days are able to reproduce again after mating with males [9], sug-
gesting that reproductive cessation in self-fertilized hermaphrodites is caused by
self-sperm depletion, rather than a bona fide decline in reproductive capability.
Therefore, although self-reproductive span is often used as a quick and rough esti-
mate of reproductive ageing, mated reproductive span is the most accurate, gold-
standard measurement of C. elegans reproductive ageing.
Late-Life Cross Progeny Production Hermaphroditic reproductive decline is
intrinsic and is independent of reproduction early in life (a “usage independent”
mechanism) [2]. Thus, instead of measuring the entire reproductive span, several
studies focus only on late-life progeny production. Rather than being mated begin-
ning at late L4 stage, in this case the hermaphrodites are mated later, such as Day
8 – Day 15 of adulthood. For wild-type hermaphrodites (N2), Day 13 is the latest
time reported to regain fertility after mating [9]. To assess reproductive capability,
daily progeny production profiles or total progeny number can be obtained for the
hermaphrodites after they are mated at various ages with males [2, 9]. Another way
to quantify late-life reproductive ageing is to calculate the percentage of worms able
to regain fertility after crossing with males [9, 10]. However, there is a discrepancy
between the fully-mated reproductive span (hermaphrodites mated on Day 1 of
adulthood) and late-life cross-fertility. daf-2(e1370) insulin receptor mutants have a
7 Reproductive Ageing 141
greatly extended reproductive span when mated early in adulthood [2, 4, 7, 8]. By
contrast, Mendenhall et al. reported that the cross-fertility of daf-2 worms is lower
than that of wild-type controls in late-life mating assays [9]. Additionally, there is
little overlap between genes that alter reproductive span using a similar late repro-
duction assay in an RNAi screen [11] with those discovered by other genetic meth-
ods. Different physiological states at late age might complicate the assessment of
reproductive capability. Therefore, although easier to perform, late-life cross prog-
eny production results should be interpreted with caution until the regulatory mech-
anisms have been further explored.
such as the DNA repair gene mlh-1, cause embryonic lethality specifically in late
progeny, suggesting that some processes, including DNA repair, become more
crucial in older oocytes [4]. Therefore, counting embryonic death rate (unhatched
embryos) with age is a simple way to measure reproductive ageing and is compati-
ble with mated reproductive span assays.
Embryonic lethality has also been used to assess the role of apoptosis in repro-
ductive ageing. Physiological apoptosis, which occurs in the germline’s apoptotic
zone prior to the germ cell nuclei’s cellularization, removes over half of the oogenic
germ cell nuclei [16], and disruption of this process through apoptosis-defective
mutants increases embryonic lethality with age, suggesting a role in oocyte quality
control [13]. Surprisingly, however, neither physiological nor DNA damage-induced
apoptosis [17] contributes to TGF-β- or IIS-mediated extension of reproductive
span [4]. Therefore, while apoptosis is necessary for the normal production of
oocytes, it does not appear to be a critical factor in the slowing of reproductive age-
ing and maintenance of oocyte quality under IIS- or TGF-β signalling of low-
nutrient conditions.
Fertilization The ability to be fertilized is a critical factor for oocytes in all spe-
cies, and is especially easy to measure in C. elegans: unfertilized oocytes are still
laid, but are evident as amorphous light “blobs” in contrast to solid eggs. This allows
relatively easy scoring of infertility with age, particularly in mated assays that have
excess sperm [4]. This assay revealed that IIS and TGF-β mutants improve the fer-
tilizability of aged oocytes [4].
To summarize, each method described above has its advantages and disadvan-
tages. There is a trade-off between accuracy and manual labour. Progeny profile
counts focus on early rather than late reproduction, and so are less informative about
reproductive ageing. Mated reproductive span, which measures the rate of oocyte
quality decline (the limiting factor for reproductive ageing in both worms and mam-
mals) is the gold standard to study reproductive ageing in C. elegans, but is labour
intensive compared to self-reproductive spans. Changes in germline and oocyte
morphology, as well as simple readouts of oocyte quality, particularly embryonic
lethality, are also good indicators of reproductive ageing, and in some cases can
identify the particular mechanism of oocyte quality control.
7.4 C
onditions, Genetic Pathways, and Tissues That Affect
Reproductive Span
C. elegans has been a great model for the study of somatic longevity [18] and was
recently established as a reproductive ageing model, as well. In this section, we will
introduce various mechanisms of reproductive span extension and discuss recent
findings on reproductive ageing in C. elegans. We will focus more on the studies
using mated hermaphrodites, which better mimic reproductive (oocyte) decline in
7 Reproductive Ageing 143
Temperature Temperature affects somatic longevity, and the neurons and genetic
pathways that are necessary for this regulation have been identified: thermosensory
AFD neurons and the downstream DAF-9/DAF-12 pathway are required in lifespan
regulation at warm temperature (25 °C) [20]. By contrast, a cold-sensitive TRP
channel, TRPA-1, is specifically involved in lifespan regulation at 15 °C [21].
Temperature also significantly affects the reproductive span of C. elegans [2]. At
15 °C, wild-type hermaphrodites have a 29 % increase in mated reproductive span.
Although the total progeny produced are similar at the two temperatures, there is
about a tenfold increase in progeny produced after Day 9 compared to worms raised
at 20 °C, suggesting that temperature affects the rate of oocyte utilization and shifts
the peak to the right. By contrast, higher temperature (25 °C) causes a 32 % decrease
in mated reproductive span, a significant reduction in total progeny production, and
decreased late-life reproduction [2]. However, which mechanism is responsible for
reproductive span regulation at various temperatures remains to be explored.
Diet
Dietary Restriction (DR) The reduction of dietary intake is known to extend the
longevity and reproduction of many animals across great evolutionary distances [2,
8, 22–25]. In C. elegans, several forms of reduced diet, including bacterial depriva-
tion on plates [26, 27], bacterial dilution in liquid cultures [28–30] and on plates
144 C. Shi and C.T. Murphy
[31, 32], and axenic and chemically defined liquid media [33, 34] extend lifespan
[32], as does intermittent fasting (IF) [35]. Additionally, most of these treatments
reduce total progeny production. However, many of these direct reductions of bacte-
rial intake have not been tested for their effects on reproductive ageing.
The genetic mutant eat-2(ad465) has been established as a model for DR [36].
eat-2 encodes an acetylcholine receptor, and its loss reduces the worms’ ability to
digest food due to reduced pharyngeal grinding [37]. eat-2 mutants produce fewer
progeny, but extend mated reproductive span more than 30 %, and increase late-life
progeny production by five to eightfold [2]. eat-2 and other forms of DR also dra-
matically increase late life cross-fertility. When mated late in life (after Day 13 of
adulthood), wild-type hermaphrodites no longer produce cross progeny, but eat-2
mutants can reproduce even when mated at Day 17 of adulthood [9]. eat-2’s
extended life span depends on the activity of pha-4, a FoxA transcription factor
[38]; pha-4 is also required for eat-2’s extended reproductive span [7].
Bacterial source: In the lab, C. elegans is typically fed the E. coli strain OP50
[39], but different bacterial diets have been shown to affect reproduction and fertil-
ity, just as different bacterial diets affect longevity [40, 41] (see also Chaps. 17 and
18). For example, nuclear hormone receptor nhr-114(lf) mutants are sterile on
OP50, but fully fertile on HT115 or OP50 + tryptophan, illustrating that amino acid
sensing affects fertility [42]. More recently, Chi, et al. found that pyrimidine salvage
pathway-deficient mutants are sterile on OP50, but fertile on HT115 or OP50 +
uridine(U)/thymidine(T), and that germline proliferation can be modulated by dif-
ferent levels of U/T in food through the GLP-1/Notch pathway [43]. Although nei-
ther study performed reproductive span assays, it is possible that the two nutrient
sensing pathways, amino acid sensing [42] and nucleotide sensing [43], might be
involved in reproductive span regulation, in addition to the known glucose/carbohy-
drate sensing of the insulin/IGF-1 pathway [2, 7].
Similarly, Sowa et al. [10] also found that different bacteria diets result in differ-
ent self-reproductive spans, with OP50, the normal laboratory diet, displaying the
longest RS, and HB101 with the shortest. The downstream genetic pathways are not
known, and no measurements of oocyte quality were performed, so the effect of
different bacterial diets on reproductive ageing is unknown. However, the fact that
sensory neurons are activated differentially by the different bacterial sources sug-
gests that, just as dietary restriction slows reproductive decline [7, 8], differences in
reproductive span lengths may be the consequence of the animal interpreting the
nutrient value of a food source, and adjusting its reproductive span (and life span)
accordingly [44].
C. elegans can also be grown on axenic media [33, 34], but the effects on repro-
ductive ageing have not yet been determined. Axenic cultivation of C. elegans leads
to reduced brood size, a prolonged reproductive period, and extended lifespan [33,
34]. However, such reproductive span extension is coupled with the Dietary
Restriction effect of axenic media, so whether the media itself affects reproductive
ageing is not yet known.
7 Reproductive Ageing 145
Life History
Post-dauer In response to harsh environmental conditions during the first larval
stage, C. elegans can enter the alternative third larval dauer stage at the second
moult, and exit dauer arrest and proceed with development to reproductive adult-
hood when favourable conditions resume [45]. A detailed discussion of the dauer
larva can be found in Chap. 3. Superficially, post-dauer adults (which enter and then
exit dauer stage) and normally developed adults (which bypass the dauer stage) are
similar. However, Hall, et al. [47] compared the transcriptomes of age-matched
young adults that had undergone these two different developmental histories, and
found that among the differentially expressed genes, the largest group is “reproduc-
tion”. Twenty-three percent of previously identified sperm-enriched genes are sig-
nificantly downregulated in post-dauer animals, and 32% of previously identified
oocyte-enriched genes are upregulated in post-dauer animals [46, 47]. Post-dauer
animals have a longer mean life span and produce more self-progeny than controls,
particularly on later days (Day 3, Day 4) [47]. Since only self-reproductive span
assays were performed, increased brood size and late progeny production could be
due to increased spermatogenesis or to improved oocyte quality. Therefore, mated
reproductive span assays would need to be performed with post-dauer worms to
determine whether the passing through the dauer stage can actually improve oocyte
quality and delay reproductive ageing.
ARD (Adult Reproductive Diapause) In addition to the dauer stage, C. elegans
can also enter the state of adult reproductive diapause (ARD) when starvation is
induced in the final stage of larval development (L4) [48]. ARD can delay reproduc-
tive onset 15-fold and extend total adult lifespan at least threefold. In starvation-
induced ARD, the germline is dramatically reduced, and at most one oocyte is
retained per germline. Upon re-feeding, the shrunken germline regenerates and
multiple oocytes can re-form. Therefore, viable oocytes are produced even after
prolonged starvation. ARD dramatically increases the reproductive period and lifes-
pan of worms if eggs or larval stages are considered as time zero. However, mean
lifespan of animals rescued from ARD after different periods of starvation is similar
(if time zero is set as the exit from ARD), and the total cross progeny produced after
ARD recovery is also similar. Therefore, whether ARD is able to extend the abso-
lute reproductive span of gravid adults (i.e., the first egg laid till the last egg laid),
particularly with mating, is still unknown.
tion factor PHA-4. Neither the tissue where PHA-4 acts to control reproductive
span, nor the possible targets of PHA-4 that specifically affect reproductive span
separately from life span are known yet.
am117 does not act through the IIS pathway, but exhibits phenotypes similar to
Dietary Restriction mutants. The mutation is positioned on the right arm of chromo-
some I, but the exact location has not yet been mapped [73].
Drugs
Ethosuximide In a screen of drugs that are FDA-approved for human use, the anti-
convulsant medicine ethosuximide was identified to extend lifespan of C. elegans in
a dosage-dependent manner [74]. The same lab later found that treatment with etho-
suximide (2 mg/ml) increases adult life span by about 17 %, and has no effect on
self-fertilized reproductive span, but increases mated reproductive span by 12 %,
with a seven-fold increase in late-life reproduction [2]. However, ethosuximide’s
effects on oocyte quality and its interactions with known RS regulatory pathways
are unknown. Although ethosuximide has been shown to extend lifespan by inhibit-
ing chemosensory function in the nervous system [75], how ethosuximide regulates
reproductive ageing is still unknown.
Metformin Metformin is commonly used to treat Type II diabetes. 50mM metfor-
min treatment leads to a 40 % median lifespan increase, but maximum lifespan is
not extended [76]. Metformin at the same concentration also extends self-fertilized
reproductive span of wild-type hermaphrodites by about 1 day [76]. Although met-
formin has been shown to induce a DR-like state to promote somatic healthspan via
AMPK, LKB1, and SKN-1 [76], mated reproductive span assays must be done to
determine whether and how it also regulates reproductive ageing, including the
downstream genetic pathways, tissues where metformin acts, and downstream
mechanisms.
Additional screens for drugs that extend reproductive span and more importantly,
increase oocyte quality maintenance with age, will be aided by the development of
high-throughput reproduction and progeny production assays, including microflu-
idic approaches. Li, et al. developed one such device to monitor the progeny produc-
tion output and reproductive timing from individual mothers [77]. Scaling up such
microfluidic approaches will allow high-throughput screens with detailed reproduc-
tive information.
In summary, reproductive timing and maintenance in C. elegans hermaphrodites
is influenced by various environmental inputs (such as temperature and diet), the
worm’s life history, and multiple signalling pathways, such as IIS, DR, and TGF-β.
External signals and signal transduction within the animal require the coordination
of signalling across different types of tissues, including neurons, hypodermis, intes-
tine, muscle, and germline. It will be beneficial to identify more conditions that
affect reproductive capability, and additional mutational and small molecule screens
may identify novel regulators of reproductive ageing. How signals are transduced
and coordinated between various tissues and under different environmental condi-
tions to influence reproductive ageing are also worth deeper investigation.
7 Reproductive Ageing 149
7.5 C
omparisons of Reproductive Ageing in C. elegans
and Humans
Can we apply what we have learned from studying C. elegans reproductive ageing
to higher animals, in particular, humans? In this section, by comparing and contrast-
ing human and worm reproductive ageing, we will demonstrate that although many
differences exist, C. elegans and humans have similar reproductive schedules, suffer
from the same major cause of reproductive decline, share cellular and molecular
features in reproductive span regulation, display similarities in transcriptional pro-
files, and are regulated by evolutionarily conserved pathways.
Originally, it was thought that C. elegans could not be used as a model of mamma-
lian reproductive ageing, because it was believed that C. elegans produce oocytes
continually throughout its life from its pool of germline stem cells, whereas humans
are born with a finite number of oocytes. However, these long-time assumptions
have been challenged by studies in both C. elegans and humans. First, if C. elegans
could continually reproduce, one would predict that later and later mating would
result in a shift of the peak progeny production to the right; instead, Hughes et al.
[2] showed that fewer and fewer progeny were produced with later mating, support-
ing a “usage independent” limitation to reproduction [2], and arguing against the
continuous production of new, usable oocytes. This model was further supported by
the findings that oocyte quality declines with age, and is the limiting factor for
mated reproductive span [4]. In humans, oocyte-producing stem cells have been
found in adult ovaries in women of reproductive-age [78], although how often these
“oogonial stem cells” (OSC) are used for oocyte renewal is unclear [78]. In any
case, human reproductive ageing, marked by decreasing rates of fertility and
increases in miscarriage and birth defects, occurs much earlier than the exhaustion
of oocyte supply, indicating that oocyte quality rather than germline stem cell pro-
duction or oocyte quantity is the major determinant of reproductive success [79].
7.5.2 O
ocyte Quality Is the Limiting Factor for Reproductive
Span in Worms and Women
and the major maternal age-related birth defects are due to chromosomal abnormali-
ties, in particular aneuploidies [79]. C. elegans also exhibits increased chromosome
nondisjunction rates with age [4, 82, 83], resulting in unfertilizable oocytes, embry-
onic lethal eggs, and increased male progeny production [4]. C. elegans mutants
with extended reproductive spans, such as daf-2 and sma-2, have significantly
reduced rates of chromosome nondisjunction [4]. In summary, C. elegans shares
many major characteristics of age-related oocyte quality decline with humans.
Worm and human oogenesis share some common features [13]. For example, devel-
oping oocytes in early meiosis share their cytoplasm: young C. elegans oocytes
reside in a large syncytium, and similarly in the early stages of human follicle devel-
opment, oocyte nuclei are also not separated by membrane boundaries. Additionally,
in both species, oocytes arrest in prophase of meiosis I and wait for a signal to
mature. The mechanisms underlying oocyte maturation are highly conserved,
and programmed cell death is involved in the process in both species [84, 85].
Genes that maintain these processes, including cell cycle arrest genes such as cyclin
b (see above) are required for extended reproductive span, fertilization, and egg
hatching [4].
7.5.4 S
hared Mechanisms Required for Oocyte Quality
Maintenance
To identify genes required for high-quality oocytes, Luo et al. [4] isolated oocytes
from young (Day 1), aged (Day 8), and reproductive span mutants (TGF-β sma-2 on
Day 8) for transcriptional profiling. In contrast to the genes required downstream of
the IIS pathway for somatic longevity [58], the genes upregulated in the oocytes of
sma-2 mutants compared to wild-type worms, and in young compared to old wild-
type oocytes, are not primarily associated with maintenance of protein and cell
health; instead, genes required for the maintenance of the cell cycle, chromosome
segregation, chromosome organization, and DNA damage response and repair are
upregulated [4]. This suggests a focus on processes required for the continued func-
tion of mitotic cells. Additionally, genes associated with mitochondrial function (a
major factor in continued function of human oocytes with age [86]), transcriptional
regulation, reproductive processes, and ubiquitin pathway genes are up in high-
quality oocytes [4].
Remarkably, most of these processes are shared with those found to be down-
regulated in oocytes from older mice [87] and in oocytes from women of advanced
maternal age [88]. A large proportion of genes significantly upregulated in sma-2
7 Reproductive Ageing 151
oocytes (indicating better, more youthful oocytes) and enriched Gene Ontology
(GO) terms associated with them are shared with genes and GO terms downregu-
lated in ageing mouse and human oocytes [4, 87, 88]. Even more striking is that
some of the same genes in the processes of chromosome segregation, cell cycle
maintenance, and DNA damage response are similarly regulated in the oocytes of
worms, mice, and humans with age, suggesting that oocyte maintenance genes are
well conserved and required for oocyte quality regardless of animal or time scale
(days, months, or years).
Luo, et al. tested 60 of the top-scoring “oocyte quality” genes using RNAi knock-
down, and found that almost half of them were required for hatching. The loss of
some, such as the condensin SMC-4 and the cell cycle regulator cyclin b (CYB-3),
caused severe defects in both hatching and fertilization. Others, such as genes
involved in DNA repair (MLH-1), are required specifically for the hatching of late
progeny [4], indicating that age-related damage may appear in oocytes even after a
few days.
Insulin/IGF-1 signalling (IIS), TGF-β signalling, and Dietary Restriction (DR) all
play critical roles in reproductive span regulation in C. elegans. All three pathways
are evolutionarily conserved and their counterparts have also been shown to be
involved in human/mammalian fertility [89].
In mice, loss of Foxo3a (the human ortholog of DAF-16) causes age-dependent
infertility and abnormal ovarian follicular development [90], and Foxo3 overexpres-
sion increases the number of ovary follicles and increases fertility 30–50 % com-
pared to wild-type littermates. At the transcriptional level, the ovaries of aged Foxo3
transgenic mice also appeared more “youthful” [91]. Additionally, ARHGEF7, a
gene that interacts with FOXO3, was identified in a GWAS study as a candidate
gene associated with age at menopause in humans [92]. Therefore, like their roles in
C. elegans reproductive ageing, IIS and FOXO are also critical for fertility and
reproductive ageing in higher animals.
Likewise, many TGF-β pathway components have been implicated in mamma-
lian fertility regulation. BMP-15 and GDF-9 participate in the transition from pri-
mordial follicles into growing follicles [93]. SMAD1 and BMPR1 are upregulated
in aged mouse oocytes [87]. AMHR2 is identified among genes involved in the
initial follicle recruitment associated with age at menopause [94]. A reduction in the
expression of genes associated with TGF-β signalling is found in the cumulus cells
of women 35–36 compared to women under 30; these cells are essential for oocyte
quality [95].
DR extends both lifespan and reproductive span across species over large evolu-
tionary distances, although usually at the cost of progeny number. DR delays repro-
ductive ageing in C. elegans hermaphrodites [2, 8], female Drosophila [25], and
female rodents [22–24]. Beyond the fact that PHA-4 is required for the reproductive
152 C. Shi and C.T. Murphy
In humans and worms, reproductive ageing occurs well before the deterioration of
other somatic tissues. However, as described in previous sections, somatic and
reproductive ageing share conserved regulatory pathways, such as Insulin/IGF-1
signalling and the Dietary Restriction pathway. Therefore, it is reasonable to ask,
are reproductive and somatic ageing always coupled? How does one affect the
other? In this section, we will discuss the relationship between reproductive and
somatic ageing.
Germline removal significantly extends the lifespan of C. elegans ([64]; and Chap.
6). The lifespan-shortening signal of the germline originates from the mitotically-
proliferating germline stem cells [97], suppressing the dafachronic acid- (DA)/
DAF-12 and DAF-16 longevity-promoting activities in the soma [98]. Germline
loss also triggers downregulation of the TOR pathway, which in turn stimulates
autophagy [99]. Several components have been identified in each of the above-
mentioned pathways involved in germline-mediated longevity regulation [100]. By
contrast, compared to the extensive knowledge of germline-to-soma communica-
tion, the reverse process, soma-to-germline communication, is less well
understood.
Luo et al. [4] demonstrated that the TGF-β Sma/Mab and insulin/IGF-1 signal-
ling pathways mediate soma-to-germline communication. Ligands (Insulin-Like
Peptides, TGF-β DBL-1) are secreted from neurons and mediate signalling to the
7 Reproductive Ageing 153
reproduction does not appear to directly influence longevity of the individual, argu-
ing against the Disposable Soma hypothesis.
7.7 Conclusion
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Chapter 8
Nervous System Ageing
Abstract In the face of ever-changing cellular environments during life and age-
ing, the nervous system ensures the coordination of behaviour and physiology. Over
time, however, the nervous system declines structurally and functionally, leading to
age-related cognitive and behavioural decline in humans. Aspects of nervous system
ageing are being studied using C. elegans as a model system. Here we review the
age-related neuronal changes that occur at the structural, cellular and functional
levels in normally ageing animals, as well as how these changes relate to lifespan in
healthy ageing and in neurodegenerative conditions. Understanding the cellular
mechanisms that result in neuronal decline in C. elegans will help identify cellular
factors that protect the nervous system structure and function during normal ageing
and in disease states. Ultimately, elucidating the molecular networks and cellular
processes underlying the ageing of the nervous system will fuel research and design
of interventions to improve human life at old age.
8.1 Introduction
C. Bénard (*)
Department of Neurobiology, University of Massachusetts Medical School,
Worcester, MA, USA
Department of Biological Sciences, University of Québec at Montréal,
Canada
e-mail: claire.benard@umassmed.edu; benard.claire.2@uqam.ca
M. Doitsidou (*)
Centre for Integrative Physiology, University of Edinburgh, Edinburgh, UK
e-mail: maria.doitsidou@ed.ac.uk
Similar to the healthy ageing human brain, the nervous system of C. elegans shows
no neurodegeneration or gross deterioration during normal ageing [5–9].
Furthermore, the overall architecture of the nervous system is preserved throughout
life ([5, 6, 8, 9], Bénard C, unpub.). However, like in humans, more subtle morpho-
logical neuronal changes do occur in ageing C. elegans. Hermaphrodites have been
used in the studies reviewed here (except in the case of male mating behaviour in
Sect. 8.4). Neuronal soma and axon diameter shrink with age [5], and some neurons
exhibit specific morphological changes, such as new branches along neuronal pro-
cesses, axon swelling, axon waviness, defasciculation, new neurite-like extensions
from the soma, and soma distortion ([6–9] and Bénard C, unpub.). These changes in
neuronal morphology arise early during adulthood, progressively worsening in mid-
(days 4–7) and old-aged animals ([6–9] and Bénard C, unpub.). The type of
Fig. 8.1 Age-related changes in the C. elegans nervous system. (a) Schematic depiction of a survival
curve for wild-type C. elegans self-reproducing hermaphrodites at 20 °C. The peak of self-progeny reproduc-
tion is during days 2–5 of adulthood. Motility declines from around day 8 onwards. (b) Timeline of selected
manifestations in nervous system functional decline. Arrows indicate the age at which decline is first observed
early in adulthood. Synaptic transmission is measurably reduced as early as day 7 of adulthood (in motor
neurons). (c) Diagrams represent examples of age-related neuronal changes at the morphological, synaptic,
and subcellular levels. See text for details on specific types of neurons affected, age of onset and rate of change
Table 8.1 Effect of longevity mutants on age-related changes in the nervous system
Insulin signalling Dietary restriction Mitochondria
166
Genotype
Wild type daf-2 daf-16 hsf-1 eat-2 clk-1
Phenotype
Neuronal Appearance of neuron- In some cases decreased Increased appearance of Increased changes In some cases similar to Decreased
specific changes with appearance of morphological morphological changes [7] [6-8] WT [8] morphological
morphology age [6-8] changes with age [6-8] changes [8]
Suppresses daf-2 effects [8]
(Touch neurons)Abnormal
branching, shape, wavy
axons defasciculation
In other cases increased [7,9] In other cases, decreased [7,9] In other cases decreased
[9]
Synapses Vesicle and puncta Synaptic puncta maintained (d18,
Number of synaptic decline (d15, 18) [6, 11] d30) [11]
vesicles and puncta
Amplitude of post-synaptic PSC decline (starting d7) PSC maintenance (up to d27) [13]
currents PSCs [13]
Mitochondria Increases (up to d4) Lower load than WT, but steady Similar to WT [36] Lower than WT (d4-8),
(in ALM) Maintained (d4-8), (d1 to 25) [36] and steady (d4 to d11)
Mitochondrial load Decreases (after d8) [36] [36]
Mitochondrial transport Decreases after d1 [36] Steady (d1 to 25) [36] Similar to WT [36] Steady (d1 to 11) [36]
Resistance to oxidative Increases (up to d4) Higher resistance, lower rate of
stress Decreases after d4 [36] decline until d22 [36]
Axon regeneration Declines from d1 Delayed decline (no decline on d5, Suppresses increased Similar to WT [60]
GABA motor neurons Abolished by d5 [5] decline by d10) [60] regeneration of daf-2 [60]
Learning and Declines (d6) Enhanced learning in young Suppresses daf-2 delayed Increased learning in Enhanced learning
Absent (d11) [97] Delayed decline in old [97] decline [96, 97] young. Delayed decline in young. Delayed
Memory with age [97] decline in old [97,
Thermotaxis learning 97]
LTAM (positive olfactory) Declined (d2) Abolished Longer in young animals (40 vs Defective in LTAM [104] Impaired in young adults
by d5 [106] 24 hr in the WT) [11]
Not extended in aged animals Improved in older
[104] animals [104]
STAM (positive olfactory) Massed learning: decline 3x longer STAM in young adults Defective in STAM, suppresses Similar to WT in young
begins d3, abolished by Maintained in older worms (no loss daf-2 STAM extension [11, adults [104]
d6. Spaced learning lasts in d5) [104] 104] Improved in older
until d7 [104] animals (after spaced
learning) [104]
Neurodegeneration, Increased Reduced aggregation and Suppresses daf-2 protective Required for daf-2 Protects from
aggregation/proteotoxicit proteotoxicity [142, 146–150] effect [142, 146–150] and dietary proteotoxicity [152]
proteotoxicity
WT wild type, d day of adulthood, PSC post-synaptic currents, LTAM long-term associative memory, STAM short-term associative memory, IT
isothermal tracking, Aβ Amyloid beta. Green boxes indicate that neuronal phenotype is improved (=more youthful, delay of aging phenotype) rela-
C. Bénard and M. Doitsidou
tive to the wild type. Red boxes indicate that neuronal phenotype is deteriorated (=less youthful, stronger aging phenotype) relative to the wild type.
Yellow boxes in the lifespan mutants indicate no change relative to the wild type. Days indicate observation points reported in the cited papers
8 Nervous System Ageing 167
morphological change, age of onset, and frequency are highly neuron-type specific.
Furthermore, the incidence and severity of these morphological changes vary among
individual worms in isogenic populations that have been age-synchronized and co-
cultured, suggesting that stochastic factors may influence these age-related neuronal
changes.
Structural changes have been most extensively characterized in “gentle touch”
mechanosensory neurons (ALM, PLM, AVM, PVM), each of which displays spe-
cific types of morphological changes. For instance, ectopic outgrowths appear from
the soma of ALM by day 4 of adulthood, and new branches along the axon of PLM
are frequent by day 8. Ectopic neurites sprouting from neuronal processes extend
and retract dynamically [6, 7, 10]. Microtubule networks are disorganized in mech-
anosensory neurons with misshapen soma (ALM [6]), and mitochondria are often
located at the sites of ectopic neurites and swellings along the process [7]. The
functional implications of these changes are unknown.
Other neurons also display age-related morphological changes, including branch-
ing from the soma of the dopaminergic neuron PDE from early adulthood onwards
[7], defasciculation of cholinergic axons in the ventral nerve cord starting at day 6
of adulthood [6], axon beading of GABAergic neurons [6], and ectopic branches
from GABAergic axons by day 5 [8]. Characterization of ageing in additional neu-
ron types (e.g. other dopaminergic neurons, chemosensory neurons, interneurons,
and motor neurons) extends the observation that age-related morphological changes
are neuron-type specific and widespread across the nervous system, but not ubiqui-
tous ([9], Bénard C, unpub). It will be important to study a variety of neuronal types
in mechanistic detail to forge a deeper understanding of the neuronal responses to
age and elucidate the factors underlying the differential susceptibility of neurons to
ageing. Such analyses will provide insights into the basis of the selective neuronal
vulnerability in neurodegenerative conditions in humans.
and dendrites through specialized transport and anchoring [34]. Thus, processes that
disturb the cytoskeletal network or mitochondrial function and transport can poten-
tially affect healthy ageing and lead to neurodegenerative disease [35]. As men-
tioned above (Sect. 8.2), such cellular events are affected in ageing C. elegans as
microtubule networks become disorganized in neurons with age [6] and mitochon-
dria localize at the base of age-related ectopic branches along neuronal processes
[7].
The effect of ageing on C. elegans neuronal mitochondria in the cell body and
processes of the mechanosensory neuron ALM was examined by Morsci et al. The
frequency and distance of mitochondrial anterograde and retrograde transport pro-
gressively declines within the neuronal processes, starting already from the first day
of adulthood, indicative of cytoskeletal transport decline [36]. Indeed, microtubules
of mechanosensory neurons were shown to disorganize with age [6] and play a role
in structural maintenance of neurons in the adult [37]. The size, density and stress
resistance of mitochondria also change with age following a phasic pattern: first
they increase during early adulthood (days 1–4), then they are maintained at high
levels in mid-adulthood (days 4–8), and finally they decline in later adulthood (days
8–15) [36]. The mitochondrial filamentous network becomes more complex and
expansive in mid-adulthood whereas at later stages mitochondria exhibit ultrastruc-
tural abnormalities, e.g. loss of cristae structures [36]. Mitochondrial fragmentation
was also observed in mechanosensory neurons and the ADF neurons [38]. By day 9
of adulthood, 50 % of the ADF neurons exhibit fragmented mitochondria.
Mitochondrial changes are affected by lifespan mutations [36]. Mitochondrial
fragmentation is attenuated in long-lived daf-2/IGF1R mutants, whereas it pro-
gresses more rapidly in short lived hsf-1 mutants. daf-2/IGF1R mutants also have an
elevated baseline oxidative stress level and do not exhibit decay in mitochondrial
trafficking with age. Long-lived mutants daf-2, eat-2 and overexpression of sir-2.1
maintain a steady mitochondrial load during mid-adulthood, in contrast to the ele-
vated levels of same age wild-type animals [36]. Since compared to the wild type,
long-lived mutants in general maintain a higher level of nervous system function at
old age (see Sect. 8.5), it appears that the mitochondrial profile of healthy neuronal
ageing correlates with steady, rather than increased, mitochondrial content. How the
interplay of mitochondrial biogenesis, degradation or fusion/fission dynamics
brings about age-related mitochondrial changes and how these changes impact ner-
vous system function, is under investigation in C. elegans and other models [35, 39].
In humans too, normal brain ageing is characterized by subtle changes in the mor-
phology of specific neurons in selective brain regions [40, 41]. For instance, den-
dritic branching and length is enhanced in some hippocampal regions in aged
8 Nervous System Ageing 171
individuals compared to young adults, and changes in dendritic spine and synapse
number are observed in the ageing neocortex and hippocampus [40, 42, 43]. Despite
the simplicity of its nervous system and the short life of C. elegans, its neurons -as
described above- undergo age-related changes that parallel some neuronal changes
in humans. Given the extensive evolutionary conservation of cellular processes
between worms and humans, elucidating the mechanisms underlying the neuronal
responses to ageing in C. elegans is expected to uncover conserved principles of
neuronal ageing.
Damaged axons have the ability to repair, which helps the nervous system to remain
functional throughout life. In C. elegans, axons can be injured by laser axotomy and
their regeneration examined with single-cell resolution. Severed axons frequently
form a growth cone and regrow [44]. Multiple types of neurons, including mecha-
nosensory neurons (ALM, PLM, AVM) and GABAergic motor neurons can regen-
erate, and the regenerative capacity differs among neuron types [45–48]. Similar to
mammals, regrowth of injured axons in C. elegans is often misguided; nonetheless,
regenerated axons appear to rewire -at least partly- into proper circuits, as demon-
strated in worms that regain mobility after regeneration of their GABA motor neu-
rons [45, 49].
Several molecular pathways that promote or inhibit axon regeneration have been
discovered in C. elegans through genetic screening [50, 51]. Mechanisms of axon
regeneration [52–56] include the PTEN and DLK-1 MAP kinase pathway and other
MAP kinase pathways [51, 57–62], Notch signalling [54], microtubule regulators
[50, 63, 64], and the IIS pathway [60]. Genetic analysis of axon regeneration has
revealed that different neuron types share some regeneration genes, but have strik-
ing neuron-type-specific dependencies on other genes for axon regeneration.
Age is a strong determinant of a neuron’s potential to drive axon repair. Young neu-
rons regenerate damaged axons, but the regenerative ability of neurons quickly
declines in early adulthood, worsening further with age [44]. Studies on the effect
of age on axon regeneration have identified age-dependent mechanisms that regu-
late regenerative potential. In the mechanosensory neuron AVM, regeneration
declines already during larval development and reaches stable levels that are sus-
tained in adults. The pathway of miRNA let-7 and its target gene lin-41 regulates a
switch from high capacity for axon regrowth in early larvae when AVM develops, to
172 C. Bénard and M. Doitsidou
low capacity for axon regrowth shortly after the developmental outgrowth of AVM
is complete [65]. In contrast, the axon regrowth capacity of GABA motor neurons
is high throughout larval stages and up to day 1 of adulthood, but steeply declines
during adulthood (severely reduced by day 5 and abolished by day 10) [57, 60]. This
decline is a result of age-related deterioration in both axon initiation and axon elon-
gation after injury. The insulin receptor DAF-2/IGF1R regulates this decline in
GABA axon regeneration by inhibiting the daf-16/FOXO transcription factor and
its downstream regulation of dlk-1/DLK and other genes of the DLK MAP kinase
pathway [60]. Thus, C. elegans regulates the regenerative capacity of neurons in
response to age.
The capacity of axons to regenerate in ageing C. elegans does not directly cor-
relate with lifespan, as not all long-lived mutants maintain regenerative capacity at
old age. For instance, long-lived eat-2 mutants and animals overexpressing sir-2.1
have the same rates of regeneration as the wild type [60]. In contrast, loss of DAF-2/
IGF1R function enhances regeneration of aged axons but not of young axons [60].
Neuron-specific expression of DAF-16/FOXO, which does not rescue lifespan, res-
cues axon regeneration in aged animals. Conversely, intestine-specific expression of
DAF-16/FOXO, which rescues lifespan, does not rescue axon regeneration pheno-
types in aged daf-2 mutant animals. Thus, the role of the daf-2/daf-16 pathway on
axon regeneration is intrinsic to the nervous system and is uncoupled from its roles
in lifespan regulation. The C. elegans adult neuronal IIS/FOXO transcriptome
revealed the forkhead transcription factor FKH-9 as a IIS/FOXO target [66]. Loss of
fkh-9 impairs axon regeneration in aged daf-2 mutants, and pan-neuronal expression
of FKH-9 in daf-2;fkh-9 mutants restored the regeneration phenotype, confirming
its neuronal site of action [66].
During axon regeneration in C. elegans both age and neuron type determine a neu-
ron’s regenerative potential, partly because of specific dependencies on molecular
pathways mediating axon regeneration. Similarly, age and neuron type strongly
influence the regenerative capacity in humans. In adults, axons in the peripheral
nervous system regenerate, whereas axons in the central nervous system do not
[67]. Intrinsic determinants of regeneration differ across the nervous system as well;
for instance, removing PTEN greatly enhances optic and peripheral nerve regenera-
tion, but has a modest effect on spinal cord axons [68–70]. These findings highlight
the importance of studying diverse neuronal types in order to gain an understanding
of regeneration, a goal that is achievable in the short term in C. elegans and that will
inform research in mammals. Molecules identified in C. elegans to function in axon
regeneration (e.g. PTEN and DLK), are conserved in mammals. Elucidating the
mechanisms that regulate adult axon regeneration and the effect of age on neuronal
regeneration will increase our understanding of how a neuron ages and inform
approaches to treat injury and disease in humans.
8 Nervous System Ageing 173
Learning and memory are fundamental biological processes that allow living organ-
isms to respond and adapt to their environment. Memory decline in ageing is a well-
documented phenomenon in humans [91] and a common feature across species [92].
8 Nervous System Ageing 175
Despite the simplicity of its nervous system, C. elegans exhibits behavioural plastic-
ity and a range of well-characterized paradigms of short- and long-term memory
[93]. These include examples of associative and non-associative memory, some of
which have been studied in detail. Genetic pathways known to affect lifespan also
affect learning and memory in different ways. In some cases, they play a role in the
formation of memory itself; in other cases, they influence how fast memory declines
during ageing. Here, we focus on some of the well-characterized models of learning
and memory in C. elegans to review the consequences of ageing on neuronal plas-
ticity and the influence of lifespan-altering mutations on age-related decline.
affects lifespan. Despite the complex roles of mitochondrial metabolism and ROS
production in ageing [99], specific mutants of the electron transport chain are known
to alter lifespan. Both isp-1 (coding for the iron sulphur protein of mitochondrial
respiratory complex III [100]) and clk-1 (coding for a central enzyme in ubiquinone
synthesis) mutants show increased thermotaxis learning behaviour in young adult
animals assessed by isothermal tracking [96]. The increased learning in isp-1
mutants is daf-16/FOXO-dependent and it is abolished in daf-16 mutants, despite
the fact that the longevity effect of these mutants does not depend on daf-16/
FOXO. Furthermore, clk-1 mutants also delay age-related decline of isothermal
tracking behaviour. In contrast, short lived gas-1 and mev-1 mutants, defective for
respiratory complex I and II, are more sensitive to oxidative stress [101, 102] and
show decreased thermotaxis behaviour, a phenotype rescued by treatment with anti-
oxidants [96].
C. elegans learns to associate volatile chemicals like butanone with food, and che-
motaxes towards them [103]. When butanone is paired with food for a single train-
ing session (massed learning) it produces a short-term associative memory (STAM).
In contrast, when worms are subjected to multiple training sessions (spaced learn-
ing) they form a long-term associative memory (LTAM) that lasts between 16 and
24 h [104]. LTAM formation declines with age, starting already at day 2 of adult-
hood and is abolished by day 5. LTAM deteriorates prior to the decline in olfactory
learning, chemotaxis and motility [104], suggesting a higher sensitivity of LTAM in
ageing. Massed learning begins to decline already by day 3 and is completely lost
by day 6 of adulthood. Spaced learning, which begins to decline on day 3, is lost by
day 7.
Lifespan mutants affect LTAM and STAM in C. elegans both in young and older
age. In young adults, daf-2/IGF1R mutants show three times longer STAM, although
their learning rate is similar to wild type. Moreover, daf-2 mutants show signifi-
cantly longer LTAM, which remains active past 40 h. Lastly, LTAM is established
after fewer training sessions in daf-2/IGF1R mutants compared to wild-type ani-
mals. These memory improvements in young animals are daf-16 dependent [104].
In older worms, daf-2/IGFR mutants retain their ability to learn for a longer time.
At day 5 of adulthood, there is no significant loss in the formation of STAM. However,
although learning is extended, LTAM is not improved in aged daf-2/IGFR mutants
compared to the wild-type animals [104].
Dietary restriction affects positive olfactory associative memory differently than
the IIS pathway. In young animals, eat-2 mutants show no improvements in STAM
compared to the wild type, whereas LTAM is reduced. In contrast, older eat-2 ani-
mals show improved memory compared to wild type, and both STAM and LTAM
persist for a longer period. Importantly, age-dependent memory loss can be allevi-
ated if dietary restriction is imposed in adult worms [104].
8 Nervous System Ageing 177
C. elegans also exhibits non-associative memory [93]. The best characterized exam-
ples are habituation to a mechanical stimulus and chemosensory habituation. These
forms of adaptation can have short- or long-term memory timescales depending on
the training regime. Age-related changes in habituation have been reported: worms
in their sixth and eighth day of adulthood habituate more rapidly to mechanical
stimulus and show slower recovery from habituation than younger adults [105].
Timbers et al. tested adaptation to mechanical stimulus in middle-aged worms and
showed that changes in habituation started at the peak of their reproductive age, as
early as the second day of adulthood [106]. This timeline is similar to the onset of
changes in positive olfactory associative learning described above. In contrast to
associative learning, the IIS pathway does not impact non-associative learning pro-
tocols involving chemosensory habituation [107].
Several forms of memory in C. elegans, for example olfactory STAM and LTAM,
decline before any morphological neuronal changes become apparent [104, 108].
As a cautionary note, an analysis of age-related morphological changes of neurons
that mediate learning and memory is still lacking. Thus, it seems that changes at the
molecular level, which precede obvious morphological defects, are responsible for
memory decline [104, 108]. Indeed, LTAM deterioration with age in both the wild
type and longevity mutants correlates tightly with crh-1/CREB expression levels
[104]. This correlation appears to be conserved in mammals: the levels of CREB in
the brain are predictive of spatial memory decline in aged rats [109] and overexpres-
sion of CREB in the hippocampus attenuates spatial memory impairment during
ageing [110].
Parallels can be drawn between memory and synapse decline during ageing in
the wild type and lifespan mutants. The complex synaptic machinery required for
memory formation is well documented [111]. C. elegans research has revealed a
correlation between synapse deterioration and STAM decline with age [11]. At the
molecular level, the anterograde kinesin motor UNC-104/KIF1A, which transports
synaptic vesicles along axons, is required for STAM maintenance in ageing. UNC-
104/KIF1A levels are reduced with age in the wild type but maintained in daf-2
mutants in a daf-16/FOXO-dependent manner [11].
The examples above demonstrate that a reduction in the IIS pathway promotes
positive associative learning and memory in ageing C. elegans, through activation
of the DAF-16/FOXO transcription factor. Neuron-specific transcriptome analysis
of DAF-16/FOXO targets in daf-2/IGF1R mutants revealed a landscape of DAF-16/
FOXO-dependent regulators of short-term memory extension, distinct from previ-
178 C. Bénard and M. Doitsidou
ously identified targets in other tissues. This analysis showed that some of the DAF-
16/FOXO neuronal targets that extend memory in daf-2/IGF1R mutants also
regulate memory in the wild type [66]. Thus, IIS pathway-dependent memory
extension is due to augmentation or maintenance of the molecular machinery that
regulates memory in the wild type, rather than the activation of an alternative mech-
anism. Similarly in mouse, FOXO6 is highly expressed in adult hippocampus and is
required for memory consolidation by regulating the expression of genes responsi-
ble for synaptic function [112].
Among the DAF-16/FOXO targets that are upregulated in daf-2/IGF1R mutants
at the whole worm level is FKH-9. It was shown that FKH-9 is required in the neu-
rons for memory enhancement in daf-2 mutants and in the somatic cells for lifespan
extension [112]. Molecular characterization of the tissue-specific transcriptional
programmes that regulate longevity or neuronal function, combined with an analy-
sis of the conservation of these programmes across phylogeny, will facilitate a more
complete understanding of nervous system ageing.
Age is the leading risk factor for neurodegenerative disease [41, 122, 123]. This
suggests that cellular changes occurring during ageing increase the vulnerability of
neurons to such conditions. Ageing cells show increased levels of oxidative, meta-
bolic and ionic stress that result in the accumulation of dysfunctional organelles,
damaged proteins and DNA. Failure of neurons to adapt to such stresses leads to
neuronal dysfunction and susceptibility to neuronal degeneration. Understanding
the molecular mechanisms underlying age-related neurodegenerative diseases and
identifying neuroprotective strategies is a major focus of modern medical research.
[135]. Finally, a number of C. elegans models exist for studying neuronal channelo-
pathies and excitotoxic cell death [136–138].
Modifier screens on the above models have contributed important insights into
the understanding of neurodegenerative disease and revealed numerous general and
disease-specific modifiers conserved in other organisms. Studies on C. elegans
models of PD led to the discovery of several neuroprotective mechanisms; for
instance, overexpression of the chaperone TOR2/TorsinA, the lysosomal P-ATPase
catp-6/ATP13A2, or human Cathepsin D were shown to ameliorate aspects of
α-synuclein toxicity [139–141] and the glycolytic enzyme GPI-1/GPI was identified
as a conserved modifier of dopaminergic degeneration [142]. Genetic screens in C.
elegans models of AD led to the discovery of many disease modifiers, e.g. orthologs
of human kinases such as kin-18/TAOK1 and sgg-1/GSK3β, and chaperone stress
response molecules (such as xbp-1/XBP1, hsp-2/HSPA2, hsf-1/HSF1 and chn-
1/CHN1) were identified as key regulators of tau toxicity [143]. A long list of simi-
lar discoveries has confirmed the validity of C. elegans as a model to study
neurodegenerative disease. Key discoveries stem not only from research on models
of disease but also from studies that enhanced our understanding of the normal
function of disease-associated genes in healthy situations. A comprehensive review
of these findings is beyond the scope of this chapter.
Acknowledgements We thank Arantza Barrios, Emanuel K. Busch and Cassandra Blanchette for
feedback on the manuscript. Research in the lab of Dr. Maria Doitsidou is supported by the
Norwegian Research Council and the Wellcome Trust, UK. Research in the lab of Dr. Claire
Bénard is supported by grant R01 AG041870-01 from the National Institutes of Health of the USA
to C.B., the Ellison Medical Foundation New Scholar Aging Award to C.B., and the American
Federation for Aging Research Award to C.B..
Dedicated to the memory of Muhammad Ali (January 17, 1942–June 3, 2016).
182 C. Bénard and M. Doitsidou
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8 Nervous System Ageing 189
9.1 Introduction
increases, the system yields and deformation becomes inelastic. Finally, application
of even greater stress force results in failure, or rupture. This would be a lethal
physiological stress.
Stress, defined as force(s) that perturb physiological systems, is a common
occurrence. Forces that cause stress can derive from fluctuations in environmental
conditions such as changes in temperature, food availability, pathogens, toxins, or
oxygen availability. Stress can also be induced by changes in internal physiological
conditions, which can result as a consequence of cellular dysfunction due to genetic
mutations, illness, or injury. Even ageing may cause stress, as cellular processes
become dysfunctional or less efficient. A general consequence of stress is to perturb
homeostasis, defined by Cannon as the ability of organisms to maintain an internal
equilibrium when external conditions are altered [2]. In Cannon’s paradigm, the
limits of homeostatic mechanisms are revealed at the point at which physiological
stress results in failure (rupture), often manifest as cellular death or damage [3].
Stress response pathways can therefore be defined as cellular and organismal
mechanisms to resist the effects of stress and/or restore homeostasis when condi-
tions change. Biological stressors come in many flavours, such as thermal stress,
oxidative stress, xenobiotic stress, proteotoxic stress, and osmotic stress. Each of
these perturb the physiological system differently, and as such a variety of stress
responses have evolved to defend homeostasis in these different conditions. In
effect, stress response pathways highlight the weaknesses in physiological networks
revealed by the application of stress. Understanding how stress-response pathways
buttress cellular physiology to maintain homeostasis and ensure survival provides
powerful insight into fundamental pro-survival mechanisms.
9.3.1 Avoidance
9.3.2 Anticipation
In some situations, animals can use prior experience or environmental cues to pre-
dict upcoming stress, enabling a more rapid and robust response to the imminent
changes. A well-studied example of this strategy is preconditioning. In precondi-
tioning a relatively mild stress improves survival upon exposure to a more extreme
stress. Hypoxic preconditioning is a well-known example conserved in mammals,
where a mild, non-lethal exposure to reduced oxygen availability improves survival
upon subsequent exposure to more severe hypoxia. Similarly, C. elegans exposed to
atmospheres without oxygen (anoxia), for a short time can survive subsequent
exposure to long periods of anoxia better than unpreconditioned controls [57]. The
effects of hypoxic preconditioning are relatively short-lived, and the mechanistic
basis that confers this protection is not clear. However, mutations in the apoptosis
factor, ced-4, but not other cell death genes, prevent hypoxic preconditioning in
these experiments [57]. Hyperosmotic stress can also stimulate a preconditioning
response, where animals produce glycerol from glycogen and are resistant to subse-
quent osmotic stress [58]. Adults exposed to hyperosmotic stress also package glyc-
erol into embryos, which are then resistant to osmotic stress [53]. This maternal
protective effect requires insulin signalling, as daf-2(e1370) mutant animals do not
increase glycerol/trehalose provisioning to embryos, which are thereby sensitive to
osmotic stress [53]. Preconditioning also improves resistance to heat stress in C.
elegans, as a short heat-shock increases survival upon subsequent exposure to high
temperature [59]. In heat shock, the preconditioning advantage is correlated with
the expression of heat-shock proteins and requires the heat-shock transcription fac-
tor hsf-1. This is an example of hormesis – where induction of a stress response
persists even after the stress is removed. The ability for sub-lethal stresses to increase
resistance to subsequent stress conditions is highly conserved [60–63].
Epigenetic effects can also enable animals to anticipate stressors to survive, by
facilitating environmentally-induced gene expression, and may contribute to effects
on lifespan. DNA methylation is not a major source of epigenetic effects, as C.
elegans do not possess cytosine methyltransferase enzymes; however, adenosine
methylation has been recently discovered in C. elegans, and it does interact with
fertility defects of the histone 3 at lysine 4 (H3K4) demethylase spr-5 [64]. Instead,
epigenetic effects are largely mediated by effects on histone proteins. Loss of func-
tion of the ASH-2/trithorax complex, which acts to methylate H3K4, and the H3K4
demethylase RBR-2 increases lifespan in C. elegans in a germline dependent man-
ner [65], whereas RNAi of utx-1, a H3K27me3 demethylase, increases lifespan in a
germline independent manner [66]. It is not clear how stress response pathways are
influenced by these epigenetic factors. However, the effects of loss-of-function of
the ASH-2 complex on lifespan persist transgenerationally [67], suggesting that
information about the environment can be stored and transmitted. The DAF-16 tran-
scription factor has been shown to physically associate with the SWI/SNF chroma-
tin remodelling complex, and this interaction facilitates efficient transcription of
daf-16 target genes in daf-2 mutant animals [68]. We have recently discovered that
9 Stress Response Pathways 197
When stress cannot be avoided or anticipated, stress response pathways are invoked
to correct the physiological disturbance and return the system to homeostasis. In
each instance, a stress must be sensed and then a physiological response mounted to
counteract or correct the damage and restore homeostasis. In the next section we
consider a few examples of well-characterized stress responses to highlight these
aspects of stress response mechanisms.
The temperature at which C. elegans are raised, similar to many poikilotherm organ-
isms, has a large effect on the ultimate lifespan of the animal. C. elegans can be
cultured in the lab across a broad range of temperatures from approximately 10–25
°C with a lifespan that is inversely correlated with temperature [8]. At lower tem-
peratures metabolic rate is decreased, leading to the hypothesis that the increased
lifespan is simply a “slowing” of normal life processes [75, 76]. However, the lifes-
pan effects of temperature are not just governed by thermodynamics, as there are
genetic components which influence the temperature dependence of C. elegans
198 D.L. Miller et al.
lifespan. For example, the TRP channel, trpa-1, is necessary for extended lifespan
at low temperatures as well as the decreased lifespan when larval animals are grown
at low temperatures [77, 78]. Temperature can also modulate the effects of some
lifespan-extending mutations. For example, mutations in the hypoxia-responsive
transcription factor hif-1 have different effects on lifespan at different temperatures
[24].
C. elegans has neuronal mechanisms to sense temperature and coordinate behav-
ioural responses to changes in temperature. C. elegans thermotax towards the tem-
perature in which they have been cultivated with food and away from high, noxious
temperatures [79, 80]. The bilateral AFD amphid neurons are the main thermosen-
sory neurons in C. elegans. AFD neurons are activated by both increases and
decreases in temperature as small as 0.05 °C [81, 82]. Although AFD is involved in
both thermotaxis and the avoidance of noxious temperature (thermonociception),
different neural circuits mediate these two behaviours. In thermonociception, AFD
neurons activate AIB interneurons, which connect to AFD by electrical junctions,
resulting in initiation of backward movement [80]. In thermotaxis, AFD activates a
neural circuit including AIY and AIZ interneurons to direct movement towards the
preferred temperature [83].
Heat stress impinges on many aspects of cellular physiology. One consequence
of thermal stress that contributes to activation of the heat-shock response is the
accumulation of unfolded or misfolded proteins [84]. Thermal stress can also induce
formation of reactive oxygen species, leading to oxidative damage of cellular com-
ponents [85–87]. For example, protein carbonylation, an oxidative modification, is
enhanced by thermal stress, perhaps because unfolded proteins are more accessible
to modification by reactive oxygen species [88]. Upon exposure to thermal stress,
cells activate the heat-shock response, which leads to increased expression of heat
shock proteins (HSPs) to defend against damage induced by thermal stress. Many
HSPs are molecular chaperones, which help to maintain proper protein folding, or
promote degradation of damaged proteins (reviewed in [89, 90]).
At the cellular level, the highly conserved HSF-1 transcription factor mediates
transcriptional responses to thermal stress to activate the heat-shock response [72].
In non-stressed conditions, HSF-1 binds to HSP-90 and sequesters it in the cyto-
plasm. Upon heat shock, cellular proteins become unstable and partially unfolded,
and these misfolded or unfolded proteins become clients for the protein chaperone
HSP-90 (DAF-21 in C. elegans), and compete for HSP-90 binding [91, 92]. As a
result, HSF-1 is released, transits to the nucleus, and trimerizes. These active HSF-1
trimers bind to heat shock elements in the genome, inducing expression of HSPs. In
addition, HSP-70 and HSP-40, two HSF-1 targets, act in a negative feedback loop
of HSF-1 activity, binding to HSF-1 and decreasing its activity [93, 94].
Several lines of evidence suggest that HSF-1 modulates organismal ageing.
Overexpression of HSF-1 increases lifespan and stress resistance [19], whereas
knockdown of hsf-1 leads to progeroid phenotypes [95]. Moreover, hsf-1 is required
for increased lifespan by reduced insulin/IGF-like signalling and at least one model
of dietary restriction [16, 18, 19]. Finally, sublethal heat stress itself can increase
9 Stress Response Pathways 199
lifespan [9, 96, 97]. The effect of hsf-1 activation on lifespan is likely a result of
increased HSP expression. Overexpression of HSP-16.2 or the chaperone HSP-70 is
sufficient to extend lifespan in C. elegans [98, 99]. Moreover, variations in expres-
sion of the small HSP hsp-16.2 predict lifespan in an isogenic, wild-type popula-
tion, with animals that express higher levels living longer [100].
All cells must respond to heat stress, and hsf-1 is expressed in most, if not all,
cells. However, there are also central regulators of the organismal response to ther-
mal stress suggesting that this cellular stress response is not autonomous. Expression
of HSPs in response to thermal stress in somatic cells can be blunted in C. elegans
by ablation of thermosensory AFD neurons [101]. Similarly, ablation of AFD fur-
ther reduces lifespan at higher temperature [102], suggesting that the thermosensory
neurons play a role in integrating the heat-shock response and lifespan. Expression
of HSF-1 in neurons increases both lifespan and thermotolerance, but the effects of
lifespan require DAF-16 in the periphery whereas increased resistance to thermal
stress depends only upon activation of HSF-1 [103]. These experiments indicate
that the thermosensory neurons regulate multiple downstream activities to integrate
organism-wide responses to different environmental stresses.
Organisms must maintain appropriate redox balance in cells, as this is essential for
the oxidation-reduction reactions necessary to maintain life. This involves keeping
the cytoplasm reducing, while performing oxidative protein folding in the endoplas-
mic reticulum. C. elegans must accomplish this in a very oxidizing environment, as
every cell is exposed to the gaseous environment [104]. The main source of oxida-
tive stress for C. elegans in nature is most likely fluctuations in environmental O2.
In the natural environment of C. elegans, rotting fruit and compost [105], O2 levels
can fluctuate from normoxia, which we define as 21 % O2 for the purposes of this
discussion, to near anoxia (operationally defined as less than 10 ppm O2).
C. elegans avoid both hypoxia (low oxygen) and hyperoxia (high oxygen) [28, 29].
In an O2 gradient, C. elegans migrate to 5–12 % O2, depending on the steepness of
the gradient [29]. At this concentration of O2, normal aerobic metabolism is main-
tained [106]. The aerotaxis behaviour to avoid higher O2 requires the soluble guany-
lyl cyclase, GCY-35, in the URX, AQR, and PQR sensory neurons and the
cGMP-gated TAX-2/TAX-4 channel [29]. URX is activated by increases in O2 con-
centration, and is required for slowing and reversal responses to increasing O2 con-
centration [107]. URX is not required for behavioural responses to O2 downshifts,
from 21 % to 10 %. Instead, the BAG sensory neurons are activated by O2
200 D.L. Miller et al.
downshifts and are required for increased locomotion and reversals [107]. Different
soluble guanylyl cyclases are required for evoked calcium currents upon upshift or
downshift of O2 [107]. Interestingly, the neural circuits that coordinate aerotaxis are
modified by prior experience and nutritional status [108–110], which suggests an
integration between these distinct stress response modalities. The neural circuits
that regulate hypoxia avoidance have not been delineated, but are distinct from the
O2-sensing neurons that mediate avoidance of high O2.
C. elegans are incredibly tolerant to a broad range of O2, from anoxia to 100 %
O2 [106], suggesting efficient mechanisms to respond and defend against oxidative
damage. Oxidative stress generally causes cellular damage as a result of increased
production of reactive oxygen or nitrogen species (ROS or RNS). ROS and RNS are
able to oxidize key cellular components such as DNA, lipids, and proteins [111–
113]. These damaged cellular components must then be degraded or repaired to
maintain cellular function. During aerobic metabolism, up to 1–4 % of O2 con-
sumed by mitochondria is released as the ROS, superoxide (O2–) due to activity of
complex III of the electron transport chain [114]. Although these endogenously
produced ROS may be damaging, they are also an important cellular signalling
molecule [115]. It is only when production of these ROS/RNS is excessive that they
cause oxidative damage.
In addition to endogenously produced ROS, environmental toxins can also
increase ROS and RNS. Exposure to heavy metals, such as lead, cadmium, chro-
mium, and arsenite, is associated with cellular oxidative damage [116, 117], and
UV and heat stress can also increase formation of ROS/RNS [85–87, 118]. Defects
in iron homeostasis can also lead to formation of ROS, particularly the highly reac-
tive hydroxyl radical, as a result of Fenton chemistry [118]. There are also several
environmental toxins or poisons that increase ROS/RNS load, including the herbi-
cides juglone and paraquat that are commonly used as experimental tools [119].
The primary cellular defence mechanisms against ROS and RNS involve the upreg-
ulation of detoxification enzymes. For detoxification of ROS, superoxide dismutase
(SOD) converts O2– into H2O2, which is decomposed to water and O2 by catalase.
H2O2 and other peroxides can also be reduced by peroxidase enzymes. Other redox-
active compounds encountered, such as xenobiotic compounds or environmental
pollutants, are often detoxified by reduction by or conjugation to glutathione (GSH)
or UDP-glucuronic acid [119, 120]. Glutathionylation is catalysed by glutathione-
S-transferase enzymes (GSTs), and conjugation to UDP-glucuronic acid, or gluc-
uronidation, is catalysed by UDP-glucuronosyltransferases (UGTs). The C. elegans
genome includes five SOD genes, three catalase genes, 44 genes annotated as GSTs,
two GSTK (kappa class) genes, three GSTO (omega class) genes, and 65 genes
annotated as UGTs (www.wormbase.org, release WS252).
9 Stress Response Pathways 201
cascade [138], but it is not clear if oxidative stress activates the MAPK pathway
through a direct or indirect mechanism. An RNAi screen of kinases found four other
kinases required for the nuclear accumulation of SKN-1 in response to oxidative
stress from exposure to sodium azide: nekl-2, ikke-1, mkk-4, and pdhk-2 [139].
Depletion of any of these kinases renders animals more sensitive to oxidative stress
than wild-type but not as sensitive as skn-1(RNAi), suggesting redundant activation
by these kinases [139]. SKN-1 is also negatively regulated by GSK-3, the glycogen
synthase kinase orthologue. GSK-3 phosphorylates SKN-1 at a conserved serine
residue and inhibits its nuclear localization and activity in response to oxidative
stress [140]. This interaction also occurs in embryogenesis, where GSK-3 is required
to inhibit the activity of SKN-1 in the C blastomere [141]. Phosphorylation by
GSK-3 requires a priming phosphorylation by the p38 MAPK pathway [140]. This
interaction between activating and inhibiting modifications could set a threshold to
ensure that SKN-1 is not inappropriately activated.
Although SKN-1 is activated by high O2, which is associated with increased
oxidative damage [127, 142], the HIF-1 transcription factor is more important for
adaptation to low O2, or hypoxia. HIF-1 is the C. elegans orthologue of the hypoxia-
inducible factor, a conserved bHLH-PAS domain transcription factor that mediates
the transcriptional response to hypoxia in metazoans [143–147]. HIF-1 protein is
degraded in the presence of O2 as a result of modification by the EGL-9 prolyl
hydroxylase and interaction with the VHL-1 E3 ubiquitin ligase [148]. HIF-1 activ-
ity is also increased by ROS/RNS and defects in mitochondrial function, though the
mechanistic basis of this interaction is not well understood [27, 149].
Oxidative stress has been predicted to contribute to ageing phenotypes since Harman
proposed the Free Radical/Oxidative Damage Theory of Ageing, which suggests
that accumulated oxidative damage resulting from ROS/RNS contributes to cellular
dysfunction that drives ageing (reviewed in [150, 151]). Consistent with this idea,
protein carbonylation, an oxidative modification, increases with age and is reduced
in long-lived age-1 mutant animals [142]. However, many more observations argue
against a role for ROS/RNS or oxidative damage as a driver of ageing. Though
many long-lived mutants have increased expression of SODs, the expression of
SODs is not generally required for increased lifespan [152]. Moreover, increased
lifespan in animals overexpressing SOD-1 is not correlated with reduced oxidative
damage of proteins or lipids [153, 154]. Even simultaneous disruption of all five
SOD genes does not reduce lifespan, though these animals are dramatically more
sensitive to oxidative stress than wild-type controls [155]. Together, these results
indicate that superoxide is not a major contributor to lifespan. In fact, low doses of
paraquat or arsenite actually increase lifespan of C. elegans [27, 156]. Similar stud-
ies suggest that peroxides do not significantly drive the ageing process.
Overexpression of catalase does not increase lifespan even when SOD-1 is also
overexpressed [153], and although deletion of ctl-2 shortens lifespan, protein
9 Stress Response Pathways 203
VHL-1 [169]. Another possibility is that in egl-9 mutant animals the level of HIF-1
stabilization is so high as to be detrimental, or that there are isoform-specific effects.
In addition to its essential role in embryonic development, skn-1 is required for
normal lifespan. Both RNAi and loss-of-function mutations in skn-1 decrease
lifespan and render animals sensitive to oxidative stressors [125, 127, 138, 139,
170]. Overexpression of SKN-1 and gain-of-function mutations that disrupt the
interaction with negative regulator wdr-23, or RNAi knockdown of wdr-23 mod-
estly increase lifespan [132, 171, 172]. However, other gain-of-function mutations
in skn-1 do not increase lifespan [173]. This could be a result of negative effects of
too much SKN-1 activity, or effects of different isoforms. There are three distinct
isoforms of SKN-1 that act in different tissues and are subject to distinct regulation.
The B isoform of SKN-1 is constitutively expressed in the ASI sensory neurons,
where it is required for increased mitochondrial respiration and lifespan in response
to bacterial-dilution dietary restriction [174]. In the intestine, the A/C isoforms are
stabilized in response to oxidative stress [125]. SKN-1 expression has also been
detected in neurons, the pharynx, and other tissues, but it is not clear which isoforms
are expressed in these tissues [156, 175, 176]. Activation of SKN-1 is also required
for extended lifespan of daf-2 mutants and by reduced TOR signalling [170, 171,
177]. In all of these situations, skn-1 acts to increase lifespan. In contrast, overex-
pression of skn-1 decreases lifespan in hypoxia [168]. Further work to unravel the
diverse roles of skn-1 in different tissues and different conditions is a ripe area of
research to reveal novel aspects of how longevity is coordinated. Further discussion
of the role of oxidative stress in ageing can be found in Chap. 10.
small HSPs of the HSP-16 family, which bind to unfolded client proteins to prevent
aggregation and facilitate refolding. Expression of hsp-16 is induced by a variety of
environmental stressors, presumably due protein folding stress, including exposure
to thermal stress, oxidative stress (exposure to hypoxia, juglone, or heavy metals),
alcohol exposure, nicotine, pathogenic bacteria, dimethylsulfoxide, and expression
of aggregation-prone proteins including human Aβ1-42 or poly-glutamine protein
[100, 179–187]. Expression of these and other HSPs help to counteract the effects
of the stress and maintain cytoplasmic proteostasis.
For membrane and secreted proteins, folding initiates in the ER. The ER UPR,
which is distinct from the cytoplasmic UPR, is activated by protein folding stress
from disruptions of the folding environment of the ER or protein maturation and
modifications in the Golgi. Defects in ER-associated degradation (ERAD), a quality
control mechanism to remove and degrade proteins from the ER, can also lead to
accumulation of misfolded proteins in the ER and induce the UPR [188]. In general,
those cells that are highly secretory are most sensitive to ER folding stress [189].
Hypoxia induces the ER stress response [190], likely due to a defect in oxidative
protein folding. Disulphide bond formation in the ER by ERO-1 requires molecular
O2, so in hypoxia these proteins can no longer fold correctly [191]. Reducing agents
such as dithiothreitol (DTT) similarly cause ER folding stress and induce the
UPR. Common chemicals produced by bacteria also induce the ER UPR, including
tunicamycin, which inhibits N-linked protein glycosylation by GlcNAc phos-
photransferase in the ER, thapsigargin, which inhibits SERCA and depletes the ER
of calcium, and brefeldin A, which blocks transport of proteins from the ER to
Golgi.
The proximal sensors of misfolded proteins in the ER are the transmembrane
proteins IRE-1, PEK-1, the C. elegans PERK orthologue, and ATF-6. ATF-6 is most
important for coordinating the constitutive UPR that is activated during normal
development, whereas IRE-1 and PEK-1 are important for the inducible UPR [192].
These pathways are somewhat redundant, as animals with mutations in any one of
these genes are viable, though sensitive to ER folding stress. However, double
mutant animals die during development, often with severe gut atrophy, suggesting
an inability to respond to normal ER stress during development [193, 194]. In
unstressed conditions the IRE-1 and PEK-1 kinases bind as monomers to HSP-4/
HSP-3, ER resident chaperones homologous to BiP, and are inactive. When unfolded
proteins accumulate they are bound by HSP-4, freeing IRE-1 and PEK-1 to
homooligomerize and leading to phosphorylation of cytoplasmic targets (reviewed
in [195]). In addition to kinase activity, IRE-1 is an endoribonuclease which, when
activated, removes a small intron from the xbp-1 transcript [196]. The spliced xbp-1
transcript is then efficiently translated, and increased production of the XBP-1 bZIP
transcription factor induces expression of ER resident chaperones including hsp-3
and hsp-4 [192]. PEK-1 phosphorylates the translation initiation factor eIF2α,
which inhibits translation initiation [194]. Together, activation of IRE-1 and PEK-1
reduces both protein folding load, by reducing the synthesis of new proteins, and
improving protein folding capacity by increasing chaperone function.
206 D.L. Miller et al.
Most mitochondrial proteins are encoded by the nuclear genome and translated
in the cytoplasm. These proteins must then be imported into the mitochondria,
folded, and then assembled into functional complexes. Any perturbation in mito-
chondrial protein import or complex assembly can cause protein folding stress in
the mitochondria [197–199]. Two chaperones, hsp-6 and hsp-60, are expressed in
the mitochondria and induced upon protein folding stress [198]. These chaperones
assist in import, folding, and assembly of mitochondrial protein complexes. There
are at least two somewhat overlapping modes for activating the mitochondrial UPR
(UPRMT), one mediated by the ATFS-1 transcription factor and the other mediated
by DVE-1/UBL-5. ATFS-1 is a bZIP transcription factor that has both a mitochon-
drial and nuclear localization signal [197]. In unstressed conditions, ATFS-1 is
imported into the mitochondria and degraded by Lon protease [197]. When the
UPRMT is activated protein import into the mitochondria is disrupted by HAF-1, an
ATP-binding cassette transporter in the inner mitochondrial membrane, and the
cytoplasmic ATFS-1 can then be imported into the nucleus [200]. Full activation of
UPRMT also requires the DVE-1 transcription factor in complex with the ubiquitin-
like protein UBL-5 [201, 202]. This aspect of UPRMT activation requires the mito-
chondrial matrix protease CLPP-1 [201]. Together, ATFS-1 and DVE-1/UBL-5
upregulate gene products, including mitochondrial chaperones hsp-6 and hsp-60,
that restore proteostasis in the mitochondria. ATFS-1 also limits the expression of
nuclear-encoded components of respiratory complex proteins, which reduces the
protein folding burden of the mitochondria [203]. In parallel to ATFS-1, the GCN-2
kinase phosphorylates eIF2α, reducing the translation of new proteins and mito-
chondrial protein folding stress [204]. In sum, these mechanisms counteract mito-
chondrial stress and improve compartment-specific protein folding capacity.
Activation of the ER UPR and the ability to survive ER stress declines with age
[205]. The ER UPR genes ire-1 and xbp-1 are required for increased lifespan from
decreased insulin/IGF signalling or dietary restriction from bacterial dilution [23,
206], and also for pathogen survival [207, 208]. These data suggest that ER UPR
may be an important mechanism to ensure long life. However, the correlation
between activation of ER UPR and lifespan is not absolute. Although ubiquitous
expression of constitutively active, spliced xbp-1 restores ER UPR activation late in
life, it does not increase lifespan [205]. Curiously, lifespan is increased if expression
of spliced xbp-1 is limited to neurons or the intestine, whereas expression of spliced
xbp-1 in body wall muscle decreases lifespan [205]. The neuronal expression of
spliced xbp-1 that increases lifespan activates the ER UPR nonautonomously in
non-neuronal cells [205], but the neuron-derived signal produced in these animals
has not been identified.
Genetic perturbation that reduce mitochondrial function increases lifespan [209–
214]. These deficiencies also activate UPRMT [197, 199, 215, 216]. Increased lifes-
pan of isp-1 mutant animals requires ubl-5, suggesting that activation of UPRMT is
involved in mediating the effects of mitochondrial dysfunction on lifespan [216].
However, atfs-1 is required for activation of UPRMT but not increased lifespan of
isp-1 mutant animals [199]. Similarly, mitochondrial stress leads to nuclear local-
ization of LIN-65 and a gross chromatin reorganization that is required for DVE-1
9 Stress Response Pathways 207
nuclear puncta formation, but which is independent of atfs-1 [217]. These results
suggest that DVE-1/UBL-5 may have roles to modulate lifespan that are distinct
from activation of UPRMT. Another possibility, which is not mutually exclusive, is
that interactions between different tissue types underlie differences between atfs-1
and dve-/ubl-5 pathways. Mitochondrial stress in neurons, from RNAi depletion of
cco-1, leads to non-autonomous activation of UPRMT in peripheral tissues [216]. As
of now, it is not known how (or if) nonautonomous activation of UPRMT and ER
UPR are related.
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Chapter 10
Oxidative Stress
Abstract The oxidative damage theory has been the dominant paradigm in ageing
research over the last 50 years. The versatile genetic nematode model C. elegans has
been used by many to put this theory to the test. C. elegans is an attractive model as
it ages fast, it has an elaborate antioxidant system which can be easily manipulated,
and many long-lived mutants are available. Recently, it became possible to visualize
reactive oxygen species (ROS) in vivo and in real-time in this transparent animal by
using genetically encoded biosensors. The data generated in C. elegans to test the
oxidative damage theory is often ambiguous and of mere correlative nature.
Experimental manipulation of the antioxidant system most often disproves this the-
ory. Over the years, it became clear that ROS, when present at normal physiological
levels, are important signalling molecules. Interference with this ROS signal may
elicit a cytoprotective programme that, in many cases, extends lifespan. It is still an
open question whether the molecular underpinnings of this hormetic response is
also of importance to the normal ageing process. Alternatives to the oxidative dam-
age theory, such as the hypertrophy hypothesis, are currently gaining wider
attention.
Oxygen became an important constituent of the Earth’s atmosphere when the pro-
cess of photosynthesis evolved in cyanobacteria about 2.2 billion years ago [1].
Although today O2 is essential to support energy metabolism in the majority of spe-
cies, it is essentially a toxic, mutagenic gas which requires appropriate cellular pro-
tection via antioxidant defences.
Molecular oxygen is a free radical – a molecule that can exist freely with one or
more unpaired electrons – and it can generate various reactive oxygen species
(ROS) by single electron transfers, usually from transition metals. The group of
reactive oxygen species contains oxygen radicals as well as non-radicals that are
oxidizing agents and/or are easily converted into radicals. Besides ROS, also reac-
tive nitrogen, sulphur and halogen species exist [2]. Molecular oxygen can be
reduced to water by four single electron transfers, generating the superoxide anion
(O2•−), hydrogen peroxide (H2O2), the hydroxyl radical (OH•), and finally, water
(H2O). ROS may also be generated in other ways, such as homolytic fission of water
via background ionizing radiation, generating two hydroxyl radicals. The reactivity
of each of these species towards biological molecules varies widely but these uncon-
trolled reactions result in oxidative damage that may impair or alter the function of
the molecule.
Superoxide can be formed at several sites in the cell by reduction of O2 with one
electron. The predominant source of superoxide in aerobic animals is the mitochon-
drial electron transport chain [3, 4]. The rate at which electrons leak from the elec-
tron transport chain to molecular oxygen is determined by the mitochondrial
membrane potential, which in turn depends on mitochondrial activity and coupling
efficiency. This way, active mitochondria may produce less O2•− than resting mito-
chondria [5–7]. Due to its negative charge, the superoxide anion cannot readily
cross lipid membranes although transport through anion channels has been described
[8]. Superoxide does not react with most biological molecules in aqueous solution
but it can quickly react with other radicals or enzymatic Fe-S clusters. Despite its
low reactivity, superoxide is an important ROS as it is the primary precursor of
many other reactive species [2].
Hydrogen peroxide (H2O2) may be generated in the cell by spontaneous or
enzyme-catalysed dismutation of O2•−. Also, some enzyme systems such as oxygen-
ases are known to produce hydrogen peroxide. This ROS is more stable than super-
oxide but it is also poorly reactive. Hydrogen peroxide is a potent but slow oxidizer:
DNA, lipids and most proteins are not oxidized directly by H2O2, even at millimolar
levels. This species can, however, inactivate some enzymes directly by oxidizing
hyper-reactive thiols necessary for catalysis [9, 10]. The biological importance of
hydrogen peroxide should not be underestimated as it can act as a signalling mole-
cule and it is the source of hydroxyl radicals [11].
The hydroxyl radical OH• is one of the most potent oxidizing agents known to
chemistry. Immediately after its formation it reacts non-selectively with molecules
such as DNA, lipids or proteins [12] and therefore is the most damaging ROS in
biological systems. It is generated by homolytic fission of H2O2 by UV light, by
reaction of HOCl with O2•−, or most often by Fenton reactions. In these reactions,
hydrogen peroxide oxidizes a reduced metal ion, usually Cu+ or Fe2+ to produce OH-
and OH•. The oxidized transition metal can return to its reduced state, possibly by
aid of intracellular reductants such as ascorbate, quinines or semiquinones, cyste-
ine, flavins and NAD(P)H [13–15]. The availability of free iron and copper in the
cell is strictly regulated to minimize OH• formation by Fenton chemistry. However,
superoxide may cause the release of iron from Fe-S clusters or ferritin [2].
10 Oxidative Stress 221
Besides these well-studied forms of ROS, other reactive species, such as carbon-
ate, peroxyl, alkoxyl and sulphur radicals, singlet oxygen and ozone, may also be
involved in oxidative damage.
10.2 Antioxidants
In living organisms, intracellular ROS levels are kept low because of reasons rang-
ing from habitat choice to intracellular molecular architecture. Many small organ-
isms avoid oxygen-rich environments (e.g. C. elegans prefers 5–12 % O2 [16]) while
larger animals only expose their epithelia to atmospheric oxygen levels. Another
way to reduce ROS formation is the organization of electron transport chain compo-
nents into an efficient respirasome [17], minimizing electron leakage to O2.
However, ROS levels and ROS-induced damage are, above all, restrained by anti-
oxidants; substances that, by definition, delay, prevent, or remove oxidative damage
to a target molecule [2]. These include enzymes and other proteins as well as small
organic molecules.
Superoxide dismutases (SODs), first discovered in 1969 [18], catalytically
remove superoxide by dismutation. These enzymes have been found in all organ-
isms and are grouped according to their metal cofactor. MnSODs and FeSODs are
found in prokaryotes and plants while animals possess MnSODs and Cu/ZnSODs.
A nickel-containing SOD (NiSOD) was found in Streptomyces and cyanobacteria
[19]. In animals, MnSOD is localized in the mitochondria, in agreement with the
prokaryotic ancestry of these organelles. Cu/ZnSOD is found in the cytoplasm or
extracellular. While most eukaryotes only have two SODs, the C. elegans genome
encodes five sod genes [20]. Two cytosolic Cu/ZnSODs are represented by sod-1
and sod-5 and the MnSODs are sod-2 and sod-3. sod-4 encodes two Cu/ZnSOD
isoforms resulting from alternative splicing: SOD-4.1 is a homologue of the mam-
malian extracellular Cu/ZnSOD while SOD-4.2 contains a C-terminal sequence
resembling a transmembrane domain and hence this unique isoform is probably
attached to the membrane [21]. SOD-1 is the most abundant C. elegans SOD tran-
script – making up about 75 % of all SOD transcripts – and it contributes most to
total SOD activity in normal worms [22]. In mitochondria, SOD-2 is the predomi-
nant isoform [22] and this MnSOD has, together with SOD-3, been localized to the
I:III:IV supercomplex of the electron transport chain, where it may stabilize the
complex and/or reduce local superoxide formation [23]. Finally, SOD-3, SOD-4
and SOD-5 are expressed at low levels in normal worms but are strongly induced in
dauers, probably via the Ins/IGF-1 like signalling pathway [20, 22]. Loss of SOD-1
activity may lead to compensatory induction of SOD-5 [24] although this was not
confirmed by another study [25].
SODs convert O2•− into H2O2, which in turn can be eliminated by catalases and
peroxidases. Catalases are homotetramers of haem-bearing subunits, each of which
can catalyse the dismutation reaction of two H2O2 molecules into H2O and O2 [26].
As this reaction requires two hydrogen peroxide molecules at a single active site,
222 B.P. Braeckman et al.
catalases are only efficient at high substrate levels. Catalases are found in prokary-
otes and eukaryotes but have been lost during evolution in a few species [27, 28].
Catalase resides in the peroxisomes where it scavenges the hydrogen peroxide that
is produced during fatty acid β-oxidation, but cytosolic catalases are also known.
The C. elegans genome contains a tandem array of three catalase genes (ctl-1, ctl-2
and ctl-3) with very high sequence similarity [29]. CTL-2 is a peroxisomal catalase
that contributes up to 80 % of the total catalase activity in the worm. CTL-1 has been
described as a cytosolic catalase [29, 30]. The details of CTL-3 are less clear but it
appears to be expressed in the pharyngeal muscle and neurons.
Peroxidases are a class of enzymes that convert H2O2 to water or hydroperoxides
(ROOH) to the corresponding alcohol (ROH) by oxidizing another substrate (e.g.
NADPH or GSH). Glutathione peroxidase (GPX) is a Se-bearing enzyme that
occurs as a monomer or homotetramer, depending on the isoform. The C. elegans
GPX family contains at least 8 members although no enzymatic GPX activity could
be detected when applying a standard assay using tert-butyl-hydroperoxide as a
substrate [31], suggesting narrow substrate specificity of the C. elegans GPXs. C.
elegans GPX-1 is a homologue of the mammalian phospholipid hydroperoxide
GPX and interacts with dipeptide transport [32]. Other C. elegans GPX family
members await detailed study. A second class of peroxidases contains the peroxire-
doxins (PRDXs), which are also H2O2 scavenging enzymes that occur as homodi-
mers with cysteines at their active sites. They are very abundant, localized in most
intracellular and extracellular compartments and can constitute 0.1–0.8 % of the
total soluble protein content. PRDX reduces H2O2 or ROOH by oxidation of a cys-
teine to a sulphenic acid (cys-SOH). The PRDX can be reduced to its original state
by thioredoxins (TRXs) or glutaredoxins (GLRXs). The C. elegans genome encodes
for two PRDXs: prdx-2 and prdx-3. PRDX-2 appears to be expressed in the cytosol
of the intestine, gonads and neurons. Intestinal expression of prdx-2 is sufficient to
support resistance against hydrogen peroxide treatment. However, loss of PRDX-2
activates the DAF-16 and SKN-1-dependent stress resistance programmes [33] (see
also Chap. 9). The mitochondrial PRDX-3 does not protect against hydrogen perox-
ide insult [34].
An overview of reactive species and antioxidant systems in C. elegans is given in
Fig. 10.1.
ROS are key players in oxidative stress and can be generated by exogenous com-
pounds as well as mitochondrial (dys)function. Their reactivity, ephemeral nature
and local gradients make it very difficult to localize and quantify these molecules
in vivo. The majority of C. elegans studies that analyse ROS make use of reduced
dyes such as dihydrofluoresceins, lucigenins, MitoSOX and amplex red [35]. The
problem with many dyes is that their uptake in live animals may vary, they often
lack selectivity, they may need a catalyst to work, they may be metabolized or have
10
Oxidative Stress
Fig. 10.1 Schematic representation of the production and removal of reactive oxygen species and the oxidative damage clearance and repair systems in the
nematode C. elegans
223
224 B.P. Braeckman et al.
poor stability, and some probes can even generate ROS by themselves and may
disturb cellular physiology [36–38]. Moreover, many dyes react with ROS irrevers-
ibly, precluding dynamic measurements. Disruption of C. elegans for ROS quantita-
tion may create oxidation artefacts as delicate cellular redox balances are disturbed.
Hence, an ideal ROS probe should be selective, sensitive, instantaneous, reversible,
compartment-specific, non-invasive and allow in vivo monitoring [39]. Some of the
disadvantages of dyes have been overcome by designing protein-linked chemical
reporters [40], or ratiometric mass spectrometry probes [41], but even these techno-
logically advanced techniques cannot tackle every problem.
The introduction of genetically encoded ROS sensors has been a big leap for-
ward in the search for reliable in vivo ROS detection. Wild-type GFP has two exci-
tation peaks – 395 nm for the protonated and 475 nm for the deprotonated form of
Y66 – while only one emission peak exists at 509 nm [42]. This dual excitation/
single emission property of GFP can be exploited for ratiometric measurements in
which emission intensity at one excitation wavelength is divided by the emission at
the other excitation wavelength. This offers the advantage of being independent on
probe expression levels and photobleaching, greatly simplifying comparison among
samples. Fluorophore protonation is dependent on interactions with surrounding
residues and therefore conformational alterations can cause a shift in fluorescence
intensity. Based on these properties, several ROS-sensitive probes have been devel-
oped [43].
10.3.1 Superoxide
The oxidative damage theory has been tested in a plethora of species of wide phy-
logenetic diversity. In this chapter, we will focus on the work that has been carried
out specifically in C. elegans, which has become a very prominent model species in
biogerontology over the last few decades [71–73].
The predicted increase of oxidative damage with age has been supported by sev-
eral C. elegans studies. Levels of protein carbonylation, the oxidation of amino acid
side-chains to carbonyl residues, have been shown to increase over time in adult
10 Oxidative Stress 227
worms [74, 75], at least in their mitochondria [76, 77]. A positive correlation was
found between adult age and DNA damage such as single-strand DNA breaks and
5-methylcytosine [78] although the latter could not be confirmed in another study
[79]. Also, the increased occurrence of mitochondrial DNA breaks in ageing C.
elegans is ambiguous [80–82]. DNA damage and ageing in C. elegans is presented
in detail in Chap. 11. 4-hydroxy-2-nonenal (4-HNE), a lipid peroxidation product
that forms as a consequence of oxidative stress, can be conjugated to proteins by the
action of glutathione S transferases. It was shown that 4-HNE protein adducts do
indeed accumulate with age in the worm [83]. Lipofuscin is a heterogeneous cross-
linked aggregate of oxidatively damaged lipids and proteins and tends to aggregate
with age in vertebrates [84]. These aggregates are also called age pigments and tend
to show a specific fluorescence spectrum. Autofluorescence with similar character-
istics has been found to accumulate in gut granules of C. elegans populations over
time and therefore has been referred to as lipofuscin and used as a biomarker of
ageing [85–87]. However, more recently it was found that gut granule autofluores-
cence is caused by anthranilic acid glucosyl esters and that, at the individual worm
level, this autofluorescence does not increase gradually with age but rather bursts at
the time of death [88]. Overall, there are many indications that oxidative damage
increases with age in C. elegans, as predicted by the oxidative damage theory, but
not all studies are consistent. However, this correlation does not imply causation,
just like greying hair in humans is not causal to ageing.
A tighter link between oxidative stress and ageing appeared when researchers
started to analyse the oxidative damage and antioxidant capacity of C. elegans
mutants with altered lifespan. Early studies showed that age-1, a long-lived Insulin/
IGF-1 signalling pathway mutant (see Chap. 4), displays enhanced catalase and
SOD activity compared to controls and antioxidant activity appeared to rise with
age in the mutant [31, 89, 90]. This rise could not be confirmed in a later study
although the levels of antioxidant enzymes were clearly increased in the long-lived
mutants [91]. Most other long-lived mutants also show increased oxidative stress
resistance [92–94]. This strong correlation has even been exploited in a screen for
longevity mutants by using oxidative stress resistance as a rapid selection marker
[95].
The relationship between oxidative damage and lifespan also extends in the
opposite direction: the complex II mutant mev-1 suffers excessive oxidative stress,
has a higher load of protein carbonyls and lives shorter than the wild-type strain [74,
96]. However, in these cases, it is more difficult to distinguish between accelerated
ageing or oxidative stress pathologies that are not linked to ageing [97].
Lifespan extension and oxidative stress resistance are strongly linked suggesting
that both processes are causally related. However, this correlation does not provide
sufficient proof that the theory is correct. Long-lived strains are usually resistant to
228 B.P. Braeckman et al.
other types of stress as well, e.g. heat, UV and pathogenic bacteria [92, 98]. Hence,
these data would equally support theories claiming that heat, UV or bacteria are
primary causes of ageing.
A more direct approach to test the causal relation between ROS and ageing is to
manipulate the intracellular ROS levels and examine its subsequent effect on lifes-
pan. ROS levels can be changed by interfering with ROS generating systems or with
antioxidant defence, either pharmacologically or genetically.
lifespan is still elusive [34]. Suppression of prdx-3 during adulthood does not influ-
ence levels of oxidative damage to proteins, nor does it alter lifespan [106]. For the
thioredoxin trx-1, mutation slightly reduces lifespan while overexpression increases
lifespan to some extent, but the effect on oxidative damage accumulation was not
tested [107, 108]. Finally, the lifespan and oxidative damage phenotypes obtained
after knock-down and overexpression of the glutathione-S transferase gst-10 are
consistent with the predictions of the oxidative damage theory [83].
Many studies have pointed out that addition of pro-oxidants shortens C. elegans
lifespan. Although this may seem to agree with the oxidative damage theory, it sup-
ports this theory only very weakly as it may reflect a toxic effect rather than an
acceleration of the ageing process [97]. Antioxidant treatments, which are supposed
to extend lifespan, have been much more instructive. Numerous studies examined
the effect of exogenous catalytic and non-catalytic antioxidants on C. elegans lifes-
pan [109]. Many of the non-catalytic antioxidants, such as Vitamin E and C, trolox,
α-tocopherol, and N-acetylcysteine, affected lifespan differently in distinct studies,
probably because of differences in dose and method of delivery [110–116]. In some
cases, the antioxidants increased oxidative stress without affecting lifespan [117].
According to the oxidative damage theory, sufficient dietary intake of these anti-
oxidants should delay the ageing process. A more interesting approach would be the
intake of catalytically active antioxidants that require much lower doses because of
their catalytic rather than stoichiometric reaction properties. EUK-8 and EUK-134
are SOD/catalase mimetics that are readily taken up in C. elegans and tend to accu-
mulate in mitochondria [118]. Initial lifespan analyses showed that both mimetics
extend lifespan in C elegans by an average of 44 % [119]. However, these results
could not be replicated in independent studies. On the contrary, the EUK com-
pounds seemed to shorten lifespan with increasing dose [118, 120, 121]. However,
these molecules directly protect against oxidative stress imposed by exogenous
compounds [118, 121, 122].
Together, these studies do not convincingly show that feeding antioxidants to
worms extends lifespan. The fact that various antioxidants can protect against exog-
enous oxidative stress without influencing lifespan suggests that oxidative stress has
no causal relation with normal ageing.
Oxidative stress causes a hormetic effect on lifespan in C. elegans, i.e. low doses
result in moderate lifespan extension while higher doses are harmful and shorten
lifespan. This effect was observed for the oxidants juglone [123] and paraquat [105,
230 B.P. Braeckman et al.
124, 125]. The hormetic lifespan increase is caused by activation of a genetic cyto-
protective programme in response to the stressor (for more details, see Chap. 9).
The major transcription factors involved in the response to oxidative stress are
DAF-16 [90] and SKN-1 [126]. DAF-16 is a Fork head transcription factor which is
part of the Insulin/IGF-1 like signalling pathway [127] involved in dauer formation,
metabolism, innate immunity and stress resistance. SKN-1, the C. elegans Nrf2
homologue, is a transcription factor involved in gut development and oxidative
stress resistance [126]. The actions of DAF-16 and SKN-1 are intertwined [128] and
these transcription factors may interact with many other factors such as BAR-1,
SIR-2.1, 14-3-3, SMK-1, and HSF-1, to elicit the expression of overlapping gene
sets with protective functions [25]. Typical downstream genes in oxidative stress
response are glutathione-S-transferases, catalases, and superoxide dismutases [126,
128, 129]. However, other cytoprotective genes, such as small heat shock proteins,
are also activated by these transcription factors.
These hormetic effects are often at play in the beneficial effects of ‘antioxidant’
plant extracts on C. elegans lifespan. Such studies have become increasingly popu-
lar over the last few years but their innovative power and contribution to the under-
standing of the ageing process is usually very limited. In most cases, the studied
extracts trigger well-known cytoprotective responses, often involving DAF-16 and/
or SKN-1, resulting in lifespan extension at low sub-toxic doses [130–134]. In many
of these studies, authors claim to have found promising anti-ageing chemicals, but
essentially a very broad range of molecules may trigger this general hormetic effect.
A similar effect has been observed with the addition of the antioxidants N-acetyl-L-
cysteine [135] and S-linolenoyl glutathione [136]. However, not all plant extracts
extend lifespan via the same genetic pathways [137].
Hormesis has also been described in cases of mild mitochondrial dysfunction.
Incremental reduction of mitochondrial electron transport chain (ETC) activity by
RNAi dilution showed that lifespan is extended by mild ETC inhibition while more
severe inhibition reduces lifespan [138] (see also Chap. 5). Interestingly, no direct
correlation could be found between levels of oxidative damage and lifespan in this
study. Some mitochondrial (Mit) mutants show increased ROS production [124,
125] and enhanced expression of antioxidant enzymes [99, 139], but the latter is
dispensable for longevity [29, 99]. However, ROS generation is required to support
lifespan extension in Mit mutants such as isp-1 and nuo-6 [125]. In the Mit mutant
clk-1 the prolongevity effect of excessive ROS production is compartment-specific
[140].
The hormetic effect of ROS generated in the mitochondria is called mitohorme-
sis [141, 142]. In the mitohormetic theory, ROS are not only damaging agents, but
instead can act as signalling molecules that initiate cell-protective programmes of
which some key players have been identified [141, 143, 144]. In the Mit mutants
clk-1 and isp-1, the hypoxia-inducible factor HIF-1 is required for longevity. Hence,
respiratory stress and increased ROS production are linked to a nuclear transcrip-
tional response that promotes longevity [124]. Inhibition of mitochondrial respira-
tion by RNAi triggers the mitochondrial unfolded protein response (UPRmt), which
is also required for longevity of these animals. However, this response does not
10 Oxidative Stress 231
occur in long-lived worms bearing mutations in the ETC genes, suggesting that
there are at least two classes of Mit mutants – genetic and RNAi - each showing
lifespan extension by independent molecular mechanisms [145]. The UPRmt is a
cell-non-autonomous response as mitochondrial perturbation in one tissue can elicit
the UPRmt in another [146]. Yet in the frataxin mutant, another Mit mutant, it was
shown that lifespan extension is mediated by the C. elegans p53 homologue cep-1
and not by skn-1 or daf-16 [147]. Although several molecular mechanisms of Mit
longevity have recently been discovered, still many gaps remain on their relative
importance and interactions [143, 148, 149].
The notion that ROS act as signalling molecules rather than being damaging byprod-
ucts of oxidative metabolism is not entirely new [157]. In C. elegans, several ROS-
mediated biological processes have been described (for a overview, see [39]).
Reduced glycolysis [115] or mild mitochondrial dysfunction [138] increase mito-
chondrial superoxide production which acts as a signal triggering a protective
response that extends lifespan (mitohormesis, see Sect. 10.7). Intracellular SOD
may convert the short-lived superoxide into the more stable hydrogen peroxide
which can oxidize cysteins of PRDX-2 monomers, forming activated homodimers.
232 B.P. Braeckman et al.
Subsequently, PRDX-2 can activate SKN-1 via a MAPK pathway, resulting in the
expression of a cytoprotective programme [158]. Interestingly, DAF-16 can be
directly oxidized by ROS, linking it to the importin IMB-2 with a cysteine disul-
phide bridge [159], enabling it to enter the nucleus. This mechanism links oxidative
stress or ROS signals directly to DAF-16 activation. The redox control of DAF-16
and PRDX-2 may be a response to relatively large cellular redox imbalances that
require the acute activation of stress programmes to maintain cellular homeostasis
and avoid cell death. However, ROS signalling also occurs on a much smaller spa-
tial scale to regulate normal household functions such as reproduction. The C. ele-
gans globin GLB-12 was recently identified as a membrane-bound superoxide
generator, which, in concert with the intracellular SOD-1 and extracellular SOD-4,
creates a hydrogen peroxide gradient over the plasma membrane of the somatic
gonad. This gradient is required for normal gonad function and the control of germ-
line apoptosis [160]. Loss of this redox signal results in complete sterility. This
indicates that, rather than being omnipresent scavengers of superoxide, SODs are
part of local signalling cascades, an idea that was already put forward earlier [100].
Despite the general notion that hydrogen peroxide easily crosses lipid bilayers,
redox signals may act very locally as was shown in mammalian cells by means of
membrane-anchored ROS biosensors [161]. These local signals may be propagated
throughout the cell by GSH, formerly considered as an omnipresent cellular redox
buffer, but now believed to be a redox signal amplifier [162].
As an alternative to the oxidative damage theory, the redox stress hypothesis
states that functional loss during ageing is caused by a progressing pro-oxidizing
shift in the cellular redox state, leading to the disruption of redox-regulated signal-
ling mechanisms [163]. This would better explain the wide-spread cellular deterio-
ration with age than does the relatively small accrual of structural oxidative damage.
However, the cause of the pro-oxidizing shift with age is still unexplained. In the
same vein, analysis of lifespan and hydrogen peroxide level in over 40 long-lived C.
elegans strains led to the conclusion that not the absolute levels but rather the fluc-
tuation of hydrogen peroxide correlates to lifespan [164]. This suggests that tight
control of ROS fluctuation is more vital than minimizing ROS levels, hinting at the
importance of redox signalling in lifespan determination.
Taking together the (lack of) evidence for the oxidative damage theory in C. ele-
gans, it seems that this theory is ageing badly and the call for paradigm shifts is
getting louder. One such radically different view is that of Mikhail V. Blagosklonny,
who proposed that ageing is a quasi-programme, a continuation of the developmen-
tal programme that is not switched off, becoming hyperfunctional and damaging
[165]. A central player in this theory is the TOR (target of rapamycin) nutrient and
mitogen-sensing pathway, a central pathway in development and anabolic growth.
Inhibiting TOR activity by mutation or caloric restriction indeed increases lifespan
in C. elegans [166, 167]. TOR-inhibition by rapamycin also increases lifespan
10 Oxidative Stress 233
There is no doubt that C. elegans research has pushed forward molecular biogeron-
tology over the last three decades. As a prime genetic model that ages fast and that
is easily subjected to large-scale genetic screens, this species enabled us to track
down genetic pathways that influence lifespan [175]. In many cases, these pathways
appeared to be conserved and relate to ageing in other species as well [176]. Due to
its complete transparency and the availability of strains expressing genetically
encoded biosensors, C. elegans is currently the most accessible organism to study
the role of ROS, in vivo and in real-time, in the ageing process of a multicellular
organism. Hence, there are many reasons to continue C. elegans ageing research
and undoubtedly thrilling discoveries about the molecular mechanisms of ageing lie
ahead of us. Yet, this optimism should go hand in hand with necessary caution. We
always need to bear in mind that some mechanisms may be private to C. elegans (or
by extension, to nematodes) rather than public (i.e. valid for every animal species).
Being an euryoxic ectotherm, C. elegans can cope well with changing environments
and has a much more flexible metabolic network than mammals. For example, C.
elegans has a fully functional glyoxylate cycle (specific to nematodes in the animal
kingdom) and this pathway seems to be important in lifespan extension of Mit
mutants and Insulin/IGF signalling mutants [47, 177, 178]. Also trehalose, a disac-
charide absent in vertebrates [179], was shown to support lifespan extension in
Insulin/IGF mutants [180]. Besides differences in biochemistry, C. elegans also
lacks several systems such as the cardiovascular and adaptive immune system, that
have been linked to age-related diseases in humans.
In conclusion, it is clear that C. elegans is not just a 1-mm human that ages 1300
times faster than us. Nevertheless, it is an ideal system for making very fast progress
in the search for important molecular determinants of the animal ageing process that
may serve as candidates for follow up studies in other models that are closer related
to humans.
234 B.P. Braeckman et al.
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Chapter 11
Genome Stability and Ageing
The nuclear and mitochondrial genomes are constantly exposed to damaging agents
from endogenous (i.e., spontaneous, e.g., reactive oxygen species) and exogenous
(i.e., environmental, e.g., ultraviolet radiation) sources. These agents cause chemi-
cal modification of DNA, impacting chromosomal replication and transcription.
DNA replication is critical during worm development and in the gonads of adult
worms, while transcription is crucial in all cells throughout life. The advent of
whole genome sequencing revealed that the mutation frequency in C. elegans is
~6.7 × 10−10 per nucleotide per cell division [1] whereas, in humans the mutation
rate is 5.0 × 10−11 per nucleotide per cell division [2]. This suggests that even with a
short lifespan, worms accumulate a significant number of mutations, analogous to
humans. Additionally, single-strand DNA breaks [3] and deletions in the mitochon-
drial genome [4] increase with age in the worm, which is also comparable to humans
(reviewed in [5–7])
The DNA repair pathways that are conserved between mammals and C. elegans
include the following [8]:
Base excision repair (BER): BER identifies and excises subtle lesions that don’t
distort the helical structure of DNA. The kinds of lesions routinely repaired by
BER are abasic (AP) sites, oxidized bases, alkylated bases, deaminated bases
and single-strand breaks.
Nucleotide excision repair (NER): NER detects and repairs numerous different
types of lesions that cause helical distortion, including UV-induced (6-4) photo-
products (6-4PPs) and cyclobutane pyrimidine dimers (CPDs), bulky adducts
formed by environmental agents such as a by-product of tobacco smoke BP-7,8-
diol-9,10-epoxide (BPDE). NER consists of two sub-pathways: Global Genome
NER (GG-NER): lesions repaired anywhere in the nuclear genome and
Transcription Coupled NER (TC-NER): repair of lesions occurring in the tem-
plate strand of an actively transcribed gene.
Interstrand cross-link repair (ICLR): ICLR or the Fanconi pathway repairs
lesions that covalently link both strands of DNA together.
Mismatch repair (MMR): MMR is a DNA repair mechanism that is responsible
for correcting base-base mismatches, insertion/deletion mismatches and small
hairpin structures resulting from misalignment that occurs during DNA replica-
tion and recombination.
Homologous recombination (HR): HR is used to repair DNA double-strand breaks
(DSBs) using a sister chromatid or homologous chromosome as a template to
acquire lost sequence information. In addition to double-strand breaks, HR is
needed for the repair of interstrand crosslinks (ICL) and for the recovery of
stalled replication forks.
Non-homologous end-joining (NHEJ): DNA double-strand breaks with two bro-
ken ends are repaired by NHEJ via a mechanism by which the two ends are
ligated together.
11 Genome Stability and Ageing 247
Table 11.1 List of conserved and absent DNA repair proteins in C. elegans [79]
Genome
maintenance
mechanism C. elegans Mammalian Function
Base excision R09B3.1a APEX1 AP endonuclease
repair(BER) (exo-3)
C29A12.3 LIG3 ATP-dependent DNA ligase
(lig-1)
R10E4.5 NTHL1 DNA N-glycosylase
(nth-1)
Y56A3A.27 NEIL3
H23L24.5 PARG Poly (ADP-ribose) glycohydrolase
(parg-2)
Y71F9AL.18 PARP1/ Poly(ADP-ribose) polymerase
(parp-1) PARP2
F21D5.5 PNKP Polynucleotide kinase 3′-phosphatase
Y56A3A.29 UNG Uracil-DNA glycosylase
(ung-1)
W03A3.2 POLQ DNA polymerase
(polq-1)
Y47G6A.8 FEN1 Flap endonuclease
(crn-1) b
(Missing) DNA polymerase
POLB
b
APEX2 Weak apurinic/apyrimidinic (AP)
endodeoxyribonuclease
b
MBD4 T:G mispair glycosylase
b
MPG 3-meA, hypoxanthine glycosylase
b
NEIL2 5-hydroxyuracil glycosylase
b
OGG1 8-Oxoguanine glycosylase
b
SMUG1 Uracil glycosylase (single-strand DNA
substrates)
b
TDG T:G mispair glycosylase
(continued)
248 A.U. Gurkar et al.
An important consideration when studying DNA damage and its role in ageing is
that repair mechanisms are differentially utilized in tissues (reviewed in [12]). For
instance, germ cells respond more strongly to DNA damage than somatic cells.
Therefore, post-mitotic adult worms are relatively resistant to ionizing radiation,
whereas germ cells are extremely sensitive. Proliferating and meiotic germ cells
repair DSBs by HR, whereas, post-mitotic somatic cells utilize NHEJ. Similarly,
GG-NER, BER and ICLR maintain DNA stability in the mitotic germ cell compart-
ment. TLS is highly active during early embryonic growth, contributing to resis-
tance to genotoxic stress during this phase of development. During development,
somatic cell genome maintenance requires HR and NHEJ, whereas the post-mitotic
adult is mostly dependent on TC-NER. These observations reveal complex spatial
and temporal regulation of DNA repair mechanisms (Fig. 11.1).
Notably, there are a few key proteins involved in genome maintenance that appear
to be lacking in the worm. γH2Ax, MDC1 and RNF8 DNA damage signalling pro-
teins have not been identified in C. elegans. Similarly, some regulators of NHEJ and
ICL repair pathway do not appear to be present in the worm (see Table 11.1).
11
Genome Stability and Ageing
Fig. 11.1 DNA repair pathways utilized during different stages of C. elegans development and ageing
251
252 A.U. Gurkar et al.
Another point to consider is that in higher organisms DNA damage can induce cel-
lular senescence [13], which has been shown to drive ageing [14]. However, it is
currently unclear if C. elegans have a cellular senescence programme [15].
Nonetheless, the importance of DNA repair mechanisms to genome stability and
organismal lifespan is well documented in the worm. We focus here on each of the
genome maintenance pathways and their relationship to ageing.
One of the earliest studies measuring DNA damage over the lifespan of worms was
reported by Klass et al. in the 1980s. The authors observed a 34-fold increase in sin-
gle-strand DNA breaks in day 15 adults compared with young, day 5 animals, using
an Escherichia coli DNA polymerase I assay. Additionally, 5-methylcytosine (an epi-
genetic marker regulated to some extent by DNA repair) was also exponentially
increased in older worms compared to larvae and young adults. These changes were
accompanied by reduced transcription [3]. These data are consistent with the notion
that incomplete repair of DNA damage leads to damage accumulation with age.
Similarly, the oxidative DNA lesion 8-oxo-7,8-dihydro-2′-deoxyguanosine
(8-oxo-dG) was measured in the short-lived mutant, mev-1 (a gene that encodes a
subunit of complex II in the mitochondrial electron transport chain) [16] causing
accumulation of dysfunctional mitochondria, reduced mitochondrial membrane
potential [17], increased ROS, and hypersensitivity to oxidative stress [18]. A sig-
nificant increase in adducts is detected in mev-1 mutants compared to wild-type
worms by high-performance liquid chromatography coupled with electrochemical
detection (HPLC-EC). mev-1 mutants also have a five to ten-fold higher mutation
frequency than WT worms, based on a fem-3 mutation assay that detects loss-of-
function mutations by measuring reversal of temperature sensitive sterility [19].
These studies are consistent with the idea that mitochondrial ROS contributes to
nuclear genomic instability and mutagenesis, and promotes ageing. However, lon-
gitudinal studies in worms measuring DNA damage accumulation over the organ-
ism’s lifespan have yet to be reported.
Radiation-sensitive mutant strains (rad) were first isolated in the 1980s, based on
their sensitivity to ultraviolet light (UV). These mutants are also sensitive to other
DNA damaging agents such as methyl methane sulphonate (MMS) and ionizing
radiation (IR) [20]. In this study, no significant differences in lifespan were observed
in the rad mutants compared to wild-type worms. Five of the seven rad mutants
(except rad-4 and rad-7) have slightly shortened lifespans after exposure to IR, but
11 Genome Stability and Ageing 253
One line of evidence that supports the theory that decreased repair capacity drives
ageing comes from humans with genetically inherited defects in DNA repair path-
ways (reviewed in [26, 27]). These defects lead to hypersensitivity to DNA damag-
ing agents, accumulation of DNA damage and accelerated ageing of one or more
tissues. The identification of such progeroid syndromes in humans led to the design
of mutant C. elegans strains that have defects in DNA repair mechanisms. Below,
we examine each DNA repair pathway and evidence that links it to ageing.
254 A.U. Gurkar et al.
C. elegans possess two AP endonucleases, EXO-3 (exo III family) and APN-1 (endo
IV family) [28]. EXO-3 (R09B3.1a) is an endonuclease required for BER that nicks
DNA 5′ to an AP site. exo-3 mRNA levels decline 45 % by day 5 of adulthood and
are maintained at low levels as worms age beyond that point [29]. RNAi depletion
of exo-3 increases ROS and mitochondrial genome deletions, which are character-
istics of aged worms. Knockdown of exo-3 also leads to other common ageing fea-
tures such as neuronal damage and reduced motility.
Pharmacological suppression of ROS in exo-3 deficient worms inhibits neuronal
damage and increases motility, suggesting that ROS is a key cause of morbidity in
the mutant worms [29]. In accordance, exo-3 RNAi leads to a reduction in both
mean (20 %) and maximum (10 %) lifespan of C. elegans. Interestingly, suppres-
sion of cep-1, ortholog of the tumour suppressor p53, rescues ageing phenotypes of
exo-3 RNAi mutants. One possible explanation is that cep-1 is known to increase
oxidative stress by inducing expression of pro-oxidant genes and repressing antioxi-
dant genes, in response to cellular stress including genotoxic stress [29]. Thus delet-
ing cep-1 should reduce ROS and oxidative DNA damage in the exo-3 mutant
worms. Interestingly, in WT nematodes, suppression of cep-1 leads to upregulation
of exo-3 and preserves healthspan (neuronal integrity and motility). This suggests
that cep-1 and exo-3 coordinately respond to oxidative or genotoxic stress and this
influences age-related decline.
Kato et al. further characterized the exo-3 deletion mutant (tm4374) and con-
firmed a reduced lifespan [30]. The short lifespan of exo-3 mutant is also suppressed
by deletion of ung-1, a monofunctional uracil DNA glycosylase. UNG-1 acts
upstream of EXO-3 in BER to remove uracil from DNA (caused by spontaneous
hydrolysis of cytosine) creating an AP site. This reveals that AP sites are more deli-
terious than uracil lesions. Additionally, the authors reported a surprising difference
between somatic versus germline cells (post-mitotic vs proliferating). In the germ-
line, exo-3 is highly expressed and loss of EXO-3 leads to a reduced brood size
(reflecting the proliferative capacity of germ cells). Interestingly, the impact of exo-
3 on brood size requires the presence of nth-1, a second DNA glycosylase that
removes oxidized pyrimidines [31]. This suggests that oxidative DNA lesions are a
major substrate of BER in germ cells, whereas deamination products are more
important in somatic cells.
Although one might predict that increased levels of oxidative DNA lesions would
promote ageing, nth-1 null mutants show a normal mean and maximum lifespan
[32]. Surprisingly, QPCR studies reveal that the rate of removal of damage caused
by oxidative and alkylating agents in the WT and nth-1 adult worms is similar [33].
This could imply that there are redundant mechanisms for removing oxidized
purines in nth-1 deficient somatic cells and that it is unlikely that nth-1 depletion
induces ROS as occurs in exo-3 mutants. It is also possible that oxidative DNA
lesions may not be a major determinant of lifespan in somatic cells. Collectively,
these genetic studies help reveal what endogenous DNA lesions are apt to contribute
to ageing and lifespan [30].
11 Genome Stability and Ageing 255
Inherited mutations affecting NER are responsible for several progeroid syndromes
in humans including Xeroderma pigmentosum (XP), Cockayne syndrome (CS),
trichothiodystrophy (TTD) and XFE progeroid syndrome [26]. These syndromes
are all characterized by accelerated age-related decline of several tissues and the
premature onset of diseases associated with old age. Many of these progeroid syn-
dromes have been recapitulated in mice, often by single DNA repair gene muta-
tions. These human syndromes fuelled several studies to interrogate whether NER
promotes healths and longevity in worms.
In C. elegans, the mechanism of repair of UV-induced DNA lesions (i.e., NER)
is very similar to humans [35]. There are several lines of evidence suggesting that
DNA lesions that are substrates for NER promote ageing in worms. Expression of
NER proteins is significantly lower in non-gravid adults (older adults) compared to
gravid adults [35], indicating that NER is important for replicative longevity. glp-1
mutants have an arrested germline and therefore enable measurement of DNA repair
exclusively in post-mitotic animals. Repair of UV lesions is slower in ageing glp-
1adults compared to young worms. This diminished repair in somatic cells however,
is not because of decreased expression of DNA repair proteins (at least at the mRNA
level). This could mean that protein translation, subcellular localization or post-
translational modification of DNA repair proteins is affected with age [35]. These
studies contribute evidence that DNA repair capacity decreases with age.
XP Complementation Group A (XPA) is required for GG-NER and TC-NER
and plays a key role before damage excision. As in humans and mice [36], rad-
3/xpa-1 worms are hypersensitive to UV irradiation and have an increased mutation
frequency in response to UV. Steady state levels of the oxidative lesions formami-
dopyrimidines (FapyGua and FapyAde) and 8-hydroxyadenine are significantly
increased in xpa-1 (ok698) mutants [37]. Human XP-A lymphoblasts also show an
accumulation of these oxidative lesions, suggesting the importance of NER in
repairing these endogenous lesions [38]. These adducts block both replication and
transcription, and are increased in several age-related diseases such as Alzheimer’s
and cancer [39].
256 A.U. Gurkar et al.
Reports on lifespan of xpa-1 (ok698) mutants vary. Hyun et al. report a ~20 %
reduction in mean lifespan [25]. Lans et al. find no lifespan shortening when look-
ing only at a population of healthy adults (but observe a shortened lifespan in an
unbiased population consisting of developmentally delayed mutants) [40, 41].
Fensgård et al. see a reduction in mean lifespan but not in maximum lifespan, when
strains are grown on standard E. coli OP50 bacteria [32]. Interestingly, nth-1 dele-
tion (BER-see above) restores lifespan of xpa-1 mutants. Furthermore, transcription
of several DNA damage response genes is attenuated in the double mutant (nth-
1;xpa-1 compared to xpa-1 alone) [32]. Taken together, these data suggest that upon
loss of xpa-1, nth-1 tries to process the lesions usually repaired by NER. However,
NTH-1 and BER is apparently unable to resolve this damage through BER and
instead causes an increased genome stress signal that culminates in a shortened
lifespan. One possible interpretation of these data is that it is not the accumulation
of DNA lesions itself that affects healthspan and lifespan but the damage-associated
stress signal that is detrimental.
Many long-lived mutants, such as daf-2 and age-1, require the FOXO transcrip-
tion factor, daf-16 for their extended lifespan [42, 43] (see Chap. 4). In the absence
of cellular stress (or presence of insulin and IGF-1) DAF-16 is hyperphosphorylated
by AKT and maintained in the cytoplasm under basal conditions. Upon stress, such
as starvation, DAF-16 phosphorylation is attenuated and this allows for nuclear
translocation and induction of several downstream target genes, including ROS
scavengers and detoxifying enzymes. DAF-16 is predominantly in the nucleus in
response to DNA damage (UV and in the xpa-1 mutant), and is required for growth
and development in the presence of genotoxic stress. As worms age, DAF-16 nuclear
translocation in response to UV radiation diminishes [44]. This would suggest that
with age the responsiveness of DNA damage-associated stress-protective genes is
attenuated. The ability to respond to stress and longevity has long been proposed to
go hand-in-hand. Thus the loss of stress responses upon genomic instability may
explain the shortened lifespan in some DNA repair mutants.
To determine if DNA repair and genomic stability is necessary for the increased
lifespan of the longevity mutants, xpa-1 was knocked-down in age-1 mutants.
Although age-1 mutants live ~1.6-fold times longer than N2, the lifespan of xpa-1
(RNAi);age-1 is similar to that of WT worms [25], suggesting that NER is critical
for longevity. However, in stark contrast, knock-down of ERCC-1/XPF-1 expres-
sion further extends the life span of daf-2 mutants. This is puzzling since ERCC-1/
XPF-1 functions downstream of XPA-1. However, interpretation of these results is
complicated by the fact that ERCC-1/XPF-1 plays a role in several DNA repair
pathways including DSB repair and ICL. Further studies, with suppression of differ-
ent NER proteins in long-lived mutants, is required to resolve this conundrum.
11 Genome Stability and Ageing 257
Despite strong evidence in humans that DNA damage increases with age and is
associated with several age-related diseases, whether it plays a causal role in driving
ageing remains contentious. C. elegans as a model system has provided critical
insights on the DNA damage theory of ageing. It is undeniable that several DNA
repair pathways examined in the worm have an effect on lifespan. However, the
11 Genome Stability and Ageing 261
mechanism remains unclear. Does chronic activation of the DNA damage response
or multi-stress response mechanisms play a role? Or is it mutagenesis caused largely
by transcription drive ageing?
Mutation accumulation has been measured in 24 different regions of the genome
to compare the relative importance of BER vs. NER vs. MMR in protecting genomic
stability [78]. This revealed that loss of MMR led to 48-fold increase in mutations,
while NER mutants caused a 28-fold increase and BER deficient worms had a
17-fold increase compared to WT worms. In contrast, whole genome next genera-
tion sequencing (NGS) in WT and 17 different DNA repair-deficient mutants
revealed no significant increase in mutation rate in the absence of various DNA
repair mechanisms [1]. These differences could stem from the number of genera-
tions examined. However, both of these studies do not reveal any significant accu-
mulation of mutations during one lifespan, suggesting minimal role of mutation
accumulation on lifespan.
Likewise, another question that can be answered in the nematode, is the relative
importance of DNA repair in proliferating versus post-mitotic cells and its effect on
lifespan. Additionally, C. elegans does not seem to have the traditional cellular
senescence and senescence associated secretory phenotype (SASP) [13]. This could
be an advantage in understanding primary mechanisms that respond to DNA dam-
age and impact cellular programming finally impacting ageing. Last, but not the
least, the one question that plagues the field is whether improving DNA repair effi-
ciency leads to longevity? This poses a challenging problem, since DNA damage
recognition and repair processes are extremely complex. However, with the advent
of newer techniques such CRISPR, generating transgenic overexpression lines in
the worm is feasible, timely and cost-effective.
In conclusion, using the strengths of C. elegans to elucidate the role of DNA
damage in maintaining healthspan and lifespan is key to understanding how evolu-
tionary adaptations has led to lifespan differences in species.
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Chapter 12
Protein Homeostasis and Ageing in C. elegans
Silvestre Alavez
S. Alavez (*)
Health Sciences Department, Metropolitan Autonomous University,
Lerma, Estado de México, Mexico
e-mail: s.alavez@correo.ler.uam.mx
12.1 Introduction
The increasing average age of the global population is an issue that affects virtually
every country around the world, because age is the single most important risk factor
for the onset and progression of a group of human degenerative diseases that repre-
sent a huge social and economic burden. Therefore, unravelling the mechanisms
underlying the ageing process in order to develop preventative therapies and inter-
ventions aimed at reducing or delaying age-related disease-associated morbidity
should be a priority for biomedical research. It is not difficult to imagine the positive
impact of such anti-ageing interventions in decreasing healthcare costs for the
elderly, increasing the healthy years of life, and possibly extending lifespan.
In consequence, the growing interest in understanding the process of ageing is
not surprising. In order to explore the basic mechanisms underlying ageing and age-
related diseases, simple models that allow researchers to answer basic questions of
why this process occurs, and to perform experiments related to this physiological
process in short periods of time, are required. During the last 30 years, the round-
worm C. elegans (C. elegans) has become a critical asset for ageing research.
Amongst the many advantages of this nematode as a model system, the relatively
short lifespan (around 3 weeks, depending on temperature) has made C. elegans
particularly suitable for longitudinal studies on ageing and ageing-related diseases.
Through studies in C. elegans, as well as other invertebrate model organisms
such as fruit flies (Drosophila melanogaster), budding yeast (Saccharomyces cere-
visiae), a large number of genes has been shown to influence the lifespan. These
genes encode a wide variety of proteins involved in the control of intracellular sig-
nalling processes, endocrine functions, metabolic functions, cell cycle checkpoint
functions, cellular stress response and protein turnover, amongst others. Despite this
wealth of information regarding mechanisms of ageing, the causes of ageing and the
reasons why ageing is a risk factor for age-related disease are still not fully under-
stood. This could be due to the multifactorial nature of ageing. Germline signalling,
oxidative damage, mitochondrial function, inflammation, DNA damage, cell senes-
cence, autophagy, and several other factors are thought to play a role in ageing (See
Chaps. 4, 5, 6, 7, 10 and 11). Interestingly, all these physiological alterations are
also related to the onset and/or progression of several diseases which suggests a
mechanistic crosstalk between ageing and disease. Possibly one of the clearest
examples of this kind of crosstalk is the breakdown of protein homeostasis which
leads to significant alterations in protein synthesis, protein folding, protein repair
and protein degradation. A failure of protein homeostasis leads to intra- and/or
extracellular protein aggregation, a common feature of physiological ageing and of
many diverse human diseases. For example, a common feature of ageing in many
species is the accumulation of a particular kind of protein aggregates (sometimes
referred to as lipofuscin) that are composed of fluorescent pigments, oxidized pro-
teins, lipids, carbohydrates and metals [1, 2]. In addition, a broad range of neurode-
generative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD),
Huntington’s disease (HD), frontotemporal dementia, and motor neuron disease are
12 Protein Homeostasis and Ageing in C. elegans 267
Protein turnover means that proteins are continuously being synthesized, degraded
and replaced with newly synthesized copies, at a rate that is specific for each pro-
tein. This process, referred to as protein homeostasis, is required to protect the func-
tional integrity of the proteome by constantly supplying functional proteins,
preventing potentially dangerous misfolded or damaged proteins from adversely
affecting the cell. Interestingly, there are significant differences in the turnover of
cellular proteins, arising from different proteins having different half-lives that
range from minutes to the whole lifespan of a cell. This variability in protein half-
life is probably due to the specific physiological function or the intracellular local-
ization of each protein. For instance, proteins located inside membranous organelles
like mitochondria or endoplasmic reticulum have very long half-lives [5]. As a con-
sequence, intracellular proteins with a long half-life will be exposed for longer peri-
ods to extra or intracellular noxious effects that could alter their conformational
structure and/or function. Furthermore, proteins that escape the surveillance of pro-
tein homeostasis mechanisms would remain in the cytosol with the consequent risk
for the cell physiology. These kind of damaged proteins tend to accumulate and this
could lead to the formation of different types of protein aggregates that, in turn, will
play a critical role in ageing and age-related diseases.
Protein aggregation arises as a result of protein misfolding and alterations in
primary structure in response to mutations, posttranslational modifications, local
changes in pH or salt concentrations and during thermal or oxidative stress [6, 7]. In
a very general sense, protein aggregates are oligomeric complexes of modified con-
formers, mainly produced by hydrophobic interactions but are often cross-linked,
that turn into large, stable complexes [6–9] (Fig. 12.1). These aggregates have a
poor solubility in water or detergent and do not exhibit the functions of their con-
stituent proteins. In the literature, overlapping terms for protein aggregates can be
found. Amongst others, the terms aggresomes, inclusion bodies, plaques, lipofuscin
and ceroid are frequently used [10].
268 S. Alavez
Fig. 12.1 Protein homeostasis. Basic mechanisms involved in the proteome preservation, the
stress response, the ubiquitin/proteasome system and autophagy. Chaperones assist in the folding
of new proteins and refold misfolded proteins while the two other proteolytic systems dispose
damaged or misfolded proteins
It is possible that physiological changes elicited during the ageing process, such
as a decrease in proteases and proteasome activity, accumulation of oxidative stress
and interaction with heat-shock proteins, amongst others, could accelerate the pro-
gression and exacerbate the effects of protein aggregates. Mechanisms involved in
the protein homeostasis response are critical for cellular defence and adaptation to
stress during ageing. Therefore, the repair mechanism (heat shock response), the
degradation system (the proteasome and the ubiquitin system) and the disposal sys-
tems (autophagy) should work in a concerted action to prevent damaged, obsolete
or misfolded proteins to aggregate and avoid cytotoxic effects (Fig. 12.1), some-
times referred to as proteotoxicity.
Misfolded proteins tend to accumulate in the cytosol where they activate heat shock
factor 1 (HSF-1), the master regulator of a particular class of proteins, the heat
shock proteins (HSPs). HSPs are stress response factors that are rapidly induced in
response to elevated temperatures and other stress stimuli by the activation, via
12 Protein Homeostasis and Ageing in C. elegans 269
Some damaged or obsolete proteins are marked for degradation by specific chaper-
ones, but most of them are marked by the covalent attachment of several units of the
small (8 kDa) protein ubiquitin (Ub) and thereby assigned to be degraded by a large,
ATP-dependent, complex called the ubiquitin/proteasome (UPS). Oxidized proteins
are preferentially degraded by the 20S proteasome [26], a multimer of 28 subunits
arranged in 4 rings staked in a cylinder-like structure, while ubiquitinated proteins
are marked for ATP-dependent degradation by the 26S proteasome, the result of the
association of the 20S proteasome with the regulatory subunit 19S [27]. Protein
ubiquitination starts when a protein substrate receives a covalent linkage of ubiqui-
tin catalyzed by a group of enzymes commonly known as E ligases. The attachment
270 S. Alavez
Fig. 12.2 Protein homeostasis alterations during ageing. Changes in the activity of the three main
mechanisms involved in protein homeostasis. Light down arrows indicate decline in function or
activity, changes in protein levels while up arrows indicate accumulation or increase in function
for some types of human cancer. Microarray experiments in human fibroblasts and
rat skeletal myocytes have shown a decrease in the transcription of several genes
encoding the 20S or the 26S proteasome subunits during cellular senescence [36]. A
decrease in free ubiquitin, downregulation of some ubiquitin-conjugated enzymes
and E3 ligase has been reported during ageing.
This decline in protein degradation during ageing leads to the formation of insol-
uble protein aggregates [37] and age-related decline in proteasome activity has been
associated with the development of several conformational pathologies, particularly
neurodegenerative diseases (Fig. 12.2). Therefore, it is highly possible that the mod-
ulation of proteasome activity plays an important role in controlling lifespan in
different species.
Several studies in C. elegans have lead to a better understanding of the role of stress
response in ageing and age-related diseases. There is ample evidence supporting
that the overexpression of chaperones, HSP induction by thermal stress or activation
of the transcriptional activator HSF-1 leads to a significant lifespan extension [16,
19, 20, 45]. In line with this, long-lived mutants present high levels of heat-shock
proteins [46]. In a recent study, it has been shown that stress response deteriorates
soon after the onset of the reproductive period in C. elegans [47]. The expression of
human proteins involved in neurodegenerative diseases produces toxic effect and
decreases lifespan (see below), suggesting that alterations of protein homeostasis
have profound impact in regulating lifespan. Over the last few years, various screens
of small molecules have been conducted to find long-sought interventions in ageing.
Recently, a series of chemicals have been identified in C. elegans that stabilize the
protein homeostasis network and extend lifespan [48].
In C. elegans, the 26S proteasome consists of a 20S protease core particle that is
capped at one or both ends by the 19S regulatory particle, which has an approximate
molecular mass of 700 kDa [49, 50]. A double stranded RNA interference (RNAi)
study of the 26S proteasome subunits has shown that a knockdown of most of these
genes produces embryonic and post-embryonic lethality, suggesting that protea-
some activity is critically required for development [51]. The entire 26S proteasome
core and, as expected, most of the regulatory subunits tested in that study were
lethal. However, loss of some of the regulatory subunits (rpt-9, rpt-10 and rpt-12)
has no effect on C. elegans survival. Recently, it has been shown that several com-
ponents of an E3 ligase family (SCF CUL-1 complex) function in the postmitotic
adult somatic tissues of daf-2 mutants to promote longevity, suggesting a role for
the proteasomal system in C. elegans lifespan [52]. In line with this, the overexpres-
sion of one 19S proteasome subunit, RPN-6, was shown to increase proteasome
activity and extend lifespan at 25 °C but not 20 °C [53]. However, mutation of a
gene encoding a different 19S subunit, rpn-10, causes reductions in the proteasome
activity but increases stress resistance and lifespan at 25 °C [54]. Since mutants with
both increased and decreased levels of proteasomal activity can have extended lifes-
pans, it is not clear how the changes in ubiquitin proteasome activity that occur
naturally with age might affect normal lifespan. In an elegant experiment,
Holmberg’s group used constitutively ubiquitinated fluorescent proteins as reporters
for proteasome activity and found a tissue-specific decline in proteasome activity,
with neurons being more affected than muscle [55]. However, an in vitro study
found an upregulation of the proteasome subunits 19S and 20S in lysates from aged
12 Protein Homeostasis and Ageing in C. elegans 273
worms [56] suggesting that the observed decrease in proteasomal activity is not due
to decreased proteasome levels. This could be explained by a tissue-specific decline
in the half-life of the proteasomal subunits with age. Other degradative processes,
like lysosomal (autophagic) and proteasomal degradation, as well as the activity of
cytosolic and mitochondrial proteases, are closely related to the proteasome to
maintain the continuous turnover of damaged and obsolete biomolecules and organ-
elles [57]. It has been proposed that the ageing process involves a decrease in pro-
tein degradation [58, 59], leading to the accumulation of damaged or obsolete
proteins and lipofuscin, as well as mitochondrial failure. In line with this, hundreds
of proteins with diverse functions were found in detergent-insoluble extracts from
old but not young C. elegans worms [2, 60]. Moreover, reduction of the expression
of many genes encoding proteins that become insoluble during ageing results in
extended lifespan consistent with a connection between the aggregation process and
ageing [2, 60].
Taken together, studies in C. elegans have demonstrated that protein homeostasis
collapses during ageing, leading to an accumulation of protein aggregates as well as
to significant changes in cell physiology. Some of these changes are similar to those
observed in human neurodegenerative diseases suggesting that C. elegans models
protein aggregation diseases could shed light on the mechanisms controlling ageing
and disease.
toxicity in this worm [86] and that polyQ aggregates affect protein homeostasis
[87]. Another model, where polyQ expression is directed to the muscle by the unc-
54 promoter, increases mitochondria degradation [88] and this effect is suppressed
by the co-expression of ubiquilin [89] suggesting the relevance of protein homeo-
stasis, particularly the proteasome, in mitochondria degradation.
Interestingly, several compounds that increase lifespan in C. elegans have also
been shown to decrease polyQ aggregation through the activation of different mech-
anisms. For example, trehalose (a disaccharide) and celecoxib (a non-steroidal anti-
inflammatory) both prevent polyQ toxicity and increase lifespan through a
mechanism that involves decreased insulin-like growth factor signalling activity and
increased stress resistance [90, 91]
The devastating effects of neurodegenerative diseases and ageing are well docu-
mented. The economic burden of treating disease symptoms, as well as the psycho-
social aspects of disease and ageing are a huge problem for modern societies. In
consequence, unravelling the mechanisms underlying ageing and neurodegenera-
tive diseases is imperative to delineate effective interventions and therapies to even-
tually prevent or cure neurodegenerative diseases, and improve healthspan and
longevity.
Protein aggregation is not just a hallmark of conformational diseases but also
plays a critical role in ageing. In this sense, C. elegans has proved to be an excellent
model for the study of ageing. However, regarding neurodegenerative diseases, it is
possible that C. elegans does not closely reflect the physiopathology of human neu-
rodegenerative diseases. Worm models of high prevalence NDs have been devel-
oped and have helped unravel mechanisms controlling these diseases. The use of
reverse and forward genetics on C. elegans models of disease has the potential to
uncover mechanisms of regulation of neurotoxicity that can potentially be con-
firmed in mammals and extrapolated to humans. Some results obtained using worm
models of neurodegenerative diseases await validation in mammalian systems, and
it is important to keep in mind that several of these results could be due to the par-
ticular physiology of this nematode. It is possible that new models of neurodegen-
erative diseases will be generated in the near future to broaden the research in this
kind of human diseases.
Additionally, these worm models of disease are an excellent platform to rapidly
test a number of compounds, thanks to the clear phenotypes that are associated with
protein aggregates (paralysis, loss of coordination, fluorescent aggregates, etc.).
Although any identified compounds will need validation in mammalian systems,
this approach greatly accelerates discovery of compounds with the potential to pre-
vent or even cure neurodegenerative diseases.
Despite the caveat and limitations of C. elegans as a system for modelling human
neurodegenerative disease, the results so far are encouraging, and it is highly pos-
sible that new proteins and mechanisms, as well as compounds mimicking those
processes, will be identified using worm models of disease. It is also possible that
new models of other conformational diseases will be developed in the near future,
opening new avenues for the knowledge of shared mechanisms between ageing and
disease.
Acknowledgements I would like to thank Dr. Regina Brunauer for helpful reading and discus-
sion. SA was supported by PROMEP UAM-PTC483.
12 Protein Homeostasis and Ageing in C. elegans 279
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Chapter 13
Translational Control of Longevity
13.1 Introduction
The production of proteins via mRNA translation is critical for organismal growth
and proper development. This process is energetically expensive and has been esti-
mated to consume as much as 50 % of the available energy pool [1]. Due to its
importance for survival and its metabolic cost, translation has evolved to be regu-
lated by mechanisms that are highly conserved among eukaryotes, including well-
studied invertebrate and mammalian models. Translation is regulated at three stages:
initiation, elongation, and termination. Across different species, mRNA translation
changes in response to developmental cues and environmental inputs. Periods of
growth necessitate rapid translation, while various forms of stress are accompanied
by an overall reduction in the synthesis of new proteins, as well as by differential
translation (i.e., translation that changes differently for different genes) that leads to
increased production of certain proteins important for withstanding stress and
restoring homeostasis. The relative importance of quantitative and qualitative
changes in translation has become a major focus of research into cellular adaptation
to stress and, more recently, to physiological ageing. A growing body of evidence
suggests that environmental and genetic interventions that result in negative transla-
tional regulation are associated with increased longevity. What remains unresolved
is how attenuating translation mechanistically promotes longevity, especially in
organisms with complex tissues of highly varied function, and whether these effects
are dominated by global changes in protein synthesis or by relative changes in trans-
lation of specific mRNAs in specific tissues. In this chapter, we present examples of
what model organisms have taught us about the biology underlying changes in age-
ing regulated at the level of translation. We will focus especially on the contribu-
tions made in work carried out in C. elegans, where the revolution in our
understanding of the genetics of ageing began and where many of the physiological
responses to translational modulation in a multicellular system were originally
characterized.
Fig. 13.1 (a) Stress signalling impinges on translation to promote longevity. A variety of abiotic
stresses can decrease insulin-like signalling (IIS) or target of rapamycin (TOR) signalling, which
leads to a decrease in ribosome subunit and initiation factor biogenesis. Reduction of TOR activity
also leads to reduced initiation factor activity. Stress can activate the kinases GCN2 and PERK
which act to inhibit translation through the ternary complex. These responses to stress lead to a
decrease in protein translation. (b) Chronic translation attenuation promotes longevity through the
global decrease in protein synthesis and/or through the preferential translation of specific tran-
scripts. The global decrease in translation can increase longevity due to enhanced proteostasis or
shifts in energy expenditure from growth to maintenance mechanisms. The preferential translation
of pro-longevity genes can lead to their relative up-regulation during periods of stress
translation to help fine-tune protein synthesis [11]. Limiting translation rates at this
stage makes sense if organismal survival is enhanced by mitigating energy expendi-
ture associated with this costly process. However, changes can also be observed
through stoichiometric alterations in the translation machinery, itself, including
changes in the abundance and distribution of ribosomal subunits [12]. Expression at
this level is modulated, in part, downstream of pathways controlling longevity that
help orchestrate organismal response to environmental conditions, including the tar-
get of rapamycin (TOR) and insulin/IGF signalling (IIS) pathways. The following
section elucidates the role of certain stress-sensing kinases and signalling pathways
that influence translational responses.
The TOR pathway integrates a number of cellular cues to influence protein produc-
tion and turnover (Fig. 13.1a). It is among the most well-documented longevity
pathways and the interplay between TOR, translation, and longevity has been the
subject of several exemplary reviews [23–25]. In brief, TOR is a nutrient responsive
kinase associated with a number of other subunits to form a complex that integrates
environmental cues with cellular responses [24]. TOR activity is downregulated
under nutrient restriction and other stresses. Decreased kinase activity of TOR leads
to reduced phosphorylation of two translation regulatory proteins, ribosomal sub-
unit S6 kinase (S6K/RSKS-1) and eIF4E binding protein (4EBP).1 Reduced phos-
phorylation of S6K/RSKS-1 leads to reduced biogenesis of ribosomal subunits [28].
In comparison, hypophosphorylation of 4EBP enhances its ability to bind to the
translation initiation factor eIF4E. When eIF4E is bound by 4EBP, it is prevented
from forming a complex with the methyl-guanosine cap at the beginning of the 5′
untranslated region (UTR). Loss of this complex abrogates cap-dependent transla-
tion, a key rate-limiting event in translation initiation [11].
The IIS pathway is a robust regulator of longevity [29] (see Chap. 4). Under
conditions that are not ideal for development, IIS signalling is reduced, stress resis-
tance is enhanced and longevity is extended [30] (Fig. 13.1a). The pro-longevity
effects of IIS are largely dependent on nuclear translocation of the FOXO transcrip-
tion factor DAF-16 in C. elegans [31], which increases transcription of several pro-
longevity genes with functions involved in regulating stress responses, metabolism,
lipid synthesis and peptide degradation [32]. Reduced IIS also results in reduced
S6K/RSKS-1 activity and overlaps with TOR signalling in this regard, which may
help explain an observed decrease in polysomes [2] (mRNA bound by two or more
1
4EBP as it pertains to translation inhibition has not been identified in C. elegans [10]. Another
notable caveat with implications for translation is 5′ UTR trans-splicing [26], in which native 5′
UTRs are replaced with a spliced-leader sequence 22 nucleotides in length. An in depth appraisal
of the merits and caveats of C. elegans use in translation research is found in Rhoads et al. [27].
290 J. Rollins and A. Rogers
Just because a transcript is manufactured does not guarantee it will be used to syn-
thesize the protein it encodes. A number of factors control the availability and pro-
pensity of a transcript for translation. These involve cis-regulation, which is
determined by sequence-specific characteristics of the mRNA, as well as by trans-
factors that help guide mRNA species to their fates (degradation, storage, or transla-
tion). MicroRNA (miRNA) and long noncoding RNA (lncRNA) are mechanisms of
post-transcriptional regulation that illustrate what C. elegans has helped teach us
about this ever-expanding mode of regulation and its influence on organismal
ageing.
miRNAs are a species of small non-coding RNA that regulate and fine-tune
expression of target genes post-transcriptionally [35]. Although evidence shows that
part of the way miRNAs may influence expression is by diminishing transcript sta-
bility, they may also impair translation prior to degradation of the RNA message
[36]. The existence of miRNAs was first discovered in C. elegans during character-
ization of the gene lin-4 [37]. In this pivotal work, lin-4 was determined to play a
role in developmental timing by down-regulating lin-14. Based on the complemen-
tation of lin-4 to sequences in the 3′UTR of lin-14, investigators suggested that lin-4
regulated translation of lin-14 through anti-sense RNA-RNA interactions. At the
same time, another study confirmed post-transcriptional regulation via repeated
sequences found in the lin-14 3′ UTR [38]. Another miRNA, let-7 was later discov-
ered to also regulate development in C. elegans [39], and was found to be highly
conserved, including in humans [40]. These studies paved the way for understand-
ing miRNA as a broadly applicable mechanism governing gene expression.
Since the discovery of their role in developmental timing, miRNAs have also
been shown to mediate longevity in C. elegans. In the case of lin-4, loss-of-function
reduces lifespan while overexpression extends lifespan in a DAF-16/IIS-dependent
manner [41]. The expression of miRNAs changes with age [42], and some miRNAs,
like mir-71 and mir-246 are predictors of lifespan in C. elegans [43]. miRNAs
respond to environmental cues to regulate longevity, as has been seen with mir-80
in response to dietary restriction [44]. Under periods of limited food, mir-80 is
downregulated in C. elegans and a deletion mutant of mir-80 was shown to be long-
lived. The longevity phenotype of mir-80 mutants was dependent on the activity of
the DAF-16 and the transcriptional co-factor CBP-1. These and other observations
led the authors to formulate a model where mir-80 represses cbp-1 under well-fed
conditions, which results in reduced transcriptional activity of DAF-16. However,
13 Translational Control of Longevity 291
under dietary restriction lower levels of mir-80 allowed the translation of cbp-1
mRNA, the product of which could then act as a co-factor to DAF-16 to promote
transcription of pro-longevity genes. The presence of miRNAs related to mir-80
have been observed in D. melanogaster and humans. Thus, research investigating
the role of miRNAs on longevity in humans [45–47] was inspired and informed by
research pioneered in C. elegans.
Another class of non-coding RNAs with emerging roles in translational regula-
tion and longevity are lncRNAs, which are distinguished from other non-coding
RNAs in that they are typically 200-bp or longer in length. There are several estab-
lished ways that lncRNAs can modulate to affect gene expression [48]. For exam-
ple, lncRNAs can promote stability and translation of target genes through extended
base-pairing with them or can elicit reduced translation via partial base-paring.
Additionally, lncRNA may play a role in alternative splicing by acting as ‘sponges’
for splicing factors [48]. A role for lncRNA in ageing was recently shown using
high-throughput sequencing of transcripts associated with polysomes [49]. In C.
elegans, long-lived daf-2 IIS-deficient nematodes exhibit significantly reduced
polysome activity compared to wild-type N2 worms [34, 49], which is indicative of
overall reduced translation. The lncRNA tts-1 (Transcribed Telomerase-like
Sequence) was found to be specifically enriched in mono- and polysomal fractions
of daf-2 mutants as compared to wild-type or daf-2;daf-16 double mutants [49].
When tts-1 was knocked down via RNAi, polysome levels were returned to near
wild-type levels. Additionally, upon reduction of tts-1 the longevity of daf-2 mutants
was significantly reduced. Although the precise nature of the interaction between
tts-1 and ribosomes has yet to be fully elucidated, results suggest that tts-1 nega-
tively influences translation in a manner that contributes to enhanced longevity in
this model.
Before there were studies directly linking translation and longevity, there was evi-
dence associating lifespan regulation with genetic and environmental conditions
that influence translation. As early as 1976, dietary restriction associated with car-
bohydrate or nitrogen (protein) restriction, both of which increase lifespan [50],
were observed to decrease protein synthesis in rat heart, lung, and liver tissue [51].2
The TOR pathway, which was already known to modulate translation [23], was
linked to longevity regulation in yeast [54, 55], Drosophila [56], and C. elegans [57]
by the mid-2000s. The timing of these discoveries regarding the TOR pathway came
on the heels of a major discovery in C. elegans related to genetic screening that
2
Results for acute or short-term dietary effects should not be confused with long-term studies
showing that protein synthesis is better maintained with age under dietary restriction [52, 53].
292 J. Rollins and A. Rogers
Over the last decade, the link between longevity and translation (as well as transla-
tion and age-related diseases) has grown substantially [63]. In order to understand
how downstream biological processes are affected to alter lifespan, efforts have
been applied towards determining associated changes in global and gene-specific
(i.e., differential) mRNA translation. One classic method of ascertaining global
translation rates is through pulsed metabolic labelling as performed in translation-
longevity regulation studies carried out by Hansen et al. [6] and Pan et al. [9]. Using
this method, the amount of protein synthesis is quantified by measuring the incor-
poration of radiolabeled methionine into de novo synthesized proteins [64]. This
labelling approach may be used in combination with 2D-gel electrophoresis to
quantify individual proteins [65]. More recently, stable isotope labelling by amino
acids in cell culture (SILAC) was developed using the incorporation of ‘light’ and
13 Translational Control of Longevity 293
‘heavy’ versions of amino acids into newly synthesized proteins [66]. When cou-
pled with mass-spectrometry, SILAC allows the identification and quantification of
newly translated proteins compared to those previously synthesized.
Although methods of proteomic analysis can be used to approximate transla-
tomic changes, most studies do not distinguish whether changes in specific proteins
arise from altered synthesis or from altered turnover. In addition, changes in protein
synthesis with respect to a particular gene may arise from changes in translation
efficiency of the mRNA and/or from transcript abundance. Distinguishing the
effects of transcription and translation on protein synthesis can be realized by com-
bining transcriptional analysis with polysome profiling [67] or ribosome profiling
[68] technologies. When coupled with microarray analysis or mRNA sequencing,
these profiling methods enable the global quantification of individual mRNAs that
are actively being translated. By comparing the abundance of a transcript in the
translated fraction against the abundance in the total RNA fraction, the propensity
of that transcript to be translated can be estimated.
Polysome and ribosome profiling are similar in that they both use ultracentrifu-
gation of lysed tissue over a sucrose density gradient to separate free mRNAs,
mRNAs that are bound by single ribosomes (monosomes), and mRNAs actively
translated by two or more ribosomes (polysomes) (Fig. 13.2a, b). It is at this point
that the two profiling methods diverge; ribosome profiling introduces an RNAase
step which degrades all mRNA not protected by the ribosome, leaving the “ribo-
some footprint” behind (Fig. 13.2c). In polysome profiling, translated mRNA is
isolated intact and quantified via mRNA-seq. While both methods can be used to
ascertain information about differential mRNA translation between sets of condi-
tions, each has its advantages with respect to resolving specific characteristics of
translational regulation.
Ribosome profiling ascertains the position of the ribosome within the mRNA as
the nascent peptide is elongated. Thus, this technique excels at determining changes
in elongation rate associated with codon usage [69]. Although translation elonga-
tion may not be a limiting factor in healthy organisms under optimal conditions, it
can be slowed or paused in response to stress and depletion of charged tRNAs. For
example, ribosome profiling was used to show that ribosomes accumulate near the
open reading frame (ORF) in response to proteotoxic stress in mouse and human
cells lines [70]. Similar results were obtained using heat shock as a stress [71]. In
addition to providing information about pauses in elongation, ribosome profiling
also facilitates identification of alternative upstream ORF usage [72]. For example,
translation at repressive upstream ORFs can be distinguished from translation at
productive ORFs by quantifying the ribosome footprints aligning to those sequences
[69]. Additionally, since each short sequencing read from ribosome profiling repre-
sents the binding of a single ribosome, more exact measurements of translation
(elongation) rates are achievable as compared to polysome profiling [69], which
provides relative abundances. However, due to the short read length of ribosome
footprints (28–32 bp), many reads are discarded due to ambiguous alignments lead-
ing to reduced coverage of the transcriptome [73].
294 J. Rollins and A. Rogers
Fig. 13.2 Polysome and ribosome profiling as diagnostics of translation. (a) Cell lysate is sepa-
rated over a sucrose gradient to resolve free RNA, monosome bound RNA, and RNA bound to
polysomes. (b) Sucrose gradients are fractionated based on the absorbance of RNA at 254 nm. A
representative profile of C. elegans lysates treated with control RNAi is on the left. The peaks
13 Translational Control of Longevity 295
Polysome profiling leaves mRNA intact, which means that the length of tran-
script reads are only limited by the sequencing technology. While typical read
lengths from high-throughput sequencers currently range from 50 to 1000 bps, the
upper limit of this range has steadily increased. Longer reads lengths can be used to
more reliably map and discover exon-exon junctions [74]. The ability to align reads
across exon-exon junctions is important in distinguishing transcript isoforms that
arise due to changes in alternative splicing. Therefore, polysome profiling is well
suited for isoform-specific quantification of mRNA translational efficiency [75]. In
addition, the sequencing of intact mRNA also preserves 5′ and 3′ UTR sequences,
which contain cis-regulatory elements that help determine mRNA stability and
translatability [76–78]. For example, binding of the trans-factor ELAVL1 to such
elements within 3′ UTRs increases their stability [79]. Despite their differences and
potential pitfalls, both ribosome profiling and polysome profiling provide a wealth
of information about the status of the translatome and have allowed researchers to
quantify global changes in translation as well as transcript specific changes.
Fig. 13.2 (continued) corresponding to 40S and 60S subunits, monosomes (80S), and polysomes
are labelled. A representative profile of C. elegans lysates treated with eIF4G/ifg-1 RNAi is shown
for comparison on the right. Knockdown of ifg-1 results in a decrease in active polysomes and an
increase in 40S and 60S subunits. (c) Samples corresponding to the translated (polysomal) frac-
tions are processed by one of two methods. For polysome profiling, full length mRNA is extracted
from polysomes and submitted for library preparation, which typically includes a fragmentation
step, and next-generation (next-gen) sequencing. For ribosome profiling, fractions are treated
using ribonucleases to digest mRNA not bound by ribosomes. The resulting RNA ‘footprint’ rep-
resents the position of the ribosome along the transcript. These fragments are then subjected to
library preparation and sequencing
296 J. Rollins and A. Rogers
theories are mutually exclusive and are included in the translation-based longevity
model in Fig. 13.1. Here, we talk about studies that helped form these theories,
along with new studies that are adding resolution to these paradigms.
13.3.3.1 Proteostasis
[92], although it was noted that the effect was dependent on the genetic context and
required known positive regulators of longevity. Although the precise role of altered
proteasome efficiency in mediating translational effects on longevity requires fur-
ther study, separating aspects of proteostatic mechanisms as done for this study will
help interrogate the relative contributions of altered homeostasis associated with
attenuating translation.
Yet another way proteostasis might be improved by attenuating translation in a
manner that increases lifespan is by improving the fidelity of folding that occurs on
a nascent peptide. For example, it was shown in bacteria that slowing translation
elongation leads to enhanced proper folding of eukaryotic proteins [93]. A recent
study to look for whether this phenomenon also exists in eukaryotic systems utilized
mammalian tissue culture to show that slowing (but not stopping) translation
dramatically improved the fidelity of protein folding [94]. Furthermore, slowing
translation actually improved function of mutant proteins that normally display a
high level of misfolding. Interestingly, the most dramatic improvements in protein
folding were obtained by slowing translation elongation [94], suggesting that the
specific mode of translational regulation is important for enhanced proteostasis con-
tributing to longevity.
signalling in yeast [97, 98]. This factor acts as a scaffold to bring together mRNA
and ribosomal subunits and is linked to cancer [99–102] and Parkinson’s disease
[103, 104] in humans. Previous studies showed that genetic suppression of eIF4G
increases lifespan in yeast [105] and C. elegans [6, 9, 62, 106]. In yeast, the level of
eIF4G was found to be negatively correlated with translational preference based on
mRNA length [107]. This correlation was shown to be preserved in C. elegans,
where a combination of polysome profiling and microarray analysis showed differ-
ential translation associated with eIF4G/ifg-1 knockdown that was biased towards
longer transcripts [83]. Certain translationally upregulated genes involved in main-
taining cellular homeostasis and responding to stress were required for fully
increased lifespan under this condition [83]. Another study showed that depleting
eIF4G, while diminishing overall protein synthesis, led to a widespread effect on
translational efficiency in yeast [108]. Together, results in both yeast and C. elegans
show that eIF4G differentially regulates mRNA translation, and furthermore, dif-
ferentially regulated mRNAs are functionally connected in a manner consistent
with effects on longevity.
As indicated earlier, TOR is a nutrient-responsive kinase with inputs to transla-
tion and other cellular processes that increases lifespan when inhibited [24]. One
study showed that translational reporters of the pro-longevity transcription factor
genes daf-16 and skn-1 accumulated under TOR inhibition in C. elegans in a man-
ner consistent with increased translation and were required for longevity through
this pathway [109]. Interestingly, daf-16 and skn-1 were also among the translation-
ally upregulated genes in response to suppression of eIF4G/ifg-1 [83]. This is con-
sistent with what is known about TOR in systems outside C. elegans, where it
regulates cap-mediated translation, of which eIF4G is a part [23]. In addition, TOR
regulates differential translation in yeast in a manner that was shown to be depen-
dent on eIF4G [110]. The links between eIF4G and the TOR pathway suggest that
differential translation mediated by the level of eIF4G may be a key player in lon-
gevity regulation through this translation factor.
The effect of translation on longevity may extend beyond protein abundance, trans-
lation error rates, and differential translation. As translation is an energetically
expensive process, its decrease could theoretically increase the energetic resources
for somatic maintenance (Fig. 13.1b). It is conceivable that some of this energy
could be redirected to ameliorate oxidative stress and DNA damage that may con-
tribute to the ageing process [111]. DNA repair and scavenging of free radicals are
both energy dependent processes [112, 113]. Some evidence of enhanced DNA
repair during lowered translation has been documented in C. elegans eIF4E (ife-1/2)
loss-of-function mutants and with cycloheximide treatment [114]. eIF4E acts in
physical association with eIF4G as a factor that binds to the 5′ methylated cap of
mRNA to help initiate translation. When eIF4E mutant worms were subject to
13 Translational Control of Longevity 299
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cc.25062
Chapter 14
Lipid Metabolism, Lipid Signalling
and Longevity
Abstract Ageing research gains more attention as the aged population increases
worldwide and ageing-related diseases become more prevalent. Model organism
research in the last three decades has shown that ageing is regulated via several
genetic pathways and environmental interventions, most of which are evolutionarily
conserved. C. elegans has been the powerhouse of ageing research since the discov-
ery of mutant strains with doubled lifespan. Interestingly, the pathways that regulate
C. elegans ageing often affect lipid biology as well. This chapter will focus on the
interaction between lipid biology and ageing by introducing well-known pathways
that regulate ageing and how lipid levels, composition or distribution change when
these pathways are defective. Last but not least, the signalling role for lipids in age-
ing will be discussed.
14.1 Introduction
Ageing is an inevitable part of life, and until recently, it was thought to be a passive
phenomenon that leads to decrease in organismal functions and fitness. However,
elaborate research in the last three decades has shown that ageing is a complex pro-
cess that is regulated by both intrinsic signalling pathways and extrinsic
environmental stimuli [1]. Studies using model organisms such as yeast, nematodes,
fruit flies and mice discovered that several genetic pathways and environmental
interventions, which regulate ageing, are evolutionarily conserved and hold great
promise for human ageing research.
Since the discovery that mutations in the C. elegans insulin receptor, DAF-2, can
lead to doubling of lifespan, worms have become the prominent force in ageing
research [1]. In addition to their short lifespan, C. elegans are transparent which
renders them beneficial for staining techniques and microscopy imaging. Last but
not least, the vast number of tools available for genetic manipulation as well as the
ease of performing high-throughput genetic screens has enabled researchers to find
several key players of signalling pathways regulating longevity and their detailed
epistasis analysis.
Interestingly, many of the pathways that regulate longevity affect lipid biology as
well. For example, C. elegans mutants that lack normal DAF-2 activity also have
increased lipid levels [2, 3]. However, there is no simple correlation between pro-
longevity pathways and increased lipid storage levels. eat-2 mutants, genetic mod-
els of dietary restriction in C. elegans, are long-lived, but have decreased lipid
storage [4]. Thus, the involvement of lipids in ageing is more complicated than
expected. Apart from their role in energy storage, lipids are also important signal-
ling molecules. Examples include, ceramides and certain fatty acids, which have
been shown to be important for ageing [5, 6]. More studies on the characterization
of the role of lipid biology in longevity will advance our knowledge in the biology
of ageing and improve strategies for therapeutic interventions for healthy ageing.
In this chapter, we will focus on the general concepts of lipid biology and
lifespan-regulating pathways with an emphasis on C. elegans longevity mutants and
their lipid metabolism. We will also provide an overview of lipid analysis method-
ologies. Finally, we will mention more recent research on the role of lipids as sig-
nalling molecules.
14.2 Lipids
Lipids are a diverse class of small, organic molecules that are either amphipathic or
hydrophobic [7]. Lipids are perhaps most associated with their roles as a source of
energy storage and accumulation during obesity. Other than storing energy, lipids
play major roles in forming membranes to mark the boundaries of a cell and to sepa-
rate cellular compartments. Besides their well-known structural functions, lipids are
also biologically active molecules providing communication within and between
cells [8]. These signalling roles have implications in several diseases, such as vari-
ous types of cancer and metabolic syndromes [9], as well as regulating healthy age-
ing [10].
14 Lipid Metabolism, Lipid Signalling and Longevity 309
Because lipids are such a broad grouping of molecules, they have been classified
into categories, each of which has multiple subclasses [7]. These categories are:
fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol
lipids, saccharolipids, and polyketides [7]. Within these groups, different lipids are
distinguished from each other by a variety of criteria, including their length, the
number and location of double bonds in their hydrocarbon tail(s), and the attached
structure(s), such as phosphates or glycerol. Four groups of lipids are focused in this
chapter to demonstrate the diversity between different lipid categories (Fig. 14.1).
Fatty acyls are a diverse group of lipids that include the major sub-grouping of fatty
acids, which are carboxylic acids with a hydrocarbon tail [11]. Based upon the num-
ber of double bonds in the hydrocarbon tail, fatty acids can be further divided into
saturated (no double bonds), monounsaturated (a single carbon-carbon double
bond), and polyunsaturated fatty acids (more than one carbon-carbon double bond).
The saturated fatty acids usually contain 14–22 carbon atoms. The monounsatu-
rated fatty acids are also similar length, but they have a carbon-carbon double bond,
commonly in cis-configuration, which means the hydrogen bonds next to the double
bond are positioned in the same direction. The presence of the double bond gives the
molecule a “kink” in its shape, which changes its biochemical properties.
Polyunsaturated fatty acids (PUFAs) contain several carbon-carbon double bonds,
and they are named depending on the location of the first double bond: they are
called ω-3 fatty acids if the bond is between the third and the fourth carbon after the
ω-carbon and ω-6 if the bond is between the sixth and seventh. Two PUFAs, linoleic
acid and alpha-linolenic acid are essential nutrients for mammals, but unlike mam-
mals, C. elegans express the desaturases (fat-1 and fat-2) that are necessary to syn-
thesize these PUFAs de novo [12].
Fatty acids are important energy fuels for the cell, which can be degraded via
β–oxidation to generate acetyl-CoA and subsequently used to generate ATP via the
citric acid cycle [13]. Fatty acids and their derivatives are crucial for cellular homeo-
stasis and organism fitness, and they can be utilized in both intracellular and extra-
cellular signalling. Research in the last decade showed that dietary ω-3 fatty acids
are involved in both neurotransmission and neurogenesis, and may also be impor-
tant for preventing age-related brain damage and neurodegenerative diseases, such
as Alzheimer’s disease [14]. Even though the complete mechanism is unclear, these
studies showed that ω-3 fatty acids regulate microglia and astrocyte activity, improve
mitochondrial functions, and reduce oxidative damage [14]. More recently, a spe-
cific monounsaturated fatty acid derivative, oleoylethanolamide (OEA), was shown
to be involved in longevity regulation at the organismal level. This C. elegans study
showed that OEA acts as a signalling molecule between the lysosomes and the
310
Fig. 14.1 Different classes of lipid species. Fatty acids, which are carboxylic acids with a hydrocarbon tail, are building blocks of many other lipid species.
Depending on the number of double bonds in the hydrocarbon tail, they are classified into three groups: saturated, mono-unsaturated (MUFA) and poly-unsatu-
rated (PUFA). Glycerolipids consist of a glycerol backbone attached to one-to-three fatty acids. For example, triacylglycerols have glycerol attached to three
fatty acids, which can have hydrocarbon tails of different sizes and saturation. Triacylglycerols are the major source of energy storage in cells. Glycerophospholipids
are lipids with a polar group attached to one of the positions on the glycerol backbone and fatty acids attached to the other two. For example, phosphatidylcholine
has a choline attached to the third position on the glycerol. Sphingolipids are membrane-associated lipids with a sphingoid base attached to one fatty acid. For
example, ceramides have sphingosine attached to a fatty acid
J. Duffy et al.
14 Lipid Metabolism, Lipid Signalling and Longevity 311
nucleus where it will activate specific nuclear hormone receptors to induce longev-
ity [10]. OEA is part of a larger group of fatty acid derivatives known as
N-acylethanolamines (NAEs). Another NAE, eicosapentaenoyl ethanolamide
(EPEA), has been shown to be able to modulate organism lifespan via dietary-
restriction [15]. Additionally, fatty acids have roles in extracellular signalling.
Several free fatty acids (FFA) have been shown to regulate insulin secretion in
mammalian cell lines via G-protein coupled receptor (GPCR) signalling [16]. More
specifically, palmitoleate (C16;1n7) acts as a lipokine derived from the adipose tis-
sue to improve insulin and glucose metabolic homeostasis in the muscle and liver
systematically [17].
14.2.1.2 Glycerolipids
Glycerolipids are lipids with a glycerol backbone with one to three attached fatty
acids [11]. There are mono-, di- or tri-acylglycerols depending on the number of
attached fatty acids. Each of the fatty acids in diacylglycerols (DAGs) or in triacyl-
glycerols (TAGs) can be different.
TAGs are the major intracellular source of energy storage, and they can be
degraded by lipases in the presence of the proper signals, such as a demand for
energy, resulting in the release of free fatty acids (FFAs). Homeostasis in lipid stor-
age, especially the level of TAGs, is essential for healthy ageing since obesity is
associated with age-related diseases such as cardiovascular disease, type II diabetes
and certain types of cancer [18]. However, as mentioned in the introduction, there
may not be a simple correlation between overall lipid levels and organism longevity.
Worms under dietary restriction have lower lipid storage [19] whereas insulin-
receptor deficient worms, daf-2 mutants, have more [20], but yet both worm strains
are long-lived. Therefore, future studies should investigate whether differential dis-
tribution of TAGs in certain tissues affect ageing. It is also possible that the compo-
sition and not the overall level of TAGs affect longevity.
DAGs, on the other hand, have a much more diverse set of roles. Many intracel-
lular pathways use DAGs as a second messenger by binding to a group of proteins
with a C1 domain such as protein kinase C [21] and indirectly regulate the activities
of G proteins [22]. Therefore, DAGs have important roles in processes such as pro-
liferation, apoptosis, differentiation, and cellular migration [23]. DAGs also affect
the physical aspects of membranes, such as their structure and dynamics, as well as
function in lipid metabolism by either being degraded to generate FFAs or added to
other lipids in order to generate more complex lipids [21]. DAG metabolism is also
involved in ageing. In flies and worms, knockdown of diacylglycerol lipase or over-
expression of the diacylglycerol kinase extends lifespan [24]. The same study sug-
gested that DAG metabolism interacts with TOR signalling to regulate longevity.
312 J. Duffy et al.
14.2.1.3 Glycerophospholipids
14.2.1.4 Sphingolipids
Sphingolipids are membrane-associated lipids that have a sphingoid base. These are
then built upon and modified to become more complex lipids, such as ceramides
[11]. Ceramide is important in multiple aspects of programmed cell death (PCD)
[28, 29]. It can affect intrinsic and extrinsic PCD-related signalling pathways as
well as both caspase-dependent and caspase–independent mechanisms [30].
Ceramide also plays a crucial role in other developmental processes such as differ-
entiation of the primitive ectoderm in embryos and asymmetric cell division [31].
As worms develop and age, sphingolipids naturally accumulate, and so inhibition of
the synthesis and accumulation of sphingolipids leads to a delay in the development
and ageing of C. elegans [32]. Interestingly, loss-of-function mutations in genes
encoding the ceramide-synthesis enzymes, lagr-1 and sphk-1, results in increased
autophagy and extension in lifespan [33].
14 Lipid Metabolism, Lipid Signalling and Longevity 313
Lipids can be either synthesized de novo or absorbed from the diet (Fig. 14.2). The
starting point of de novo synthesis is acetyl CoA, which will be extended into
malonyl-CoA by acetyl-CoA carboxylase and further into palmitic acid by fatty
acid synthase [34]. Then, palmitic acid, a 16 carbon saturated fatty acid, can be
further elongated by the enzymes ELO-1, ELO-2, and LET-767, and/or desaturated
by the enzymes FAT-1 to FAT-7. Desaturation is possible in C. elegans, but possible
only to a limited extent in mammals [12, 34]. These fatty acids then serve as the
base for many lipids. For example, coenzyme A-bound fatty acids (acyl-CoA) can
be combined with glycerol-3-phosphate, a phosphorylated glycerol, to generate
lysophosphatidic acid (LPA). A second acyl-CoA can be added to LPA to generate
phosphatidic acid, which can have its phosphate removed to generate DAG. A third
acyl-CoA can be incorporated to then generate TAG [35].
While de novo synthesis is extremely important, not every organism can synthe-
size every lipid. This makes the dietary intake of lipids a key aspect in maintaining
lipid homeostasis and general organismal functions. One notable example of the
importance of dietary intake of lipids in C. elegans is cholesterol, which is a key
component of membrane structures and signalling pathways [36–38]. Unlike mam-
mals, C. elegans cannot synthesize cholesterol and relies on dietary cholesterol for
its normal development and functions. Mammals, on the other hand, require the
dietary intake of two polyunsaturated fatty acids, linoleic acid and linolenic acid, in
order to synthesize more complex lipids, such as arachidonic acid [39].
Since lipids are a great source of energy, they often need to be stored for an
extended period of time. Neutral lipids, such as TAG, are predominantly stored in a
conserved organelle called a lipid droplet (LD). LDs are formed when there is a
localized accumulation of TAGs within the lipid bilayer of the endoplasmic reticu-
lum (ER), leading to the eventual budding off of a LD [40]. They are surrounded by
a phospholipid monolayer, structural proteins called perilipins that protect the LD
from cytoplasmic lipases, and other proteins involved in multiple aspects of LD
biology, including lipases for lipid degradation/mobilization [41]. Interestingly,
C. elegans lack perilipins, but still maintain a tight control over LD degradation
[42]. Active on-going researches in different laboratories are addressing the funda-
mental mechanisms underlying LD maintenance and dynamics in C. elegans.
In order to metabolize the lipids stored within LDs, two pathways are used, lipol-
ysis and lipophagy (Fig. 14.2). In lipolysis, cytoplasmic and LD-associated lipases
degrade the neutral lipids within the LDs. First, ATGL cleaves TAG to generate
DAG and a FFA. The DAG can then be degraded by hormone sensitive lipase to
generate monoacylglycerol (MAG) and another FFA. MAG can then be degraded
by MAG lipase to generate a FFA and glycerol [40]. In C. elegans, ATGL-1, which
is localized to LDs, is the lipase necessary for lipolysis [42]. In lipophagy, a branch
of autophagy, autophagosomes are used to mobilize the lipids stored within LDs. In
normal autophagy, cellular contents are engulfed by the autophagosomes that will
fuse with lysosomes, resulting in the degradation of the autolysosomal contents and
314 J. Duffy et al.
Fig. 14.2 Lipid synthesis, storage and degradation. Fatty acid de novo synthesis begins with
the extension of acetyl CoA to malonyl CoA by the acetyl CoA carboxylase (POD-2), which is the
rate-limiting step of fatty acid synthesis. Malonyl CoA is then extended by fatty acid synthetase
into a 16 carbon saturated fatty acid, palmitic acid. Palmitic acid can then be elongated by the
elongases (ELO-1, ELO-2, and LET-767) and/or desaturated by the desaturases (FAT-1 to 7).
These fatty acids can then be used to synthesize more complex lipids, such as glycerolipids, glyc-
erophospholipids and sphingolipids. These complex lipids are often degraded via specific enzymes.
Triacylglycerides, for example, are degraded via two different mechanisms: lipophagy and lipoly-
sis. In lipophagy, all or part of a lipid droplet is engulfed by an autophagosome, which then fuses
with a lysosome, resulting in the degradation of triacylglycerols and the release of free fatty acids.
In lipolysis, specific enzymes sequentially remove fatty acids from triacylglycerols. Once free fatty
acids have been generated by lipolysis or lipophagy, they are cyclically shortened by two carbons
via β-oxidation. This results in the production of acetyl CoA, which can then enter the citric acid
cycle to generate the reduced electron carrier proteins used by the electron transport chain, NADH
and FADH2
subsequent release of building blocks, such as amino acids and FFAs. In lipophagy,
LDs are targeted and either completely or partially engulfed by the autophagosomes
[43]. The LD-filled autophagosomes then fuse with lysosomes, resulting in the deg-
radation of LDs and subsequent release of FFAs, which are then further degraded by
β-oxidation.
14 Lipid Metabolism, Lipid Signalling and Longevity 315
β-oxidation occurs via the same reactions in both mitochondria and peroxi-
somes (Fig. 14.2) [44], but the identity of the enzymes used in these reactions are
different between the two organelles [45]. Accordingly, while most fatty acids can
be degraded by both organelles, certain fatty acids, such as very long chain fatty
acids, prefer peroxisomal degradation [45]. In β-oxidation, the hydrocarbon tails of
saturated FFAs are cyclically degraded by two carbons at a time to generate acetyl
CoA [44], which is utilized to generate energy via the TCA cycle and oxidized
electron carriers, which can be used in the electron transport chain (ETC).
Unsaturated fatty acids require additional enzymes, such as isomerases and
dehydrogenases, to process the double bonds before the β-oxidation pathway can
degrade the fatty acids [46].
Since lipids play such a variety of vital roles in cellular homeostasis and organismal
fitness, it is important that we have effective methods to study their storage, compo-
sition and distribution. In C. elegans, lipids are stored in the intestine, the hypoder-
mis and oocytes. The intestine of C. elegans provides the function of multiple
organs/tissues, such as digestion like the mammalian intestine, detoxification like
the liver, and fat storage like the adipose tissue [47]. Additionally, lipids are synthe-
sized in the intestine and transported to oocytes by vitellogenin proteins, where they
play major roles in oocyte and embryo development [38, 48, 49].
Biochemical assays are powerful methods to study lipid levels and composition.
These can provide knowledge about the relative amounts of different lipid species
within a sample, which can be important when examining lipid metabolism at the
molecular level. There are two methods that are commonly used to analyse lipids
biochemically, mass spectrometry (MS) [3] and nuclear magnetic resonance (NMR)
spectroscopy [50]. MS analyses the mass/charge ratio of the molecules in a sample
which have to be ionized prior to analysis [51]. Before the samples are analysed via
MS, a separation technique, such as gas chromatography, liquid chromatography or
capillary electrophoresis, is often performed. The samples are then ionized through
one of several techniques. Two of the more common techniques are electron-spray
ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). These
charged molecules will then be separated based upon their mass-to-charge ratio via
techniques such as time-of-flight or ion traps, before reaching a detector. Each lipid
molecule will give specific peaks in the MS spectrum, which can be detected and
analysed.
NMR is based upon the magnetic properties of hydrogens in the compounds,
which can be affected by the bonds and connected structures near the hydrogens.
This can help to provide physical and chemical details about the lipids analysed
[52]. As with every analysis, there are pros and cons for both NMR and MS. Even
though MS is more sensitive than NMR [51], NMR is quantitative and does not
require extra sample preparation steps. NMR is also a non-destructive technique
316 J. Duffy et al.
where the sample can be recovered and used for further analyses. MS is a destruc-
tive technique but requires a much smaller amount of sample than NMR needs [53].
Even though these techniques are useful for detecting different kinds of lipid spe-
cies at the molecular level, they lack spatial information of lipid distribution.
The cellular/tissue distribution of lipids and their transportation between cells/
tissues are very crucial for their functions. The transparent nature of C. elegans
makes it an ideal model for visualizing lipid storage with subcellular resolution at
the whole organism level. There are several stains that are commonly used to visual-
ize the lipid stores of C. elegans. Two of these, BODIPY-labelled fatty acids and
Nile Red can emit fluorescence when labelling LDs; while two other stains, Sudan
Black and Oil Red O, appear blue-black and red colour respectively when enriched
in LDs [54]. However, both fluorescent and non-fluorescent-based methods require
fixation, and are commonly associated with a higher degree of variability.
Alternative to the staining techniques, chemical imaging methods are established
in several laboratories for visualizing lipid species and different metabolites [20,
55–59]. These two, relatively new methods are coherent anti-Stokes Raman scatter-
ing (CARS) and stimulated Raman scattering (SRS) microscopy, both of which are
based upon stimulated Raman scattering. In both of these methods, chemical bonds
are stimulated with two lasers, one of which is fixed at a certain wavelength, while
the wavelength of the other can be adjusted accordingly to the vibrational frequency
of the chemical bond of interest. If the frequency difference of the laser beams
matches the vibrational frequency of the chemical bond, the molecular vibration
transitions to an excited state. As a result, anti-Stokes signals are emitted and the
beam intensities will change, which can be detected and quantified as a measure for
the level of the chemical bond of interest [60]. CARS was first demonstrated in 1982
as a viable microscopy method, but was not really used until 1999 [61]. Later, in
2008, SRS was shown to be an improvement over CARS by reducing the non-
resonant background, providing easier quantification [20], and quicker, more sensi-
tive imaging [60]. SRS microscopy can visualize lipid storage at diffraction-limited
spatial resolution and with 3D imaging capacity in living cells and organisms [62].
More recently, by administering deuterium-labelled [63] or alkyne-labelled [64] lip-
ids to C. elegans or mammalian cells and using SRS microscopy to detect the spe-
cific signals from these labels, this technique was proven to be useful also for
analysing the incorporation, synthesis and degradation of lipids.
In 1993, the discovery that loss-of-function mutations in daf-2 could double the
lifespan of C. elegans accelerated the field of ageing research. Since then, consider-
able effort has been put into elucidating the genetic pathways involved in the regula-
tion of organism ageing and longevity. These pathways often have common
components and crosstalk. For example, nhr-80 is a downstream effector for both
glp-1 and lipl-4. In this part, we are going to discuss the important longevity regulat-
ing signalling pathways in C. elegans and their effects on lipid metabolism.
14 Lipid Metabolism, Lipid Signalling and Longevity 317
daf-2 is a key player in the insulin/IGF-1 signalling (IIS) pathway and its role in
ageing is discussed in Chap. 4. It encodes the C. elegans homologue of the insulin/
IGF-1 receptor [2], which is a receptor tyrosine kinase. When bound to an activating
ligand, the DAF-2 receptor activates AGE-1, the C. elegans homologue of phos-
phoinositide 3-kinase (PI3K). AGE-1/PI3K then phosphorylates phosphatidylinosi-
tol 4,5-bisphosphate to generate phosphatidylinositol 3,4,5-trisphosphate, which
then activates a kinase cascade culminating in the phosphorylation of multiple pro-
teins, including DAF-16/FoxO [65]. DAF-16/FoxO is a transcription factor and
when phosphorylated as a result of the active IIS, it is sequestered in the cytoplasm
along with several other transcription factors [65]. When IIS is low, DAF-16/FoxO
translocates to the nucleus and promotes longevity by regulating the expression of
genes involved in biological processes including both fat metabolism and ageing
[66, 67].
There are a number of daf-2 mutant alleles that lead to lifespan extension [68]. In
addition to the longevity phenotype, mutants with the hypomorphic daf-2(e1370)
allele, have increased lipid storage as shown by Nile Red, Oil Red O staining [69]
and CARS/SRS microscopy analyses [20], and elevated de novo lipid synthesis
assayed by 13C isotope labelling strategy [3]. However, different lifespan-extending
alleles of daf-2 can have different effects on lipid synthesis and storage. For exam-
ple, the m577 and e1368 alleles showed no increase in de novo lipid synthesis or
total lipid storage [3]. This has lead to more detailed analyses of daf-2 mutants at the
transcriptional, metabolic and protein levels. The daf-2(m21) mutant was found to
downregulate the expression of several of vit/ lipid transport genes and upregulate
several fat/ fatty acid desaturase genes [70]. Proteomics analysis of the daf-2(e1370)
mutant also revealed that intermediary metabolism is reorganized, and some of
these changes might be related to increased lipid storage and longevity in this
mutant allele [71]. Furthermore, several daf-2 alleles were also subjected to metabo-
lite profiling, and amongst a variety of metabolite changes, choline metabolism was
specifically reprogrammed, possibly due to altered phospholipid metabolism [72].
to being required for eat-2 longevity, NHR-62 has been shown to play a role in other
forms of dietary restriction, such as using a diluted bacterial diet [4]. A more detailed
discussion of dietary restriction can be found in Chap. 16.
In various species, germline signals have been linked with longevity regulation
[78–80]. In C. elegans, ablation of germline precursor cells leads to more than 50 %
lifespan extension [81], and is discussed in detail in Chap. 6. Similar longevity phe-
notypes were also observed in the loss-of-function mutant of glp-1, which lacks
germline stem cells [82]. glp-1 encodes one of the two DSL-family Notch receptors,
is expressed in germline stem cells, perceives signals from their niche provided by
the distal tip cells, and is required to maintain the germline stem cell pool [82, 83].
Beside its longevity phenotype, the glp-1 mutant also displays increased lipid stor-
age, as shown by Oil Red O staining, MS analysis [54], and CARS microscopy [19].
This intestinal lipid accumulation is thought to be largely due to absence of lipid
transfer from the intestine to oocytes when germline development is arrested.
Several factors have been implicated in the regulation of the longevity conferred by
germline deficiency, including the nuclear receptors, daf-12, nhr-49 and nhr-80, the
transcription factor, daf-16, and the lipase, lipl-4.
1. daf-12 encodes a nuclear receptor that is required for the longevity conferred by
removal of germline stem cells. DAF-12 binds to the endogenous cholesterol
derivatives Δ1,7-dafachronic acid (DA), Δ7-DA and 3α-OH-Δ7-DA [50], and reg-
ulates the expression of a variety of genes involved in development and metabo-
lism [84, 85]. Gain-of-function alleles of daf-12 occur in the ligand-binding
domain of the protein, resulting in increased activity and lifespan extension [86].
On the other hand, daf-12 loss-of-function alleles are mostly in the DNA binding
domain, and show shortened lifespans. Knockdown of daf-12 results in a slight
decrease in lipid levels [87], but gain-of-function alleles of daf-12 display
increased fat content (unpublished results). DAF-12 functions with the corepres-
sor DIN-1S, which regulates lipid storage [88]. daf-12 knockdown affects the
expression of several lipid metabolic genes, such as lipl-4, lips-17, and fard-1,
and these genes are required for the longevity phenotype of the glp-1 mutant
[89].
2. nhr-80 encodes a nuclear hormone receptor that is also required for the longev-
ity phenotype of glp-1 mutants [90]. Once bound to its ligand, NHR-80 can use
other nuclear receptors, such as NHR-49 and DAF-12, as cofactors to regulate
the expression of its target genes, which include lipid metabolic genes such as
acs-2 and fat-6 [91, 92]. Loss of nhr-80 function does not affect the lifespan of
wild type animals, but completely abrogates the lifespan extension of the glp-1
mutant [90]. nhr-80 mutants have no changes in their total amount of lipids
stores, but do display changes in the relative lipid composition of their lipid
stores [91, 92].
14 Lipid Metabolism, Lipid Signalling and Longevity 319
Beside their well-known functions as energy fuels and structural building blocks,
lipids play important roles in both intracellular and extracellular signalling. While
some of these signalling functions have been thoroughly established, others are still
being discovered and elucidated. Emerging studies have revealed the significance of
signalling lipid molecules in the regulation of organism longevity, and have discov-
ered the involvement of protein chaperones, transporters and receptors in shuttling
lipid molecules between compartments, as well as recognizing and transducing
lipid signals.
[99]. Additionally, free fatty acids and their derivatives play roles in proper neuro-
transmission [100] and lifespan [10], amongst other processes.
Last but not least, sterols play an important role in biological signalling. Sterols,
which include cholesterol and its derivatives, are lipids with four carbon rings and
auxiliary components. Cholesterol is the basis for many of the hormones used in
mammals, and plays a major role in C. elegans biology. Sterols cannot be generated
de novo in C. elegans, so their dietary inputs play a key role in the evaluation of
environmental quality. A key transcription factor, SBP-1, the conserved homologue
of the mammalian sterol regulatory element binding protein, SREBP-1, controls the
expression of several of the fat/fatty acid desaturase genes, along with other fatty
acid synthesis genes [34, 101]. However, it is not known whether SBP-1 also regu-
lates sterol metabolism in C. elegans as SREBP-1 does in mammals.
There are several lipid-binding chaperones in C. elegans, and two major families are
lipid-binding proteins (LBPs) and vitellogenins. There are 9 lbp genes in C. elegans,
which have varied tissue and developmental expression patterns. Three of these,
lbp-1, lbp-2, and lbp-3, have secretory signals suggesting a role in extracellular
signalling [102]. Other LBPs play important roles in intracellular signalling. For
example, LBP-5 is involved in multiple aspects of metabolism, such as β–oxidation,
fat storage, and glycolysis [103], and LBP-8 mediates lysosome-to-nucleus com-
munication [10]. The other family of lipid-binding chaperones is the yolk proteins
encoded by 6 vitellogenin, or vit genes [104]. These yolk proteins are produced
exclusively in the intestine of hermaphrodites [105] and function to transport lipids
from the intestine to oocytes.
Both families of lipid-binding chaperones have been linked to lifespan regula-
tion. For example, LBP-8 was recently shown to shuttle OEA from the lysosome to
nucleus where OEA binds to and activates NHR-80. When lbp-8 is overexpressed,
the increased shuttling of OEA and subsequent activation of NHR-80 results in a
longer lifespan [10]. Additionally, when knocked down, vit-5 results in lifespan
extension [106], and in long-lived daf-2 mutants, all six of the vit genes are down
regulated [70].
As signalling molecules, lipids can bind to and activate G-protein coupled receptors
(GPCRs) and nuclear hormone receptors (NHRs). There are almost 2000 GPCRs in
C. elegans most of which are expressed in individual ciliated chemosensory neurons
to sense their environmental cues [107], which includes the lipid derivatives,
14 Lipid Metabolism, Lipid Signalling and Longevity 321
daumones [108]. Several GPCRs are required for the daumone-induced dauer-
formation response, such as SRBC-64/SRBC-66 [109], SRG-36/SRG-37 [110], and
DAF-37/DAF-38 [111]. One of these receptors, DAF-37, is specific for the ascaro-
side#2, but can mediate different responses to ascaroside#2 depending upon if
DAF-37 is activated in the ASK or ASI chemosensory neurons [111].
C. elegans have 284 NHRs, which are transcription factors with a DNA-binding
domain and a ligand-binding domain that is often activated by small hydrophobic
molecules such as lipids and lipid derivatives [50, 112]. Several NHRs regulate the
expression of genes important in lipid metabolism and/or longevity pathways, such
as DAF-12 in the germline regulation of lifespan [81], NHR-49 in lipid metabolism,
lipid storage, and lifespan [113], NHR-80 in germline-mediated longevity and the
associated lipid metabolism and storage changes [90], and NHR-62 in caloric
restriction-induced longevity [4]. These receptors respond to signals that are usually
generated within the organism. For example, DAF-12 is activated by three endoge-
nous derivatives of dafachronic acid (DA), Δ1,7-DA, Δ7-DA and 3α-OH-Δ7-DA
[50], and NHR-80 is activated by the endogenous fatty acid derivative OEA (10).
As we age, changes in our metabolism occur. One noticeable change is in our lipid
metabolism, especially the localization and quantity of lipid storage. These stor-
age locations respond to ageing and disease states differently. As we age, the sub-
cutaneous storage of fat tends to decrease, but visceral fat tends to remain and
increase [128]. The accumulation of visceral fat is associated with several disease
states, such as insulin resistance and cardiovascular disease [129]. In addition to
the proportion of fat stored in visceral fat in aged individuals being important for
healthfulness, it is becoming more evident that the profile of the lipids stored is
important as well. In some neurodegenerative diseases, such as Alzheimer’s, the
metabolism of certain lipid species, such as arachidonic acid, has been shown to
be altered, which may play a role in the progression of the disease [130]. In obese
people, the rate of fat breakdown is decreased. Relatedly, lipid turnover is also
decreased and inversely correlated with insulin resistance in both obese people and
people with familial combined hyperlipidemia [131]. This points to the impor-
tance not just of the composition and tissue distribution of lipids, but also how
long they are stored.
Hopefully key insights will be gained into the role of individual lipids/lipid spe-
cies in modulating ageing, the differences between subcutaneous and visceral fat
that leads to both decreased subcutaneous fat storage and increased insulin resis-
tance, the molecular mechanism behind lipid turnover’s relationship with insulin
resistance, and more. Using animal models such as C. elegans, which have multiple
lipid storage tissues, will end up being a critical aspect of this future knowledge.
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Chapter 15
Autophagy and Ageing
Malene Hansen
M. Hansen (*)
Program for Development, Aging and Regeneration, Sanford Burnham Prebys Medical
Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA, USA
e-mail: mhansen@sbpdiscovery.org
1
Nomenclature: Yeast genes/proteins are stated first, followed by the mammalian and C. elegans
names, if different.
Fig. 15.1 (continued) This occurs through a multi-step process that includes induction, membrane
nucleation, phagophore formation, autophagosome elongation, lysosome fusion, and degradation.
Numbers refer to b. (b) Autophagy is controlled by at least five protein complexes: (1) the Atg1/
ULK1/UNC-51 initiation complex; (2) the PI3-kinase nucleation complex; (3) the PI3P-binding
complex, which directs the distribution of Atg9, a transmembrane protein that appears to be impor-
tant for lipid delivery to the membrane; (4) the Atg5–Atg12 conjugation system; and (5) the Atg8/
LC3/LGG-1/2 conjugation system. In the latter system, Atg8 is cleaved by Atg4 to form Atg8-I,
which is then conjugated with phosphatidylethanolamine to form Atg8-II. This conjugate is incor-
porated into pre-autophagosomal and autophagosomal membranes. For simplicity, only the names
of the yeast gene products are given (See text for details)
15 Autophagy and Ageing 333
A B
(1) Atg1/ULK Atg1
Initiation
Complex Atg17 Atg13
(3) PI3P
Binding
Atg18
Complex Atg9 Atg2
Atg16 Atg5
(4) Atg12
Conjugation
Atg12
System
Atg5
Atg7
Atg10
Atg12
(5) Atg8/LC3
Conjugation Atg8-II
System
Atg3
Atg4
Atg7
Atg8 Atg8-I
Fig. 15.1 Overview of the macroautophagy process. (a) During macroautophagy (referred to as
autophagy), cytoplasmic material (i.e., cargo) is sequestered in double-membrane vesicles, or
autophagosomes, which subsequently fuse with acidic lysosomes where the cargo is degraded.
334 M. Hansen
As it does in many organisms, autophagy plays important roles during the develop-
ment of C. elegans [11], and accumulating evidence supports its direct role in the
ageing process [27]. The majority of evidence for the role of autophagy in ageing
has come from long-lived C. elegans mutants (Fig. 15.2 and Table 15.2). As
reviewed in the following sections, all C. elegans longevity models investigated to
date require autophagy genes for lifespan extension and often display increased
autophagy gene expression. The models include longevity paradigms with con-
served lifespan-promoting effects, such as disrupted insulin/insulin-like growth fac-
tor (IGF1) and mTOR signalling, dietary restriction, germline removal, and reduced
mitochondrial respiration, as well as pharmacological manipulations such as sper-
midine supplementation and resveratrol treatment. A number of additional long-
lived mutants have been analysed and show similar genetic links. In contrast to
observations with long-lived animals in which inhibition of autophagy genes during
adulthood abrogates lifespan extension, several labs have reported that adult-only
RNAi of autophagy genes has no or relatively little effect on the lifespan of wild-
type animals [28–31] (Table 15.1).2 Collectively, these observations suggest that
increased autophagy plays a critical role in ensuring lifespan extension in C. ele-
gans. In this section, the specific lines of genetic evidence linking ageing and
autophagy in C. elegans are reviewed, noting the relevance to other species where
applicable.
2
Paradoxically, one report has suggested that adult-only RNAi inhibition of several autophagy
genes can result in lifespan extension in C. elegans [32]; however, this study was performed on a
very small number of animals in the presence of 5-fluoro-2′deoxyuridine, and results were not
analysed by survival statistics.
15 Autophagy and Ageing 337
Fig. 15.2 Overview of genetic mutants and pharmacological interventions that modulate ageing
via autophagy in C. elegans. Experiments from C. elegans suggest that the longevity paradigms
shown here are at least partly dependent on autophagy. Specifically, all of the listed paradigms
require autophagy genes for lifespan extension in C. elegans (see also Table 15.2), and several have
been analysed using steady-state autophagy markers, which are increased in the tested long-lived
animals. *gfat-1, glutamine-fructose 6-phosphate aminotransferase (See text for details)
Reduced insulin/IGF-1 signalling (IIS) has been shown to extend the lifespan of a
number of model organisms [33], including C. elegans, where mutations in the
daf-2 insulin receptor can double the lifespan [34]. A detailed discussion of the role
of IIS in C. elegans longevity can be found in Chap. 4. Although extensive research
has been carried out over the last decade to define the downstream effector mecha-
nisms in these long-lived animals, a seminal paper connecting autophagy and insu-
lin signalling was published in 2003 by Melendez et al. [35]. The authors showed
that daf-2(e1370) mutants have altered levels of autophagy, as reflected by
increased numbers of autophagic vesicles (by electron microscopy) and GFP::LGG-
1-positive punctae, a marker for pre-autophagosomes and autophagosomes, in the
hypodermal seam cells during larval development. In addition, daf-2(e1370)
mutants subjected to whole-life ATG6/Becn1/bec-1 RNAi (i.e., mothers injected
with bec-1 dsRNA) had significantly shorter lifespans than animals injected with
control RNAi. Similar effects were later observed in daf-2(e1370) mutants sub-
jected to whole-life atg-12 or atg-7 RNAi [36] (see Table 15.2 for summary).
Taken together, these observations indicate that elevated autophagy levels are criti-
cal for the long lifespan of daf-2 mutants.
338 M. Hansen
Table 15.2 Direct links between longevity paradigms and autophagy in C. elegans
The mechanisms by which daf-2 mutants regulate autophagy are unclear, but
they could include post-translational and transcriptional regulation [20]. For exam-
ple, the catalytic subunit of the energy regulator AMPK (AAK-2 in C. elegans) is
essential for lifespan extension in daf-2(e1370) mutants [23], and it regulates
autophagy in both C. elegans and mammals [37]. It is possible that Ampk/aak-2-
regulated autophagy contributes to lifespan, since AMPK overexpression is suffi-
cient to increase longevity of Drosophila in an Atg1/Ulk1/unc-51-dependent manner
[25]. daf-2 mutants may also regulate autophagy at the transcriptional level. As
noted above, the C. elegans TFEB homologue HLH-30 translocates to the nucleus
of intestinal cells following mTOR inhibition [38], and mTOR and insulin/IGF-1
signalling are intrinsically linked [39]. Moreover, daf-2(e1370) mutation and RNAi-
induced mTor/let-363 inhibition do not extend C. elegans lifespan in an additive
manner [40], suggesting that they mediate lifespan extension through at least par-
tially overlapping mechanisms. Indeed, daf-2(e1370) mutants require hlh-30 for
their long lifespan, display nuclear-localized HLH-30, and have elevated levels of
several autophagy-related and lysosomal genes [38], supporting the possibility that
autophagy contributes to the long lifespan of these animals.
The daf-2(e1370) allele was originally reported to extend lifespan via the FOXO
transcription factor DAF-16 [34], and FOXO transcription factors are known to
regulate autophagy in other organisms [20]. Nevertheless, the role of DAF-16 in
autophagy regulation in C. elegans remains to be conclusively established. DAF-16
regulates at least one lysosomal gene (csta/C08H9.1, a cathepsin A homologue),
and short-lived daf-16(mu86); daf-2(e1370) double mutants have increased num-
bers of GFP::LGG-1 positive punctae, similar to daf-2(e1370) mutants [29].
However, since an increase in this steady-state reporter does not distinguish between
autophagy induction and inhibition, additional methods are needed to conclusively
evaluate whether daf-16(mu86); daf-2(e1370) double mutants have elevated or
reduced autophagy levels.
Since daf-2 mutants appear to induce autophagy, it will be interesting to identify
the cargo being recycled in a seemingly beneficial manner by these mutants. A
recent study suggested that mitophagy is induced in daf-2(e1370) mutants because
mitochondria accumulate upon bec-1 and mitophagy gene inhibition and daf-
2(e1370) mutants require mitophagy genes, i.e., the adaptor protein Bnip3/dct-1, the
E3 ligase Park/pdr-1 and the kinase pink-1 for full lifespan extension [16]. However,
a more recent paper reported that daf-2(e1370) mutants have decreased protein
turnover rates compared to wild-type animals, and the authors speculated that
autophagy may turn over a small and select set of targets, possibly in a tissue-
restricted fashion [41]. This idea remains to be addressed. To date, only larval hypo-
dermal seam cells have been investigated using electron microscopy and
GFP::LGG-1 marker analysis, and it will be important to analyse autophagic activ-
ity in additional tissues of adult daf-2 mutants.
15.2.5.1 p53/CEP-1
adult-only bec-1 RNAi has no significant effect on wild-type animals but signifi-
cantly shortens the long lifespan of cep-1(gk138) mutants [30]. Thus, it was pro-
posed that the lifespan-extending effects of cep-1 inhibition are mediated by
autophagy, consistent with the transcriptional regulation of autophagy by p53 in
mammalian cells [69].
15.2.5.2 SIRT1/SIR-2.1
15.2.5.3 Calcineurin/TAX-6
15.2.5.4 miR-34
Ceramide and its metabolites are complex lipids with important roles as structural
components of biological membranes and as functional regulators of cell growth
(see also Chap. 14). Multiple ceramide synthases exist; in C. elegans, these include
hyl-1, hyl-2, and lagr-2. C. elegans deficient in both hyl-1 and lagr-1 (hyl-
1(ok1766); lagr-1(gk331) double mutants) are long lived and have increased num-
bers of GFP::LGG-1 punctae in the hypodermal seam cells. Notably, both of these
phenotypes are dependent on the autophagy gene atg-12 [78]. Additionally, several
transcription factors with roles in dietary restriction-mediated longevity are impor-
tant for the lifespan extension and elevated GFP::LGG-1 punctae in hyl-1(ok1766);
lagr-1(gk331) double mutants. These animals also have reduced pharyngeal pump-
ing and reduced progeny production. Collectively, these observations suggest that
hyl-1(ok1766); lagr-1(gk331) mutants may experience a form of dietary restriction
[78]. If so, these mutants represent a link between ceramide synthase function,
autophagy, and dietary restriction.
have increased numbers of GFP::LGG-1 foci in the hypodermal seam cells and
display increased expression of the LGG-1-II isoform. In turn, gfat-1(dh468)
mutants have reduced numbers of p62/SQST-1::GFP foci, which are a substrate
for autophagy. Finally, atg-18 is required for the long lifespan observed in gfat-
1(dh468) mutants [79]. Consistent with these findings, glucosamine have been
reported to induce autophagy in human and other mammalian cells [80, 81].
15.3.1 Spermidine
15.3.2 Resveratrol
Studies conducted primarily in C. elegans have not only revealed a number of con-
served longevity pathways but also indicated that the cellular process of autophagy
may be a key common downstream effector mechanism. As reviewed here, all long-
lived C. elegans mutants investigated to date show a requirement for autophagy-
related or lysosomal genes for lifespan extension, and many of these mutants show
phenotypes consistent with autophagy induction, such as increased expression of
autophagy-related and lysosomal genes. Collectively, these data strongly indicate
that C. elegans lifespan extension is at least partly mediated by the beneficial induc-
tion of autophagy.
Additional research from other long-lived organisms supports this notion. In
mice, heterologous overexpression of Atg5 is sufficient to stimulate autophagy, pro-
mote a youthful appearance, and extend lifespan [86]. Similarly, overexpression of
Atg8/LC3/LGG-1/2 in the neurons and muscle of adult flies extends their lifespan
[87, 88], and neuron-specific overexpression of Atg1/ULK1/UNC-51 in adult
Drosophila induces autophagy both cell autonomously and non-cell autonomously
and causes lifespan extension [25]. Consistent with these observations, levels of
autophagy gene transcripts decrease with age in Drosophila brain and muscle [87,
89, 90], rat liver [91, 92], rat spinal cord [93], and mouse hypothalamus [94],
whereas lysosomal protease activity declines with age in C. elegans [95]. These
data are in keeping with multiple lines of evidence that autophagic capacity
decreases with age. For example, quantification of proteolysis of long-lived proteins
in the livers of rats indicates an age-dependent decline in autophagic function and
lysosomal degradation [96, 97]. Notably, dietary restriction has been shown to pre-
vent this decline [98, 99]. Additionally, electron microscopy of rat livers shows an
increase in autophagic vacuoles with age, and flux assays suggest that aged animals
have a decreased ability to turn over autophagic vesicles [96]. Although two recent
C. elegans studies argue instead for an age-dependent increase in autophagic activ-
ity [100, 101], both studies used steady-state experimental methods that did not
conclusively evaluate autophagic activity [11]. Further effort is clearly needed to
conclusively determine how autophagy changes during normal organismal ageing.
Such information will be critical for future efforts aimed at targeting autophagy in
higher organisms, including in humans, where many age-related diseases are asso-
ciated with autophagy dysregulation [1].
Several key questions also remain about how autophagy is regulated in healthy,
long-lived animals. Post-translational, transcriptional, and epigenetic mechanisms
of autophagy regulation have been proposed [20], and it will be interesting to deter-
mine how each of these modes of regulation are employed by different long-lived
animals. A specific post-translational candidate factor is the Hippo kinase Ssp1/
STK4/CST-1, which regulates autophagy in multiple organisms, including in C.
elegans [102]. CST-1 overexpression extends lifespan in C. elegans [103], yet it
remains unknown if this effect is autophagy dependent.
15 Autophagy and Ageing 347
Acknowledgments I wish to acknowledge Hansen lab members and Dr. Anne O’Rourke for
feedback on the manuscript, and Dr. Caroline Kumsta for help with Table 15.2. MH was supported
by NIH/NIA (R01 AG038664 and R01 AG039756) and a Julie Martin Mid-Career Award in Aging
Research supported by The Ellison Medical Foundation and AFAR.
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Chapter 16
Dietary Restriction in C. elegans
Abstract Ageing increases risk for multiple chronic diseases. Dietary restriction
(DR), reducing food intake without malnutrition, is a potent intervention that delays
ageing and onset of age-related diseases from yeast to mammals. Research using
model organisms such as C. elegans can therefore be used to elucidate mechanisms
underpinning DR that might have therapeutic potential. In this chapter, we discuss the
advantages and disadvantages of using C. elegans to study how DR modulates healthy
ageing. We provide a comprehensive summary on the different methods of DR used
to date, and the effects of DR on healthspan and models of age-related diseases. We
focus on the molecular mechanisms and physiological processes used by DR to pro-
mote longevity, highlighting advantages of using C. elegans as a model to discover
novel mechanisms that can be translated to anti-ageing interventions in humans.
16.1 Introduction
Until the twentieth century, old age was a privilege only experienced by the fortu-
nate. For the majority however, mortality rates were high, and most didn’t make it
past childhood or middle age. Remarkably, in just a hundred years we have added
25–30 years to average life expectancy of people in developed countries, with devel-
oping countries showing similar trends. This trend is set to continue such that while
in 2010 43 million people in America were 65 or older, by 2060 this number is
projected to be 103 million [1]. This striking rise in survival is overwhelmingly due
to advances of public health, leading to reductions in childhood mortality and death
from communicable diseases. However, success has come at a cost; increased
survival has uncovered age related non-communicable diseases never before seen.
In fact, patient age is the single biggest risk factor for the majority of complex dis-
eases. As a result, age-onset diseases including cancer, neurodegenerative diseases,
type II diabetes, cardiovascular disease, stroke, and osteoporosis are generating a
public health burden, which is rapidly becoming insurmountable [2, 3]. If the suc-
cess of public health in the twentieth century was bestowing us with advanced age,
its challenge in the twenty-first century is to reduce the extent to which age is a risk
factor for disease.
The best studied and most conserved intervention to promote overall healthspan
and reduce the effect of age on disease risk is dietary restriction (DR), the reduction
of food intake below ad libitum, but without malnutrition [4, 5]. First shown to slow
ageing in rats over 80 years ago [6], DR has now been shown to extend lifespan in
nearly all organisms in which it has been tested, from single celled organisms to
non-human primates [5, 7]. Along with robustly increasing longevity, DR also has
broad efficacy on reducing age-related pathologies. In the majority of murine mod-
els of chronic disease, the most effective treatment to reduce symptom severity is
simply to restrict food intake to 20–40 % less than what is consumed given free
access. DR has been shown to improve health outcomes in diseases including those
most detrimental to public health such as cancer [8], neurodegenerative diseases [9],
metabolic diseases [10] and cardiovascular diseases [11]. However, although DR
has such a profound effect on ageing and associated pathologies, its use as a thera-
peutic for humans is challenged by compliance along with negative pleiotropic side-
effects, such as hypotension, sex hormone dysregulation, bone thinning, cold sensi-
tivity and muscle loss [12]. Elucidating the molecular and genetic mechanisms
underpinning the beneficial effect of DR on ageing might therefore allow us to
harness the pro-health effects of DR without the associated detrimental side effects
16 Dietary Restriction in C. elegans 357
or the need for dietary changes. Given the pioneering use of C. elegans as a genetic
model to understand conserved mechanisms of the ageing process (discussed in
detail in this book), and recent advances in the genetic tool kit available in the
worm, nematodes represent a useful system to delineate causal effectors of DR lon-
gevity. Here we will review the pros and cons of using C. elegans as a tool to study
DR (Table 16.1), along with the current understanding of how DR protects against
age-onset diseases, and how work in the worm can lead us to new avenues to posi-
tively impact human health.
In the 80 years since the first DR studies in rats [6], the pro-longevity benefits of
reduction of food intake have been shown in over 20 organisms in laboratory studies
[5, 7], making DR the most conserved mechanism to slow ageing known to date.
However, despite this conservation, vast differences in species-specific ecology and
husbandry have resulted in ‘dietary restriction’ becoming an umbrella term that rep-
resents highly variable interventions across different organisms. Indeed, even in
murine systems used most widely to study DR, ‘DR’ can refer to a reduction in calo-
ries per day, every other day feeding/fasting or varying degrees between the two.
Whether reduction of calorie intake per se or specific nutritional components is
most critical to longevity is also an unsettled debate in invertebrates and vertebrate
studies alike, discussed in more detail below. Therefore, heterogeneity as to what
DR stands for remains as high in C. elegans as it is in rodents.
Although DR was first shown to increase lifespan in worms as far back as 1977
[13], the last 10 years have seen an explosion in the numbers of methodologies used
to apply DR in C. elegans, raising to at least 20 at the last count as more labs modify
existing protocols or add additional regimens (Table 16.2). A key benefit of having
multiple approaches to study DR in a genetically tractable system is the ability to
test causal molecular modulators of DR across many regimens. Strikingly, while
many of these methods extend lifespan, genetic epistatic analyses have begun to
unveil that different DR methods use different downstream mediators to achieve
lifespan extension. Such findings highlight that DR is not mediated by one linear
‘master’ pathway, but rather a network of interconnected pathways affected by
nutrient availability. Therefore, rather than multiple DR regimens being a negative
for the use of C. elegans as a tool to study DR, instead we are generating striking
insight into this most complex group of interventions, which will be invaluable as
we translate work in model systems toward personalized therapeutics that mimic
beneficial effects of DR on human pathology. Here we first summarize the main DR
methods in worm, along with current information as to how known longevity path-
ways interact with various DR regimens, before discussing key pathways linked to
DR in worms in more depth below.
358
5 BDR [16] Use small OP50 NGM plates to Day 2 of 20 °C Carbenicillin, 100 μg/mL for 39.1 % in males,
volumes of adulthood, then adulthood tetracycline and the first 2 weeks ~69 % in
S-basal treat with FUDR kanamycin hermaphrodites
medium with for 1 day (inferred from
gentle shaking Fig. 4 of Mair
et al. [16])
6 BDR [29] Use small OP50 NGM plates to L4 Day 2 of 20 °C Carbenicillin, 100 μg/mL for 149–268 %
volumes of stage, then treated adulthood tetracycline and the first 2 weeks
S-basal with FUDR for 2 kanamycin
medium days
7 BDR [71] Bacteria are OP50 NGM plates with Day 3 of 20 °C Ampicillin, 100 μg/mL 61–84.5 %
treated with live OP50 for 3 adulthood tetracycline
Dietary Restriction in C. elegans
haemoglobin
(continued)
360
13 eat-2 [25] eat-2 mutants Not On plates Hatch 20 °C Not specified No 25–31 %
have reduced specified
pharyngeal
pumping and
therefore
reduced food
intake
14 BD [32] Adult worms OP50 NGM plates with Day 2 of 20 No 50 uM 50 %
are transferred UV-killed OP50 adulthood
to bacteria-free
NGM plates for
the rest of the
Dietary Restriction in C. elegans
lifespan
15 BD [33] Adult worms OP50 Fed OP50 at 25 Day 1 of 25 No Concentration 43 %
are transferred °C until L4 adulthood not clear
to bacteria-free
plates for the
rest of the
lifespan
16 IF [36] Adult worms OP50 or Fed live OP50 Day 2 of 20 Not specified 200 ug/mL 57 %
are shifted HT115 during adulthood
between fed development
and fasting
every 2 days
(continued)
361
Table 16.2 (continued)
362
DR Bacteria Time of
method Method used in Conditions during DR Percentage
category Reference description DR development initiation Temperature Antibiotics Used FUDR? lifespan extension
17 Peptone [23] Peptone is OP50 Fed on NGM Hatch 16 Not specified No 33 %
dilution omitted from plates
NGM to
prevent
bacteria growth
18 sDR [38] Serially diluted OP50-1 Fed on NGM Day 4 of 20 Not specified No 29 %
UV-killed plates adulthood
bacteria are
placed on
NGM plates
19 sDR [95] Serially diluted OP50 Fed on NGM Day 1 of 25 Carbenicillin 5 mg/mL 74 %
bacteria are plates adulthood
placed on
NGM plates;
peptone is
omitted from
the media
20 sDR [40] Serially diluted OP50 Fed on NGM Day 1 of 20 Carbenicillin and 100 ug/mL 77 %
bacteria are plates adulthood kanamycin
placed on
NGM plates
and then
antibiotics are
added to
prevent
bacteria growth
BDR bacterial dilution in liquid, ADR axenic media, BD bacterial food deprivation, IF intermittent fasting, sDR bacterial dilution on solid plate
Y. Zhang and W.B. Mair
16 Dietary Restriction in C. elegans 363
16.2.1 Liquid DR
Since standard C. elegans husbandry uses E. coli as food source, most DR assays
involve reducing availability of bacteria. The earliest DR studies used worms grown
in liquid culture with different bacterial concentrations, known as ‘bacterial dietary
restriction’ (BDR). Decreasing food concentration increases lifespan and reduces
fecundity across various dilutions [13]. The decrease in fecundity as lifespan
increases is a key signature of fitness tradeoffs in DR. This trade-off can be used to
distinguish a DR regimen from one which simply dilutes some toxicity in culturing
conditions, thus increasing both lifespan and reproduction as the toxicity is reduced
(Fig. 16.1). Not long after the establishment of BDR, the insulin/IGF-like signalling
(IIS) pathway was discovered to be a potent modulator of lifespan in C. elegans
[14]. Given that insulin signalling is a conserved nutrient-sensing mechanism, it
was hypothesized that DR extended lifespan via reduced IIS (rIIS). This idea proved
to be oversimplified however, since even the extremely long-lived mutants of the
daf-2 gene, which encodes the insulin receptor in C. elegans, respond robustly to
BDR [15]. Further, BDR is able to increase lifespan in worms lacking the FOXO
transcription factor, DAF-16, while such worms are completely refractory to rIIS
longevity [15]. This opens the question as to whether any ‘master regulator’ of DR
exists: A factor can be defined as a putative ‘master regulator’ of DR, when its
absence completely suppresses the ability of DR to increase lifespan, as opposed to
an intervention that mimics DR by increasing lifespan in a food dependent manner
(Fig. 16.1). Given the graded response of lifespan across different levels of food
restriction (Fig. 16.1), a true master regulator can only be defined if it blocks all
lifespan extension across multiple grades of DR (Fig. 16.1) [16]. The first factors
shown to block DR across a range of DR levels in any organism were identified in
C. elegans, using serial dilutions of liquid BDR. One was PHA-4, a homologue of
the FOXA family of forkhead transcription factors [17]. Loss of PHA-4 activity
completely blocks lifespan extension by BDR across different bacterial dilutions.
Interestingly, PHA-4 and DAF-16 regulate genes with overlapping functions, sug-
gesting DR and rIIS regulate overlapping target pathways to achieve longevity [17].
In the same issue of Nature, a second transcription factor that also mediates BDR
was reported: SKN-1, the homologue of the NF-E2-related factors (Nrfs) [18].
Mutants of skn-1 show no lifespan increase when subjected to a variant of BDR that
houses worms in six well plates containing solid standard nematode growth media
(NGM) below variable dilutions of liquid bacteria [18]. Moreover, the function of
SKN-1 in mediating DR longevity was narrowed down to the chemosensory ASI
neurons [18]. This study was the first report that lifespan extension via DR can be
regulated cell non-autonomously, and that lifespan can be regulated by only two
neurons.
Another type of liquid DR uses semi-defined, bacteria-free axenic media (ADR).
One typical such medium contains soy-peptone, yeast extract and haemoglobin
[19]. Similar to BDR, worms grown under ADR conditions show significantly
delayed development and reduced fecundity [19]. Worms grown in ADR media in
364 Y. Zhang and W.B. Mair
liquid live up to twofold longer than controls [19]. ADR can also be used in place of
NGM in agar plates (solid ADR). Worms kept on solid ADR plates without bacteria
show lifespan extension compared to bacteria-feeding controls [20]. Genetic analy-
sis found that liquid ADR does not require SKN-1 to extend lifespan, but the SKN-1
target gene cup-4 and the CREB-binding protein cbp-1 are required for the full
longevity of ADR animals [21, 22]. However, despite ADR increasing lifespan, the
caloric content of this media is very high, suggesting that lifespan extension occurs
via reduction to a nutritional component of E. coli not in ADR, or non DR factors
such as lack of microbe/ host interaction or liquid husbandry.
Because of technical challenges of BDR/ADR, and that swimming in liquid cul-
ture is a potential stress for worms, researchers have tried many ways to limit food
using the standard agar plate-based husbandry methods. One such method uses
diluted concentrations of bactopeptone in agar plates to limit bacterial growth [23].
Reduced peptone levels in plates leads to increased lifespan. However, these effects
are complicated by the fact that peptone is toxic to the worms [23]. Since reproduc-
tion increases as peptone levels are reduced, diluting peptone may not only be limit-
ing bacterial availability, but also reducing peptone toxicity (Fig. 16.1).
Reproduction
Ad libitum
Food Intake
Fig. 16.1 Effects of dietary restriction (DR) on lifespan and reproduction. Black line: median
lifespan of wild type animals under different levels of food intake. As food intake decreases from
high levels (ad libitum) to lower levels (DR), lifespan increases. When food intake continues to
decrease into malnutrition range, lifespan begins to decrease. Orange line: median lifespan of a
mutant lacking a putative master regulator of DR under different food intake levels. Such mutants
should not show significantly different lifespan between ad libitum and DR. Green line: median
lifespan of animals with mutations/drugs that mimic dietary restriction. The curve is shifted to the
right such that at ad libitum levels, these animals should have increased lifespan compared to wild
type animals, mimicking the effects of DR without actually reducing food intake. Reproduction
(dashed line): reproduction keeps decreasing as food intake lowers. A key feature of DR is lowered
reproduction compared to ad libitum, representing a tradeoff instead of simply reducing general
toxicity from high food intake
16 Dietary Restriction in C. elegans 365
A widely-used agar-based method uses ‘eat’ mutants that show defects in the phar-
ynx, which lead to slower pumping rate and reduced food intake [24]. Mutants for
many eat genes live 10–30 % longer than wild type [25]. eat-2, which encodes a
ligand-gated ion channel required for normal pharyngeal muscle function, gives the
most robust lifespan extension when mutated and is the most commonly used
genetic mimic of DR [25]. Supporting that eat animals live longer due to reduced
food intake and not pleiotropic effects from the mutations, eat-2 mutants do not live
longer when subjected to BDR [16]. Furthermore, feeding animals with a different
bacteria, Comamonas sp., which are smaller than E. coli such that the ingestion
defects of eat-2 animals is negated, abolishes eat-2 lifespan extension [26]. Similar
to BDR, the longer lifespan of eat-2 mutants is independent of daf-2 and daf-16, and
BDR fully requires the ubiquinone biosynthesis enzyme clk-1 [25]. Despite the
caveat that the degree of food restriction is fixed to the levels caused by the eat-2
mutation and cannot be manipulated, eat-2 animals are a useful DR model, espe-
cially as they are easily combined with RNAi by bacteria feeding. This convenient
DR method has been used to identify important factors in DR longevity, including
the FOXA family transcription factor PHA-4 [17], the autophagy machinery [27,
28] and the nuclear hormone receptor NHR-62 [29]. Indeed, whole genome reverse
genetic RNAi screens have been performed for genes whose knockdown specifi-
cally blocks or modulates eat-2 longevity [30].
Fasting provides benefits against many chronic diseases in rodents and humans
[31]. C. elegans can survive when bacterial food source is permanently removed
during adulthood. Chronic bacterial deprivation (BD) extends lifespan by 50 % and
increases resistance to heat, oxidative agents and proteotoxic stressors [32, 33].
Starvation has different effects when initiated at different points of reproduction.
When initiated as L4s, BD worms arrest and only show a modest increase in lifes-
pan [32]. Interestingly, when starved as L4s in a crowded environment, a subpopula-
tion of worms arrest in an adult reproductive diapause (ARD) for up to 30 days.
These arrested adults remain youthful during starvation. As soon as feeding is
resumed, these animals reset their longevity, adding a regular adult lifespan to the
time spent in diapause, resulting in a total longevity up to threefold more than non-
starved animals [34]. While BD started at the beginning of reproduction shortens
lifespan, it extends lifespan at various time points after the second day of adulthood,
even when initiated after the reproductive period or very late in life [32, 33]. The
longevity benefits of BD are independent of the daf-2/daf-16/insulin signalling
pathway [32], but require the heat shock transcription factor HSF-1 [35].
366 Y. Zhang and W.B. Mair
Several methods of DR have been developed using diluted E. coli on solid agar
plates (sDR). When the amount of bacteria seeded on agar plates is reduced starting
from day 4 of adulthood, worms eat less and live longer [38]. sDR requires DAF-16
and AMP-activated protein kinase (AMPK) to extend lifespan [38], which are dis-
pensable in many DR methods [39]. At the same time, key factors in other DR
methods such as PHA-4, SKN-1 and HSF-1 are not required by sDR [39].
Furthermore, a similar method that initiates DR in adulthood, at day 1, shows partial
dependency on DAF-16, but fully requires decreased levels of DRR-2, a homologue
of human eukaryotic translation initiation factor 4H (eIF4H) [40].
Early studies using mammals focused on the effects of total calories, since DR was
often carried out by limiting the amount of food available to a fraction of what’s
eaten by the ad libitum group without changing the nutrient composition. For that
reason, DR was often referred to as caloric restriction (CR), especially in mamma-
lian studies. In the last 10 years, the effect of restricting specific nutrients during DR
has been re-examined. Studies in flies and rodents showed that iso-caloric modula-
tions of protein (even specific amino acids), carbohydrates and lipids confer differ-
ent responses to health and lifespan [41–43]. Further, the ratio of nutrient components
is as critical as total amount of any one component, with a low protein:carbohydrate
ratio seemingly giving the strongest effects on lifespan in flies and mice [44, 45].
That nutrient composition plays a significant role independent of calories might
explain some seemingly conflicting results when DR does not have consistent
effects on lifespan [46, 47]. Avoiding such problems requires full control over
dietary composition, ideally with food sources made entirely from chemically
defined components. Although some attempts at defined diets have been made in C.
16 Dietary Restriction in C. elegans 367
elegans, these diets often contain some semi-defined components such as milk pow-
der [48]. Early attempts to develop a fully defined ‘C. elegans Maintenance Medium’
(CeMM) are now rarely used [49]. Much investment in CeMM was made by NASA
as part of its testing of the effects of space travel on physiology, which was termi-
nated by the tragic atmospheric breakup of the Space Shuttle Columbia (that C.
elegans in CeMM survived [50]). To fully utilize the strengths of C. elegans genet-
ics to dissect out the effects of specific nutrients and uncouple DR effects from the
‘two organism problem’ [51], a return to studies using CeMM or a similar fully
defined medium, as has recently been achieved in Drosophila [52], would be
warranted.
Emerging studies suggest that fasting and other DR methods reduce age-related
diseases and even decreases mortality rate in humans [31, 53]. DR studies using C.
elegans have been very useful in the identification of molecular pathways that are
potent regulators of ageing. It has become clear from C. elegans research that
instead of one linear “DR pathway”, multiple nutrient-sensing pathways form an
interconnected network that promotes healthy ageing during DR. Alternate DR
paradigms utilize this network and nodes within it differentially to initiate the pro-
longevity transcriptional and physiological response to DR. Furthermore, C. ele-
gans with alternate genetic backgrounds can respond differently to DR. These
varied effects of DR on health are also seen in mice of different genders and genetic
backgrounds [54]. In the new era of personalized medicine, such differential
responses to DR suggest diet might be “personalized” for a specific genome to
maximize beneficial effects. C. elegans will therefore be a key model to test the
interaction between diet and genetics, as we push towards translating DR research
for human health benefits.
The IIS pathway was the first genetic pathway identified to modulate lifespan in any
species. For a more extensive discussion on additional identified mediators and tar-
gets of IIS see Chap. 4. For the purposes of this chapter we will focus on the role of
IIS in DR. Mutations in daf-2 [55] and age-1 (a catalytic subunit of PI3K) [56]
dramatically increase lifespan. Longevity by reduced IIS (rIIS) completely requires
DAF-16 [55]. When IIS is active, DAF-16 is phosphorylated by Akt and sequestered
368 Y. Zhang and W.B. Mair
TSC2
TSC1
RAGA-1
DAF-2
RHEB-1 RAGC-1
TORC2
AGE-1
TORC1
4EBP
SGK-1
Protein translation
Mitochondrial
Fat metabolism Stress resistance microRNAs Autophagy
respiration
In vitro regulation in mammalian cells Promotes longevity Promotes or decreases longevity in different contexts
In vivo genetic/biochemical
interactions Decreases longevity Homolog not found in C. elegans
Fig. 16.2 Genetic and physiological pathways that mediate the benefits of dietary restriction in C.
elegans. The TSC complex and 4EBP are not found in C. elegans, but have been shown to modu-
late lifespan in D. melanogaster. Green boxes: inhibition blocks the lifespan extension of DR, or
activation extends lifespan. Blue boxes: inhibition extends lifespan, or activation blocks lifespan
extension by DR or mutants that mimics DR. White boxes: modulation can lead to longer or shorter
lifespan under different conditions. Solid line: interaction verified by genetic epistasis. Dashed
line: AMPK’s role as an upstream inhibitor of the TORC1 pathway has not been verified in
C. elegans
in the cytoplasm by binding to 14-3-3 proteins. During rIIS, AKT activity is reduced,
allowing DAF-16 to translocate to the nucleus and activate target gene expression
[57, 58]. Although the corresponding phosphatases for AKT and FOXO is still
under investigation [59], calcineurin has been suggested to directly dephosphorylate
DAF-16 and coordinate with it to modulate lifespan [60].
Although IIS has a key function in nutrient sensing, it is not universally required
for all DR methods to extend lifespan. Dietary restriction by BDR [17], ADR [15],
eat-2 [25] and BD [32] all extend the lifespan of daf-2 hypomorphic mutants and
daf-16 null mutants. However, DAF-16 is required for sDR [38]. Interestingly, rIIS
interacts with a high sugar diet: addition of glucose into NGM shortens lifespan in
a daf-16-dependent manner and suppresses the long lifespan of daf-2 mutants [61].
Localization and activity DAF-16 are subjected to many levels of regulation,
which remains an important area of study. Recently, several key factors involved in
16 Dietary Restriction in C. elegans 369
its regulation have been identified, including the putative transcriptional cofactor
SMK-1 [62], the chromatin remodeller SWI/SNF [63] and the RNA helicase HEL-1
[64]. In addition to phosphorylation by AKT, DAF-16 is subjected to multiple post-
translational modifications, with its modifiers all impacting longevity, including the
deubiquitylase MATH-33 [65], AMPK [38], Ca2+/calmodulin-dependent kinase
type II (CaMKKII)/calcineurin [60] and the sirtuin homologue SIR-2.1 [66]. Much
effort has also been invested into finding pro-longevity targets of DAF-16 [58 and
others, reviewed by 67].
16.3.2 Sirtuins
16.3.3 AMPK/CRTCs
AMPK is a nutrient-sensing kinase that is activated when energy levels are low [76].
To promote ATP production and counterbalance energy stress, AMPK inhibits bio-
synthetic processes and stimulates catabolic processes, such as glucose uptake, oxi-
dative phosphorylation and autophagy [reviewed by 77]. The important role of
AMPK in maintaining energy homeostasis, as well as the widely available pharma-
cological agents that activate it [77], makes AMPK a promising target of DR to
study. Indeed, AMPK is required for some forms of DR: null mutations in aak-2,
which encodes a catalytic subunit of AMPK, blocks lifespan extension by sDR [38]
and significantly dampens the response to one form of BDR [71]. AAK-2 is not
required for longevity by several other protocols of BDR [16, 39], ADR [22], eat-2
[39], or IF [36].
Intriguingly, direct AMPK activation mimics the effects of DR and increases
lifespan whether it is achieved by overexpressing wild type AAK-2 [78], an active
form of AAK-2 [79], or an active form of a regulatory subunit of AMPK [38]. Given
that AMPK is a master regulator of metabolism and has numerous direct and indi-
rect targets [76], it is critical to identify the specific downstream processes it modu-
lates to impact ageing. Greer et al. [38] showed that DAF-16 is activated by AMPK
and required for the lifespan extension by AMPK activation. The same study also
identified AMPK phosphorylation sites on DAF-16, although the effects of these
sites on DAF-16 activity remain to be tested [38]. DAF-16 also acts in a feedback
loop to increase AMPK activity by increasing the expression of a regulatory subunit
[80]. Similar to FOXO/DAF-16, CREB-regulated transcriptional coactivators
(CRTCs) are also key regulators of metabolism in mammals [81]. Mair et al. [79]
identified a single CRTC orthologue in C. elegans, ‘CRTC-1’. CRTC-1 is directly
phosphorylated by AMPK, which inhibits CRTC-1 activity by promoting its nuclear
exclusion [79]. Mutations in these phosphorylation sites block the effects on
CRTC-1 nuclear exclusion and lifespan extension by AAK-2 overexpression [79].
Further, Burkewitz et al. [82] found the effect of AMPK in ageing is cell non-
autonomous and specific to its inhibitory effect on CRTC-1 function in neurons.
This study also showed that AMPK requires the nuclear hormone receptor NHR-49
to extend lifespan [82]. Another key target of AMPK is the TOR complex 1 (TORC1)
pathway, a master regulator of cellular metabolism with antagonistic effects to
AMPK [77]. Since direct TORC1 inhibition is sufficient to extend lifespan (dis-
cussed in Sect. 16.3.4 below), it remains unclear whether the pro-longevity effects
of AMPK activation is largely mediated by the resulting reduction in TORC1 activ-
ity. Interestingly, genetic studies using C. elegans show that AMPK is required for
longevity by TORC1 suppression [83, 84]. Therefore, more work is needed to delin-
16 Dietary Restriction in C. elegans 371
eate the relationship between AMPK and TORC1 in ageing and unravel the down-
stream factors of AMPK that contribute to its role in longevity.
16.3.4 TOR
The TOR kinase can be recruited into two different complexes: TORC1 and TOR
complex 2 (TORC2). TORC1 is activated by high levels of nutrients to regulate a
broad range of metabolic processes [85]. Specifically, TORC1 responds to changes
in amino acid levels and growth hormones, making it an ideal candidate as a media-
tor for DR benefits. Although the precise mechanisms that regulate TORC1 activity
are still under active investigation, the core components of TORC1 signalling have
been identified: high amino acid levels activate the Rag family of small GTPases,
which recruit TORC1 to the lysosomal surface; growth factor stimulation sup-
presses the TSC complex to release activity of another small GTPase, Rheb, to
directly activate TORC1 at the lysosome [86]. While the majority of the new and
traditional TORC1 components are conserved in C. elegans, the TSC complex
seems to be absent. However, Ral GAPs, another family similar to TSCs, which are
present in C. elegans, have been found to regulate TORC1 through Rheb [87].
Mechanisms regulating TORC2 have been less well studied. Nevertheless, reduced
TORC2 activity increases lifespan [88], although the effects of TORC2 on ageing
and metabolism are variable and depend on the bacteria food source and tempera-
ture [89, 90].
TORC1 is involved in many types of dietary restriction. Due to limited
phosphorylation-specific antibodies to TORC1 targets in C. elegans, evidence is
scarce on the effects of different DR methods on TORC1 activity. However, genetic
epistatic analyses show that the capacity to change TORC1 signalling is required for
lifespan extension by eat-2 [70], sDR [91] and IF [36]. More intriguingly, genetic
and pharmacological TORC1 inhibition mimics the effects of DR on lifespan exten-
sion and age-related diseases [92, 93]. The downstream mechanisms modulated by
TORC1 to regulate lifespan include SKN-1 and DAF-16 [88], PHA-4 [94], HIF-1
[95], HSF-1 [96], protein translation [70] and autophagy [28, 97]. Interestingly,
mutants of rsks-1/S6 kinase, which is directly phosphorylated by TORC1 to increase
protein translation, require AMPK for lifespan extension [83, 84]. Furthermore, it
has been shown that the arginine kinase ARGK-1, which is homologous to mam-
malian creatine kinases, is upregulated in rsks-1 mutants to activate AMPK [98].
Since ARGK-1 is expressed predominantly in glial cells [98], it is possible that
TORC1 modulates lifespan via neuronal mechanisms. Given the pivotal role of
TORC1 as a master regulator of multiple processes including metabolism, gene
expression, and proteostasis, more studies are needed to identify the tissue-
specificity and downstream mechanisms that are specific for its effects on ageing,
rather than other pleotropic effects.
372 Y. Zhang and W.B. Mair
16.3.5 PHA-4/FOXA
PHA-4 was first discovered for its role in development of the pharynx and the intes-
tine [99]. Since DAF-16 is not required for DR to extend lifespan, Panowski et al.
[17] performed a targeted RNAi screen of forkhead transcription factors and found
that PHA-4 is required for the lifespan extension by BDR and eat-2. PHA-4 specifi-
cally responds to DR but not reduced insulin signalling, although its targets overlap
with DAF-16 [17].
One mechanism regulated by PHA-4 is autophagy, a process that degrades mac-
romolecules and organelles, and provides energy when nutrient levels are low [100].
Since DR creates an environment with limited resources, autophagy serves to recy-
cle materials for synthesis of key molecules for survival. Besides many direct phos-
phorylation events that can activate autophagy [100], PHA-4 is the first identified
transcription factor that is required for autophagy activation under nutrient stress
[28]. Moreover, PHA-4 is required for TORC1, a potent regulator of autophagy, to
regulate lifespan [94]. Interestingly, deletion of S6K, a branch downstream of
TORC1 that is well-known for its role in modulating protein translation, also
requires PHA-4 to extend lifespan [94]. Recently, PHA-4 was shown to act in a
feedback loop with two microRNAs, miR-71 and miR-228, which together regulate
DR lifespan [101]. To further understand the role of PHA-4 in ageing, more efforts
are needed to identify PHA-4 target genes in low energy conditions, especially in
ageing animals, and the upstream mechanisms that regulate PHA-4 activity and
specificity.
16.3.6 SKN-1/Nrf
SKN-1 is a bZIP transcription factor that has broad functions in embryonic develop-
ment, stress resistance, metabolism and ageing [reviewed by 102]. A critical role for
SKN-1 in ageing was first discovered by the finding that mutations in the skn-1 gene
block lifespan extension by BDR [18]. skn-1 is mainly expressed in two distinct
tissues: intestine (the major metabolic tissue in C. elegans) and ASI neurons (sen-
sory neurons that transmit nutrient signals to regulate physiology). Specifically, DR
directly activates SKN-1 in ASI neurons; rescuing SKN-1 activity specifically in
ASI neurons but not in the intestine is sufficient to restore lifespan extension and
increased respiration upon DR [18]. Further studies showed that SKN-1 is also
required for longevity by BD [32] and a form of BDR [71].
SKN-1 responds to many types of stress and nutrient signals to regulate ageing,
including rIIS [103], suppression of TORC1/TORC2 [88, 90], inhibited protein
translation [104] and several ageing-related microRNAs [101]. Targets of SKN-1
are largely different from DAF-16, including many classic phase 2 detoxification
genes [103]. Studies using gain-of-function alleles further showed that SKN-1 acti-
vation leads to a gene expression profile that is largely reminiscent of starvation
16 Dietary Restriction in C. elegans 373
[105], activating genes that function to mobilize energy stores and specifically fatty
acid oxidation [106]. Furthermore, Ewald et al. [107] delineated the role of SKN-1
in daf-2 mutants by showing that SKN-1 is specifically required for longevity under
conditions that do not induce dauer-related mechanisms. Under such conditions,
SKN-1 robustly promotes expression of collagens, which are also required for lifes-
pan extension by various longevity models besides daf-2, including eat-2 [107].
16.3.7 HSF-1
When cells are under stress conditions that induce a large amount of damaged or
misfolded proteins (such as elevated temperature), the heat shock response increases
expression of molecular chaperones to help refold proteins and prevent aggregation.
HSF-1 is a conserved master regulator that orchestrates this protective mechanism
[108]. In C. elegans, hsf-1 overexpression is sufficient to extend lifespan [109]. Loss
of HSF-1 completely blocks the long lifespan of IIS mutants [109]. HSF-1 is acti-
vated by reduced insulin signalling to induce expression of heat shock proteins,
which contribute to longevity [109, 110]. HSF-1 is also required by reduced TORC1
[96] and BD [35] to extend lifespan and reduce protein aggregation.
The role of HSF-1 as a cell non-autonomous regulator of ageing has also been
reported. Overexpressing hsf-1 specifically in neurons, muscle and intestine are all
sufficient to increase longevity [111]. Neuronal HSF-1 activates expression of heat
shock proteins in peripheral tissues via DAF-16 [112]. A recent study also found
that activating an HSF-1 variant in neurons promotes longevity independently of
chaperones, by increasing the integrity of muscle actin cytoskeleton [113].
16.3.8 HIF-1
significantly prolongs lifespan [95, 116], which requires the unfolded protein
response (UPR) mediator IRE-1 [95]. Stabilizing HIF-1 at 25 °C via deletion of the
egl-9 gene, which encodes a PHD protein, suppresses lifespan extension by eat-2,
sDR, and deletion of rsks-1 [95]. At both 15 and 20 °C, however, animals with a
hif-1 loss-of-function allele do not live longer than wild type and show significant
defects in vulva integrity [116]. At 20 °C, mutants with null alleles of hif-1 and/or
vhl-1, which encodes a VHL protein, did not block the lifespan extension by BD;
hif-1 RNAi also failed to block eat-2 lifespan extension [115]. These results suggest
that when temperature is different, the same DR method can be mediated by differ-
ent mechanisms.
Strikingly, increasing HIF-1 levels also extends lifespan. RNAi of the upstream
HIF-1 inhibitors VHL-1 or EGL-9 at 20 °C extends lifespan [115]. HIF-1 is also
activated in long-lived mutants with reduced mitochondrial respiration [117]. An
intriguing recent study showed that neuronal-specific HIF-1 stabilization is suffi-
cient to extend lifespan [118]. Further, Leiser et al. [118] showed that neuronal
HIF-1 cell non-autonomously increases expression of an intestinal flavin-containing
monooxygenase gene, fmo-2, and loss-of function mutation in fmo-2 blocks the
increase in lifespan by sDR. Further, fmo-2 overexpression is able to fully recapitu-
late the lifespan extension from HIF-1 activation or dietary restriction [118].
16.3.9 NHRs
Nuclear hormone receptors (NHRs or NRs) are a family of transcription factors that
respond to lipophilic hormones. The human genome encodes more than 48 NRs
with diverse ligand-specificity and target genes [119]. Mammalian NRs are required
for a broad spectrum of key functions. Specifically, several metabolic NRs (for
example, peroxisome proliferator-activated receptors/PPARs) play important roles
in metabolism and age-related diseases [120].
The responsive nature of NRs to lipid metabolites and their function in regulating
metabolism and stress resistance make them ideal candidates to mediate physiologi-
cal effects of dietary restriction. Evidence exists in mammals that suggests a role for
NRs in DR and ageing: first, PPAR agonists cause CR-like transcriptional changes
[121]; second, genetic inhibition of PPARs via activation of the corepressor SMRT
causes premature ageing and metabolic diseases [122].
Recent work in C. elegans, in which the function of NHRs is conserved, evalu-
ated whether NRs play a causal role in DR and ageing. Studies show that DAF-12,
which is activated by insulin and TGF-β signalling, modulates ageing [123].
Furthermore, Heestand et al. [29] used RNAi to screen 246 of the 284 NR genes for
those required for eat-2 lifespan. This screen identified that RNAi of one NR, nhr-
62, fully blocks lifespan extension of eat-2 but has no deleterious effects on control
animals [29]. Metabolite profiling and RNA-seq confirm a role for NHR-62 in lipid
metabolism and autophagy [29]. Interestingly, nhr-62 mutation does not block lon-
16 Dietary Restriction in C. elegans 375
gevity from rIIS or reduced mitochondrial respiration, nor does it fully suppress
BDR, suggesting that specific NRs are required by different methods of lowered
energy levels [29]. Indeed, under starvation conditions, NHR-49 is activated to pro-
mote fat mobilization and produce energy [124]. NHR-49 is required in neurons for
global AMPK activation to extend lifespan and maintain youthful peripheral mito-
chondria morphology [82]. Evidence so far suggests that NRs are indeed mediators
of organismal response to low energy, and that different NRs specifically respond to
select upstream signals to modulate lifespan.
16.3.10 microRNAs
microRNAs are small, non-coding RNAs that are conserved regulators of post-
transcriptional gene expression. The genes targeted by microRNAs belong to a very
broad spectrum of processes, including development, metabolism and cell death
[125]. Recently, a vast number of studies using microarray and next-generation
sequencing generated data showing that expression of microRNAs change with age
in many tissues and cell types in rodents, primates and human [126]. All of these
data call for a model to test causality of microRNAs in ageing.
C. elegans has been a major driving force in microRNA research. The first
microRNA was identified in C. elegans: the non-coding RNA lin-4, together with its
target gene lin-14, which encodes a putative transcriptional regulator, were found to
regulate timing of events during development [127, 128]. Interestingly, lin-4 and
LIN-14 were also found to modulate ageing. Boehm, Slack [129] found that loss-
of-function of lin-4 shortens lifespan, while overexpressing lin-4 makes worms live
longer. All of these effects in ageing were dependent on LIN-14 [129]. Since then,
microRNAs which function to either shorten or extend lifespan have been subse-
quently identified. Such “age-associated microRNAs” are predicted to target genes
that directly regulate longevity, including those that function in metabolism [130,
131], IIS, as well as the DNA damage response [132].
Dietary restriction was found to be effective in modulating the levels of age-
associated microRNAs. Mori et al. [133] showed that in both mouse adipose tissue
and in C. elegans, expression of Dicer (or the worm orthologue DCR-1), the enzyme
that cleaves pre-miRNAs into mature miRNAs, significantly decreases with age.
This decrease is rescued in mice under caloric restriction and in eat-2 worms [133].
DR was also found to inhibit expression of a specific microRNA miR-80. In turn,
mir-80 mutants are long-lived and show various health benefits associated with DR
[134]. Furthermore, using modENCODE data, Smith-Vikos et al. [101] examined
transcription factor binding sites for ageing-related microRNAs. They found miR-
71 and miR-228 form a transcriptional feedback loop with SKN-1 and PHA-4, the
two transcription factors that are critical for DR longevity. Indeed, mir-71 and mir-
228 are required for lifespan extension by sDR [101].
376 Y. Zhang and W.B. Mair
16.4.1 Healthspan
Efforts are only beginning to be made in murine studies to accurately quantitate how
DR affects the frailty of animals at old age [137]. Although, much data exists on
how DR impacts rodent physiology, onset of age-related pathology and cause of
death [4], the biological relevance of C. elegans models of human disease is less
clear. However, a key advantage of using worm as a preliminary tool to examine the
effects of DR on disease and physiology is the ease with which causality can be
examined. Although data is limited, a handful of studies have examined the effects
of DR on disease models and age-related decline in worms. In a study of proteotoxic
stress, DR by either BD or eat-2 prevents paralysis caused by expression of the
aggregation-prone polyglutamine or Aβ peptides [35]. In a gld-1 mutant cancer
model, where worms die a few days into adulthood due to germline overprolifera-
tion, daf-2 mutation completely rescues the early death by activating apoptosis and
reducing cell proliferation in the germline [138]. eat-2 animals and mutants with
reduced mitochondria function also show some protection and decreased prolifera-
tion, although they do not increase apoptosis [138]. In an olfactory associative
memory assay, eat-2 animals do not retain long-term memory as well as wild type
when they are young. However, while wild type animals lose memory capacity rap-
idly with ageing, eat-2 animals maintain their memory capacity and even perform
better than wild type at old age [26]. Studies such as these need to be expanded to
include more methods of DR, as well as more disease models and function assays.
It has long been hypothesized that DR increases lifespan by reducing overall meta-
bolic rate. However, studies that measure metabolic rates under various DR condi-
tions suggest the picture is more complex. Traditionally, respiration can be quantified
using Clark electrodes to measure oxygen consumption rates using a large number
of animals. Strikingly, respiration in eat-2 animals is not lower than wild type when
worms are grown on agar plates [139]; further, when raised in liquid culture, eat-2
animals have higher respiration than wild type [139]. Wild type worms subjected to
ADR [19] or BDR [18, 139] also show increased oxygen consumption.
378 Y. Zhang and W.B. Mair
Recently, Seahorse Analyzers have been used to more accurately measure oxy-
gen consumption rate using a small number of animals. Moroz et al. [71] used this
method to show that a modified BDR method decreases oxygen consumption rate,
which is in contrary to results obtained using the traditional method. The same study
also showed, by addition of a mitochondrial uncoupler, that DR animals have an
increased spare respiratory capacity, suggesting that their mitochondria are more
efficient in energy production [71]. It remains unclear how the differences in meth-
odology contribute to the contradictory results. More studies are needed to examine
whether changed respiratory capacity contributes to the delayed ageing under DR.
16.4.4 Autophagy
Inhibiting protein translation via loss of initiation factors or the ribosomal protein
kinase S6K are all sufficient to extend lifespan [70, 83, 146, 147] (see Chap. 13).
Hansen et al. [70] linked protein synthesis to DR by showing that eat-2 animals
16 Dietary Restriction in C. elegans 379
have reduced protein synthesis rates and decreased expression of several ribosomal
protein genes. Ching et al. [40] further showed that eat-2 animals have decreased
expression of the translation initiation factor DRR-2. Increasing the level of DRR-2
blocks the lifespan extension in eat-2 background and diminishes the effects of a
method of sDR [40].
The mechanisms by which reduced protein translation extends lifespan remain to
be fully understood. Intriguingly, Rogers et al. [148] used polysomal profiling to
show that when global translation is decreased by inhibition of the translation initia-
tion factor IFG-1, a subset of mRNAs maintain high levels of translation. Many
products of such mRNAs are stress-responsive proteins required for prolonged
lifespan [148]. This is consistent with the finding in fruit flies that DR increases
expression of the translation repressor 4EBP, which extends lifespan via selective
translation of mRNAs for mitochondrial proteins [149].
Caloric restriction reduces body fat in mammals, however, it remains unclear how
lowered adiposity contributes to longevity [150]. Similarly, eat-2 worms have
reduced lipid content from Oil Red O (a lipophilic dye) staining and decreased tri-
glyceride levels [29]. Gas chromatography (GC) analysis of fatty acids revealed that
different FA species were differentially regulated by DR [29], suggesting that lipid
composition, rather than total lipid content, regulates lifespan. RNA seq identified
“unsaturated fatty acid metabolism” and “lipid modification and transport” as sig-
nificantly altered pathways by DR [29]. While mechanisms that mediates the reduc-
tion in body fat under DR conditions in general remains to be fully characterized,
multiple key factors have been identified under fasting conditions, which dramati-
cally depletes lipid stores [151]. Many lipid/cholesterol synthesis genes are acti-
vated by SREBP-1/2 transcription factors. Walker et al. [151] found that the activity
of the worm SREBP orthologue SBP-1 quickly diminishes when worms are fasted.
Mammalian SREBPs are directly deacetylated by SIRT1, which increases SREBP
degradation, and sir-2.1 mutant worms fail to mobilize their body fat under fasting
[151]. To investigate the mechanisms that underlie fatty acid breakdown upon fast-
ing, Van Gilst et al. [124] measured the expression of genes in fatty acid and glucose
metabolism pathways and found that fasting specifically changes fat metabolism.
Fasting induces expression of genes involved in mitochondrial oxidation and fatty
acid desaturation, and leads to increased polyunsaturated fatty acids [124]. NHR-49
is required for the expression of many such “fasting response” genes [124]. Loss-of-
function mutation in nhr-49 increases body fat and shortens lifespan [152].
Lipases are another family of enzymes important in lipid metabolism during fast-
ing, as many of these enzymes hydrolyze fat from lipid droplets during lipophagy.
O’Rourke and Ruvkun [153] showed that lysosomal lipase genes are up-regulated
during fasting. Double mutants of two lipase genes, lipl-1 and lipl-3, cannot mobi-
lize fat when fasted [153]. A targeted RNAi screen of transcriptional regulators
380 Y. Zhang and W.B. Mair
Mitochondria are important sites in the cell for energy production and for coping
with stress [155] (see also Chaps. 5 and 10). Damage in mitochondria increases with
age [155]. DR increases mitochondria biogenesis in mice and human [156, 157]. In
fruit flies, DR extends lifespan by increasing translation of proteins in the electron
transport chain (ETC) [149]. In C. elegans, DR increases mitochondria respiration
efficiency [71], while inhibiting the ETC using pharmacological agents abrogates
lifespan extension by DR [18]. Early studies on the role of mitochondria in ageing
focused on ROS production: an increase in mitochondrial biogenesis can potentially
provide more efficient entry of electrons into the ETC, thereby reducing electron
stalling and decreasing reactive oxygen species (ROS) generation [158]. However,
the effects of directly modulating ETC activity in C. elegans are not consistent with
observations in mammals; reducing ETC activity extends lifespan in C. elegans
[159]. Further, ROS production was reported to have a signalling role that is benefi-
cial. Loss-of-function mutations in ETC genes [117, 160] and glucose restriction
[161] both produce an increase in ROS production, which is required for lifespan.
Besides biogenesis and ROS production, the dynamics of the mitochondrial net-
work is also under tight regulation by nutrient availability [162]. Regulated by spe-
cific protein factors, mitochondria can undergo fission, fusion and mitophagy.
During starvation, mitochondria fuse into an elongated state to increase respiration
efficiency and resist mitophagy; under nutrient overload such as obesity,
mitochondria move to a fragmented state (fission) [162]. One can therefore hypoth-
esize that a highly dynamic mitochondrial network that responds readily to nutrients
and stress is essential for healthy ageing. Indeed, impaired mitochondria dynamics
has been causally linked with metabolic diseases [163] and altered lifespan in yeast
[164–166]. In C. elegans, mitochondrial morphology can be visualized using fluo-
rescently tagged mitochondrial proteins. Pharmacological agents that increase
NAD+ levels prolong lifespan and increases fusion [75], while short-lived nhr-49
16 Dietary Restriction in C. elegans 381
Research using C. elegans has been fundamental in changing our perception of age-
ing and the capacity to target conserved longevity modulators to promote health in
old age. However, despite genetic modulators of longevity discovered in worm
showing conserved effects in other species including mouse, using C. elegans as a
tool to uncover mechanisms by which DR promotes health is still met with scepti-
cism by traditional murine DR researchers. In part, this is due to the ever-increasing
numbers of DR methodologies in worm; if genetic mechanisms mediating one
worm DR protocol are not even conserved to another, what can they tell us about
mammals and ultimately people? However, the genetics of ageing field is based
upon the premise that conserved modulators of ageing across species exist, and this
premise is as true today as it has ever been; discovery in genetic systems continues
to push boundaries and generate ideas for work in mammalian studies in a cost and
time effective manner. Arbitrarily deciding that we have now generated enough
knowledge using invertebrate systems dogmatically closes vast possibilities for new
discovery.
Small molecules first identified using invertebrate systems that promote healths-
pan and mimic DR have now been shown to extend lifespan in mice, and many more
are in trials at the intervention testing program project sites [167]. Moreover, the
FDA has now given approval for studies testing the ability of metformin to target the
ageing process in humans [168]. These are exciting times indeed for those translat-
ing early work in model systems like C. elegans to usable therapeutics in humans.
However, the pipeline of discovery is far from dry, and the next 10 years of work in
worm will uncover new depths of understanding as to how DR promotes health. For
instance, we are just beginning to understand how different cell types coordinate to
orchestrate systemic ageing [82], how specific metabolites might be used to mimic
DR [169], how neuronal perception of DR might be as important as DR itself [18,
170], and how host and microbe genomes communicate to modulate the response of
the meta-organism to DR [51]. As the CRISPR revolution permeates fully into C.
elegans ageing research and the C. elegans intervention testing program accelerates
small molecule discovery, worms will continue to be at the forefront of our under-
standing of dietary restriction. Exciting times lay ahead.
Acknowledgements We thank members of the Mair lab for helpful discussion and critical read-
ing of the manuscript. We would like to apologize to those whose work could not be cited here due
to space limitations. W.M. is funded by the Ellison Medical Foundation and the NIH/NIA
R01AG044346.
382 Y. Zhang and W.B. Mair
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16 Dietary Restriction in C. elegans 391
Abstract Over the last 25 years it has become evident that single gene mutations
can result in remarkable increases in lifespan. Of the gene mutations identified, the
most potent at extending life- and healthspan are those that alter the quantity of food
ingested (Avery, Genetics 133 (4):897–917, 1993) and those that disrupt the animals
perception of the amount of food ingested (Gottlieb and Ruvkun, Genetics 137
(1):107–120, 1994; Dorman et al, Genetics 141 (4):1399–1406, 1995; Kimura et al,
Science 277(5328):942–946, 1997; Lee et al, Curr Biol 11 (24):1950–1957, 2001).
These mutations promote longevity, animal health and capacity for stress adaptation
(Honda and Honda, Faseb J 13 (11):1385–1393, 1999; Scott et al, Science 296
(5577):2388–2391, 2002; Garsin et al, Science 300 (5627):1921, 2003; Lithgow
and Kirkwood, Science 273 (5271):80, 1996), but importantly reveal that an intri-
cate molecular and genetic network exists to integrate diet availability, utilization
and animal physiology (Curran and Ruvkun, PLoS Genet 3 (4):e56. doi:10.1371/
journal.pgen.0030056, 2007; Dillin et al, Science 298 (5602):2398–2401.
doi:10.1126/science.1077780, 2002; Hamilton et al. Genes Dev 19 (13):1544–1555,
2005; Hansen et al, PLoS Genet 1 (1):e17, 2005; Lee et al, Nat Genet 33 (1):40–48,
2003; Tacutu et al, PLoS ONE 7 (10):e48282. doi:10.1371/journal.pone.0048282,
2012).
17.1 Introduction
In order to survive, animals must be able to uptake and utilize diverse food sources
from their surrounding environment. The body’s main source of intracellular chemi-
cal energy, ATP, is generated through the catabolism of macronutrients – carbohy-
drates, lipids, and proteins in that food source. The nutritional quality of the diet is
directly related to the macronutrient composition and that formula has potent
impacts on animal physiology and lifespan [16]. In most multi-cellular organisms,
food intake is not constant and animals must be able to store dietary energy that can
be easily mobilized when necessary. Therefore, the ability to adapt to changing
environments and food sources is of critical importance. C. elegans are bacteriovo-
res that have evolved the capacity to effectively utilize diverse bacterial diets in the
wild for sustenance [17]. Surprisingly, worms are capable of effectively using many
of these microorganisms to sustain life and reproduce. Regardless of the bacteria
ingested, C. elegans have evolved a remarkable capacity to adapt to the food source
provided. Recently, hints towards understanding the molecular mechanisms under-
lying this dietary adaptation have emerged using worms harbouring single gene
mutations being fed the two most commonly used bacterial diets in the laboratory
(E. coli B—OP50 and E. coli K12—HT115) [18–22]. The phenotypes that manifest
from these gene mutations are variable on these two similar E. coli diets, which
provides evidence for diet-gene pairs; or genes that are essential on one diet type but
dispensable on others [20]. Although both diets are E.coli based, it is clear that they
are not nutritionally equivalent and feeding of these diets is known to differentially
affect organismal metabolism [23, 24].
In general, limiting worms’ food consumption has been shown to increase their
lifespans, which is a conserved response in rodents and monkeys [4, 25–27]. Calorie
restriction (CR) is a technique that reduces the amount of calories allowed in the
diet to about 60–70 % of an ad libitum diet [28]. However, it has become increas-
ingly clear, across all organisms, that it is not simply the number of calories that
matters, but the composition of the diet, which has led to the study of dietary restric-
tion (DR) where the quality of the diet is altered [23]. A synthetic diet that facilitates
normal developmental timing, reproduction, and lifespan for worms has yet to be
synthesized, which makes DR studies difficult to design. However, C. elegans can
eat a variety of bacteria sources with varied dietary complexities, which provides an
alternative approach to assess diet composition on animal physiology. As the spe-
cific topics of CR, DR, and endocrine signalling are discussed in Chaps. 4 and 16 of
this book, this chapter will focus on how different diets can affect worm physiology,
the key players that integrate these signals, and how the implications of these find-
ings, which could only have been uncovered in C. elegans, will impact our under-
standing of human ageing, health and disease.
C. elegans is constantly on the lookout for possible food sources and has two pairs
of neurons that function to discern attractive odorants, the AWA and AWC [29, 30].
In addition, the main neuronal pair used to sense chemicals and pathogens that the
worm wants to avoid is called AWB [29, 31]. C. elegans in the wild are exposed to
many types of bacteria in its daily adventures. Some of these bacteria can be used as
food sources but some can be pathogenic. It is important for these animals to be able
17 Integration of Metabolic Signals 395
Lifespan ROS
OP50 diet
E. coli B ? Abnormal mitochondria
ATP morphology
? NMUR-1
SKN-1 LET-363
ALH-6 AAK-1/2
? MXL-3 MDT-15
Normal
Mitochondria
Fig. 17.1 Sensory neurons along with metabolic regulators may induce differential cellular phe-
notypes depending on the diet eaten, however, whether the signal is a direct response to a specific
diet or if it is a host regulatory mechanism remains unknown (?)
In the laboratory setting, C. elegans are normally fed with monoxenic bacteria
cultures that have been plated and allowed to dry on a petri dish containing nema-
tode growth medium (NGM) with agarose [59, 60]. Although only one type of
bacterial diet is customarily provided to the worm at a time, differences between
laboratories in culturing these bacterial strains can pose problems with replicating
phenotypes seen by other groups [61]. The recent appreciation that bacteria type has
an effect on animal physiology has facilitated the development of new and exciting
tools to examine diet-gene interactions. The C. elegans community canonically
used an E. coli B strain named OP50 as its standard food source. However, when
performing RNA interference (RNAi) experiments an E. coli K-12 strain named
HT115 is routinely used. As it was previously alluded to, these stains have strong
influence on organismal physiology and intriguingly, led to the discovery of diet-
gene pairs [19–21]. One such example of a diet-dependent phenotypes is found in
the examination of worms lacking alh-6, which is a conserved mitochondrial
enzyme involved in proline catabolism, were found to have a shortened lifespan on
an OP50 diet yet a normal lifespan on the HT115 diet [20] (Fig. 17.1). The diet-
dependent progeria phenotype was a result of deregulated mitochondrial function –
morphology, diminished ATP production, and increased ROS generation. While
these mutants were identified in a classical genetic screen based on their ability to
activate the cytoprotective transcription factor SKN-1 (discussed below) when these
animals were raised on the OP50 diet [20, 21, 62] it is important to note that these
phenotypes would never have been discovered using RNAi screening approaches,
as this diet is a potent of suppressor of the negative physiological consequences of
alh-6 loss. Similarly, another diet-gene pair was discovered through the utilization
of the HT115 diet during an RNAi screen for genes essential for germline develop-
17 Integration of Metabolic Signals 397
ment. The nuclear hormone receptor, NHR-114, was found to play a protective role
in germline stem cells maintenance by suppressing the accumulation of division
defects and ultimately sterility but only in the context of the HT115 diet [63]. Taken
together, these studies show how genes can be fundamentally needed on one diet,
yet nonessential on another and have opened a new and exciting quest to uncover
the potentially thousands of diet-gene pairs that may exist and possibly explain the
variability of ageing rates in humans.
While under the stress of starvation, animals change their metabolic programmes so
they can adapt to their specific environmental conditions in an attempt to survive the
famine until the next feast arrives [2, 64–76]. When starved, animals no longer have
access to dietary carbohydrates and instead must rely on intracellular lipids and
proteins for fuel [77]. In order to satisfy energy requirements, lipolysis and fatty
acid oxidation are increased to break down lipids and proteins are oxidized into
amino acids. Critical to this adaptation response are mediators of metabolic homeo-
stasis because they are able to swiftly adjust an animal to effectively and efficiently
handle their current environment. Recently, the cytoprotective transcription factor
SKN-1 has been linked to this adaptation response, which provides an intriguing
model where a critical regulator of stress resistance has the capacity to tap into the
cellular metabolic pathways to pay for this costly response.
SKN-1 has been shown to be central to a variety of stress responses [21, 62,
78–89]. SKN-1 is a bZip transcription factor canonically known for defending
against oxidative stress but has recently accumulated fame for its roles in detoxifica-
tion, immunity, proteostasis, and metabolism [21, 62, 78, 79, 81, 83–96]. Recent
work on SKN-1 identified the first two gain-of-function(gf) alleles of skn-1, which
result in the altered expression of genes related to metabolism, starvation adapta-
tion, growth, and reproduction [21, 62]. Intriguingly, when SKN-1gf animals are
subjected to a bacterial dilution (bDR) mechanism of CR [97], which leads to an
increase in lifespan for wild-type animals, it resulted in an absence of attenuation of
longevity. In addition, the SKN-1gf animals have diminished larval stage 1 (L1)
survival when starved. When taken together these findings suggest that constitu-
tively active SKN-1 leads to a perceived state of starvation even when the animals
are fed ad libitum. Amazingly, depending on the diet eaten immediately before star-
vation, SKN-1 and its co-regulator MDT-15 can establish an organism’s response to
food deprivation [21]. It was no surprise that MDT-15, a subunit of the conserved
transcriptional coregulator complex called the “Mediator,” was involved in this
response as it had been previously implicated to regulate the transcription of genes
involved in fatty acid metabolism and ingestion-associated stress responses [98,
99]. Notably, these findings support the importance of actual diet availability, per-
ceived dietary status, and the genetic pathways underlying diet sensing and utiliza-
tion. The ability to trick our bodies into believing we are nutritionally restricted
398 D.A. Lynn and S.P. Curran
while maintaining the ability to eat what we want remains a fantasy, but perhaps
SKN-1 and its co-factors are pieces of that puzzle.
Dietary stressors can come in many flavours. Society has placed particular inter-
est on the effects that a ‘Western Diet’ full of carbohydrates can have on an organ-
ism. In C. elegans, when wild-type animals are fed an OP50 diet supplemented with
2 % glucose, deemed a high carbohydrate diet (HCD), they significantly induced a
250 % increase in intestinal lipid stores compared to their fat content on regular
OP50 diets and this diet has obvious negative impact on life and healthspan [100,
101]. Remarkably, SKN-1gf animals fed this HCD did not accumulate more stored
intestinal lipids versus SKN-1gf animals on a regular OP50 diet [21]. This is remark-
able because constitutive SKN-1 activation can protect against dietary insults that
would normally cause fat accumulation. Using a Keap-1 knockdown mouse model,
which induces Nrf2 (the mammalian homologue of SKN-1) activity, researchers
have shown that this inhibits lipid accumulation even when the animals are given a
high-fat diet [102]. These findings support the idea that we can genetically manipu-
late an organism’s physiology in response to less than ideal diets and when com-
bined with the fact that this lipid metabolic role of SKN-1 is also shared by its
human homologue Nrf2 [21], it makes this even more tantalizing. Ultimately, these
findings may have larger clinical implications because Nrf2 agonists, for which
many have been identified [103–108], could be useful for combating certain meta-
bolic diseases.
Another way to induce dietary stress is through impairment of glucose metabo-
lism, which causes an increase in oxidative stress [100, 101]. Concerning oxidative
stress, both SKN-1 and Nrf2 are activated in response to compounds like H202,
paraquat, and juglone [78, 88]. There is, however, controversial data in regards to
reactive oxygen species (ROS) and their effects on physiology and signalling path-
ways [101, 109–113]. Originally thought of as harmful, high levels of ROS have
been linked to cellular damage but it has been recently shown that when animals are
only mildly stressed, secondary messengers such as ROS can alter signalling path-
ways in order to allow the organism to respond to stressors in a timely and appropri-
ate way. The mitochondria are primary sources and targets of ROS, which at
low-levels, promotes health and longevity through its activation of increased stress
resistance factors [114, 115]. This type of adaptive response has been termed “mito-
hormesis” because of the stress-induced stress resistance. Controversially, elevated
levels of oxidative stressors have been linked to an increased risk for certain cancers
and degenerative diseases because they can cause damage to cellular materials like
DNA, proteins, and lipids. Along these lines, deregulated Nrf2 has been linked to
several aggressive types of cancer [103]. However, excessively low levels will also
leave the body more susceptible to cancers and infections because cellular protec-
tion pathways, which include apoptosis and phagocytosis that rely on ROS signal-
ling, become compromised [116]. Taken together it is clear that SKN-1/Nrf2 is a
central regulator of metabolic responses, which we can manipulate, but we must
maintain the ability to dial its activity up and down as needed to ensure cellular and
organismal health.
17 Integration of Metabolic Signals 399
When there are available nutrients, a crucial governor of many anabolic processes,
target of rapamycin (TOR), let-363 in C. elegans, is activated in order to help facili-
tate biosynthetic processes like protein synthesis and nutrient storage [117, 118].
Intriguingly, when LET-363/TOR is inhibited this results in lifespan extension
[119–121]. This phenomena ties into dietary restriction models of lifespan exten-
sion as TOR is potently suppressed during fasting and nutrient limitation. Conditions
that inhibit TOR derive in part from an imbalance between energy usage and nutri-
ent consumption, specifically when cells exhibit an increased AMP:ATP ratio and
coordinate the use of AMP-activated protein kinase (AMPK), which is a well-
conserved sensor of cellular energy levels [95, 122–124]. In addition, these energy
shortage conditions also upregulate autophagy in order to recycle things like mito-
chondria, proteins, and stored glycogen for cellular energy. A discussion of each of
these exceptionally complex and essential processes can be found in Chaps. 15 and
16 of this book.
While many of the downstream effectors of metabolic adaptation have been
identified, albeit not to saturation, many of the intricacies upstream of the response
have yet to be identified. Adult hermaphrodites have only 302 neurons in their ner-
vous system, yet the inner-workings are quite complex. The chemical signalling
involved in the C. elegans nervous system includes neurotransmitters for dissemi-
nating signals across synapses and neuropeptides for cell to cell communications
[125]. A targeted screen of C. elegans carrying mutations in certain neuropeptide-
like genes, neuropeptide receptors, or G-coupled protein receptors was conducted to
assess potential differences in lifespan on an OP50 diet versus an HT115 diet [19].
Specifically, they discovered that most neuropeptide signalling pathways did not
affect the lifespan when animals were raised on the two diets; however mutation of
one gene nmur-1, did have an effect and was one of the first described diet-gene
pairs to be identified in C. elegans. nmur-1 mutant animals lived long on the OP50-
based diet but did not receive any lifespan benefit when fed the E.coli K-12 HT115
diet. Additional roles for NMUR-1 integration of diet and animal physiology were
revealed when double mutants for both alh-6 (discussed above) and nmur-1 were
fed an OP50 diet and were found to no longer display the aforementioned short
lifespan and mitochondrial deregulation phenotypes. This finding importantly
revealed that neuroendocrine signalling is required for maintaining an organismal
response to the OP50 diet. Therefore, NMUR-1 is integral in communicating dietary
information to downstream effectors.
The NMUR-1 protein has significant homology to mammalian neuromedin U
receptors (NMURs), which are conserved across evolutionary boundaries. In verte-
brate model systems, NMU is a highly conserved neuropeptide that has key roles in
many physiological processes, including feeding and energy homeostasis [126].
Fruit flies have four NMU receptors that are activated by pyrokinin neuropeptides
[127, 128]. The C. elegans genome also encodes four NMU receptor homologues
and an in silico search for the C. elegans pyrokinin-like peptide precursor genes
400 D.A. Lynn and S.P. Curran
This chapter has identified recent discoveries made in C. elegans that coordinate
diet and animals physiology. These findings are of particular importance to our
understanding of human physiology and when combined with the fact that these
pathways identified in worms are remarkably well conserved in humans (Table 17.1),
supports the continued and even increased use of C. elegans as a model for studying
human disease. Regulating and maintaining cellular homeostasis not only involves
nutrient and energy sensing, but the animal must be able to prevent the buildup of
toxic metabolic byproducts. Many human diseases, like cancer, obesity, and diabe-
tes, have underlying metabolic dysfunctions [134, 135]. In some cases, particular
diets can act as therapies or as accelerants for these diseases [136, 137]. For instance,
obesity and type-2 diabetes can manifest due to a person’s long-term dietary choices.
Diabetes mellitus affects hundreds of millions of people worldwide and the number
of people affected is steadily increasing each year. Unfortunately, the World Health
Organization projects Diabetes to be the seventh major cause of death by the year
2050 [138]. Diabetes and non-alcoholic fatty liver disease are hallmarked by
impaired glucose and insulin homeostasis which can damage tissues and cells,
impair cellular function though formation of advanced glycosylation end (AGE)
products, and generate oxidative stress through the overproduction of reactive oxy-
gen species (ROS) [139]. Pharmacological maintenance of insulin is one approach
for people with defects in the production of insulin but importantly, many aspects of
this disease can be ameliorated by diet. For example, low sugar and diabetic
“friendly” meals are readily available to consumers.
402 D.A. Lynn and S.P. Curran
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Chapter 18
Microbiota, Probiotic Bacteria and Ageing
Abstract The number of bacteria in the human intestine roughly equals the number
of cells in the entire human body. This community of bacteria and a much smaller
number of unicellular eukaryotes and prokaryotic archaea is referred to as the
microbiota. It is becoming increasingly clear that the composition of the microbiota
is important for human health and has an impact on obesity, diabetes, various bowel
diseases and likely ageing. The microbiota is composed of pathogenic, commensal
and beneficial bacteria, the latter often referred to as probiotic. Several studies have
reported that the composition of the microbiota changes during ageing. Although
recent developments in DNA sequencing technologies have allowed researchers to
more accurately determine the composition of the microbiota, little is known about
the mechanisms by which the microbiota mechanistically influences the host, not
least during ageing. This limits the use of probiotic bacteria to prevent and treat
diseases. Researchers are using C. elegans to study both pathogenic and probiotic
bacteria, which have opposing effects on lifespan. C. elegans is also successfully
being used as screening platform to identify novel strains of probiotic bacteria.
Since the natural diet of C. elegans is bacteria and the longevity pathways are well
characterized, the nematode is particularly well-suited for this purpose. In this chap-
ter we will review how the microbiota and particularly probiotic bacteria influences
ageing in C. elegans.
The human gastrointestinal tract was recently estimated to contain ~4 × 1013 bacte-
ria nearly equaling the estimated ~3 × 1013 human cells in a 70 kg “reference”
human [1]. Although this 1:1 ratio between bacterial and human cells is lower than
the 10:1 estimate previously proposed, and widely referenced, humans do contain a
staggering number of microorganisms. In addition to bacteria, the human gastroin-
testinal tract also hosts unicellular eukaryotes and prokaryotic archaea; collectively
these microorganisms are called the microbiota. The combined gene pool of these
microorganisms constitutes the microbiome. The largest population of bacteria is
found in the gastrointestinal tract, where they vastly outnumber other microorgan-
isms. Therefore, the term microbiota is often used to describe the bacterial commu-
nity in the intestine.
Dictionary
Probiotics Live microorganisms that, when administrated in adequate
amounts, confer a health benefit on the host.
Prebiotics Supplements that favour growth or activity of probiotic
bacteria
Commensal Bacteria that are part of the normal microbiota, and which
benefit from the symbiosis with the host, but without being
beneficial or harmful to the host.
Microbiota The communities of bacteria, unicellular eukaryotes and pro-
karyotic archaea hosted in the human body. These can be com-
mensal, symbiotic/beneficial or pathogenic.
Microbiome The collected genomes of the microorganisms in the microbi-
ota. Microbiome and microbiota are sometimes used inter-
changeable in the literature.
LAB Lactic acid bacteria
beneficial effect on their host can thus be called probiotic bacteria. Strains in the
Lactobacillus and Bifidobacterium genera are often considered probiotic and a
number of studies have shown that certain strains of these species can prevent and
treat a range of conditions including intestinal diseases, obesity, metabolic disorders
and various infections [10, 11]. Most of these studies are descriptive and mainly
identify associations between specific microbes and health or disease rather than
causal relationships. Nevertheless, probiotics and prebiotics (supplements favour-
ing growth or activity of probiotic bacteria) are growing industries with many areas
of application including drugs, foods, dietary supplements, and animal feed.
The idea that the microbiota could influence ageing was put forward by Ilya Ilyich
Metchnikoff more than a century ago [12]. Metchnikoff suggested that health could
be improved by altering the microbiota with help of probiotic bacteria found in
yogurt. Today many yogurt-based probiotic products are commercially available
claiming various beneficial effects, although little is known about their mechanisms
of action. However, regarding microbiota influencing human ageing, it seems that
Metchnikoff might have been on the right track, since variations in gut microbiota
composition between young and elderly have been reported in several studies [13–
19]. Most of these studies are of correlative nature and causal mechanisms are
largely unknown. The strong track record for uncovering longevity pathways and
underlying molecular mechanisms has made C. elegans a popular model system for
studying ageing and life history traits. Since bacteria are the natural diet of C. ele-
gans, the nematode is particularly well-suited for understanding the effects of pro-
biotic bacteria on ageing. Although it is a relatively young field of research, several
studies have found that feeding C. elegans with probiotic bacteria increases lifespan
and resistance towards bacterial infections (Table 18.1). Before we discuss these
studies in more detail we need to look at some of the differences and similarities
between C. elegans and humans with respect to shaping and hosting a microbiota.
In humans the vast majority of bacteria are found in the intestine and likewise in C.
elegans the intestine is where most bacteria are found. The intestine is the largest
somatic organ in C. elegans (see Chap. 2), and it carries out a variety of functions
including nutrient uptake and storage, lipid accumulation, elimination of waste
products, and protection against harmful substances and pathogens [20]. Unlike
humans, C. elegans is a bacterivore and therefore bacteria are necessary food
sources, part of the microbiota and potential pathogens.
Table 18.1 Probiotic bacteria used in C. elegans
414
Bacterial strain Group Effect on lifespan Genetic dependence Pathogenic resistance Reference
L. salivarius FDB89 I Increase Dietary restriction N.D. [52]
LAB consortium from cheese I Decrease nhr-49, pept-1, tub-1 N.D. [51]
Lactobacillus JDFM60, II Increase N.D. S. aureus [71]
JDFM440, JDFM970,
JDFM1000
L. helviticus
L. plantarum
L. rhamnosus
B. infantis
B. longum II Increase N.D. Salmonella enterica [72]
L. plantarum CJLP133
L. fermentum LA12 II Increase N.D. N.D. [53]
L. reuteri II N.D. clec-60, clec-85, reduced bacterial ETEC JG280 [57]
enterotoxin expression
L. acidophilus NCFM II, IV No effect pmk-1, tir-1, bar-1 Gram-positive [54]
pathogens
B. megaterium II, IV No effect glp-4 (BM) P. aeruginosa [26]
P. mendocina pmk-1 (PM)
L. zeae II N.D. Reduced bacterial enterotoxin ETEC JG280 [58]
expression
B. subtilis GS67 II N.D. Secreted fengycin reduces Gram-positive [56]
colonization of pathogen pathogens
L. reuteri CL9 II No effect N.D. Salmonella [68]
L. casei CL11 typhimurium
L. reuteri S64
E. coli GD1 (Q-less) III Increase Bacterial respiration N.D. [39]
K.V. Christensen et al.
18
E.coli HT115(DE3) aroD III Increase Bacterial folate synthesis N.D. [59]
mutant
E.coli Metformin disrupts folate III, IV Increase skn-1, aak-2 N.D. [65]
in E. coli
L. gasseri SBT2055 IV Increase skn-1, pmk-1 N.D. [62]
B. infantis IV Increase pmk-1, skn-1, vhp-1 N.D. [63]
B. licheniformis IV Increase tph-1, bas-1, ser-1, mod-1 N.D. [73]
L. rhamnosus CNCM I-3690 IV, II Increase daf-2, daf-16, skn-1 N.D. [64]
B. subtilis (NO) IV, III Increase daf-16, hsf-1, hsp-16, hsp-70 N.D. [60]
B. subtilis V Increase N.D. N.D. [45]
B. amyloliquefaciens JX1 V Increase N.D. N.D. [33]
Variovorax sp. JX14
B. megaterium JX15
P. fluorescens Y1
Microbiota, Probiotic Bacteria and Ageing
When maintained in the laboratory C. elegans nearly always feed on a single bacte-
rial strain, typically the gram-negative bacterium Escherichia coli (E. coli) OP50.
Other E. coli strains are also commonly used for maintenance, e.g. HB101 and
HT115, used for an extra nutritious diet and RNAi, respectively. These different
food sources have different effects on lipid deposition, development, metabolism,
and lifespan [21–24].
In the wild C. elegans feed on various types of bacteria and thus, they have a
diverse bacterial flora in their gut lumen [25–27]. Like all multicellular organisms,
nematodes must also choose what to eat when faced with a wide range of bacteria
in the wild. C. elegans is able to navigate through these and avoid pathogenic bac-
teria [28–30] in the search for high quality food, namely bacteria supporting growth,
which is partly driven by previous food experience [31]. It has been reported that C.
elegans prefers to consume soil bacteria, such as Bacillus mycoides and Bacillus
soli [32]. Others have suggested that the feeding preferences of C. elegans are
affected by bacterial respiration and growth rates [33] as well as odour attraction
[34]. Sensing of food is discussed in more detail in Chap. 17.
E. coli OP50 was originally chosen as food source because it is a uracil auxo-
troph, growing to a nicely defined lawn on NGM plates making it easier to perform
experiments in the laboratory [23]. OP50 is often considered non-pathogenic but
studies have suggested that it is in fact mildly pathogenic as the lifespan is increased
when C. elegans is fed UV-killed or antibiotic treated OP50 bacteria [35, 36]. The
metabolic state of the bacteria is also important for the development and lifespan of
C. elegans. Growth in axenic medium is associated with slow and asynchronous
development together with reduced fertility, and the worms are believed to enter a
state of dietary restriction [37]. Interestingly, addition of live bacteria reverts the
development back to normal when worms are cultured axenically. Addition of dead
bacteria does not have an effect [38]. Furthermore, respiratory deficient bacteria
lacking either Coenzyme Q or ATP synthase prolongs the lifespan [39, 40].
The bacteria consumed by C. elegans are first exposed to the pharyngeal grinder
[41] (See Chap. 2). In young animals, the grinder effectively crushes the food, leav-
ing no bacteria to pass through alive. As the worm ages the effectiveness of the
pharyngeal grinder is declining and in young adults bacteria starts colonizing the
intestine, thereby creating a microbiota [42]. The proliferating bacteria in the intes-
tine will eventually become harmful for its host and old worms can get severe con-
stipation due to bacteria blocking the lumen of the intestine. Hindering bacterial
proliferation increases lifespan associated with reduced bacterial packing [35, 36].
It has been suggested that intestinal colonization might be a general mechanism that
18 Microbiota, Probiotic Bacteria and Ageing 417
Dietary restriction has long been known to strongly increase lifespan of many
organisms including C. elegans. For a detailed review of the effect of dietary restric-
tion on lifespan see Chap. 16. Different bacterial diets have been found to affect
lifespan as well, possibly through dietary restriction or due to different macronutri-
ent composition. Macronutrient analysis of some of the most common feeding
strains for C. elegans, OP50, HT115, HB101 and DA837, revealed a significant
difference in their amount of carbohydrates and fatty acids. Nevertheless, there did
not seem to be a significant difference in lifespan of worms grown on these different
bacterial diets [22]. Other studies, however, have observed a significant increase in
lifespan of worms grown on the E. coli strain HT115 compared to worms grown on
E. coli OP50 [21, 24, 49]. Intriguingly, one study has found that feeding with HT115
shortens lifespan compared to an OP50 diet [50]. This could perhaps indicate that
the bacterial strains differ between laboratories due to a high forward mutation rate.
Humans have a very diverse microbiota, and one of the concerns arising from using
C. elegans as a model organism is their maintenance in the laboratory on bacterial
monocultures, which results in the absence of a complex microbiota in their intes-
tine. However, the use of monocultures can also be seen as an advantage because it
is possible to directly link specific bacterial strains to specific host responses (Table
18.1). A few studies have investigated the effect of feeding C. elegans multiple bac-
terial strains simultaneously [26, 27, 34, 51]. These studies follow the overall strat-
egy that bacterial species residing in the worm intestine can be isolated and analysed.
When analysing mixtures of multiple bacterial strains there is currently no way of
eliminating a bias towards enrichment of bacteria that grow easily in the laboratory.
418 K.V. Christensen et al.
There is also a risk of completely missing for example anaerobic bacteria that can-
not grow in the presence of oxygen.
Studies of C. elegans living on rotten fruit, mimicking their natural environment,
have isolated several bacteria species from their intestine indicating that they are
capable of hosting a microbiota [26, 27]. If this actually mimics the natural life of
C. elegans, this also suggests that the worm would have evolved all the response
mechanisms to host a microbiota, containing both beneficial and pathogenic bacte-
ria. This is further supported by the presence of the innate immune system in C.
elegans.
In an elegant study it was shown that “you are not what you eat”, at least if you
are a C. elegans nematode [27]. Germ free L1 larvae were allowed to develop to
adulthood on three types of soil with different bacterial compositions. When the
microbiotas of these worms were analysed based on deep sequencing of 16S rDNA
it revealed that they resembled each other despite arising from different microbial
environments. Thus, it seems that the host plays an active role in shaping its micro-
biota. From this follows that one should be able to identify mutants with altered
microbiotas. Unfortunately, such mutants were not presented in the study. However,
with mutants readily available in C. elegans such mutants will likely be identified in
the future and help uncover how the host determines its microbiota.
Whereas studies addressing complex microbiotas in C. elegans are still rare,
numerous studies have tested the effect of different monocultures including probi-
otic bacteria.
C. elegans has been used to both screen for new potentially probiotic bacteria and to
test the effect of known probiotic bacteria on nematode lifespan and resistance to
pathogenic infections (Table 18.1). Lactic acid bacteria (LAB) of either the
Lactobacillus or the Bifidobacterium genus are the most widely studied species.
Although evolving rapidly, the field of studying probiotic bacteria in C. elegans is
relatively new. Hence, the mechanistic insights into the effects of feeding probiotic
bacteria are still rather limited. However, based on the current knowledge of how
probiotic bacteria affect the worm, we have divided the bacteria into five different,
but overlapping groups: (I) changes in nutritional value, (II) antimicrobial effect,
(III) changes in bacterial metabolism, (IV) direct activation of host signalling path-
ways and (V) unknown effect (Fig. 18.1 and Table 18.1).
Several strains of probiotic bacteria can be placed in more than one of these
groups as they exert multiple effects on the host. For example, many bacterial strains
that influence the immune functions of the host are placed both in group II and
IV. Other bacterial strains have very specific effects on the host and only belong to
one group. As our knowledge improve new groups representing novel mechanism
of action are likely to be identified.
18 Microbiota, Probiotic Bacteria and Ageing 419
Fig. 18.1 Probiotic bacteria can exert their beneficial effects via different mechanisms
Different LAB strains have been shown to affect worm lifespan by regulating the
metabolism of the host. Lactobacillus salivarius was found to increase lifespan
probably through dietary restriction [52]. A LAB consortium obtained from cheese
containing a mixture of three different species decreased lifespan and regulated
expression of genes involved with lipid metabolism [51]. These studies demonstrate
the importance of investigating whether an effect on lifespan from feeding probiotic
bacteria solely arise from either calorically restricting the worms or from changing
the composition of available macronutrients as is the case for OP50 versus HT115
discussed previously (see also Chap. 17). Studies related to this group are very lim-
ited, thus it is difficult to conclude on the underlying mechanisms. More work in the
future is needed to address this lack of knowledge.
worm following infections [26, 39, 53–55]. Such growth inhibition is strain-specific
with regard to both the probiotic and the pathogenic bacteria. For example, L. aci-
dophilus and B. subtilis specifically protects against gram-positive pathogens, but
not gram-negative [54, 56].
L. zeae and L. reuteri protect against enterotoxigenic Escherichia coli (ETEC)
infection by decreasing expression of certain toxins. However, they do not affect
pathogenic colonization in the intestine of the worm [57, 58]. These are examples of
probiotics that can directly change virulence factors expressed by pathogenic bacte-
ria. However, so far only one study has been able to identify the bacterial compound
that inhibits pathogenic infection. B. subtilis was found to produce an antifungal
lipopeptide complex fengycin, which specifically inhibited the growth and intesti-
nal colonization of the pathogenic B. thuringiensis and S. aureus [56]. Bacteria
defective in fengycin production could no longer protect against infection, and
administration of purified fengycin inhibited the bacterial growth and cured infected
nematodes.
Probiotic bacteria have also been demonstrated to activate immune responses in
the worm, enabling them to overcome infections. Preconditioning C. elegans with
L. acidophilus specifically upregulated expression of genes associated with combat-
ing gram-positive pathogen infections through upregulation of the immune path-
ways containing the mitogen-activated protein kinase PMK-1 orthologous to human
p38, the Toll-Interleukin 1 Receptor domain adapter protein TIR-1 and the beta-
catenin BAR-1 [54]. P. mendocina also regulates pathogen infection through
PMK-1, as its protective effect against P. aeruginosa was abolished in pmk-1
mutants, and downstream targets of PMK-1 were upregulated in response to P. men-
docina [26].
These studies of antimicrobial effects of probiotic bacteria are extremely impor-
tant. There is an alarming spread of multidrug-resistant bacteria, which is claimed
by WHO to be a major future threat to global human health. To prevent this dysto-
pian scenario it is necessary to reduce the use of traditional antibiotics and develop
new antibiotics. The identification of interactions between specific probiotic and
pathogenic bacteria offers the possibility of developing new antibiotics as well as
new treatment strategies based upon pro- and prebiotics.
bacteria, which have a positive effect on C. elegans. A study by Gusarov et al. found
that worms feeding on B. subtilis lived longer due to bacterial production of NO,
compared to a NO deficient B. subtilis strain [60]. This lifespan extension was
dependent on both daf-16 and hsf-1, and NO upregulated the expression of the heat
shock proteins hsp-16 and hsp-70 and increased thermotolerance. In a recent study
it was shown that NO produced by B. subtilis also activates the p38 MAPK and
thereby protects against pathogenic bacteria [61]. This is a nice illustration of how
commensal bacteria are important for the host.
Although several of these bacteria are not from the traditionally considered pro-
biotic strains, such as LAB and Bifidobacterium, and not directly classified as pro-
biotic, these studies help to shed light on the complicated interplay between the
microbiota and the host. It can be speculated, that probiotic bacteria might employ
some of the same mechanisms as these commensal bacteria to elicit their beneficial
effect on the host.
A few studies have identified some of the underlying mechanisms activated in the
host by probiotic bacteria that extends C. elegans lifespan. A recurring factor is the
bZip transcription factor SKN-1, which seems to be required for the life extending
effect of several probiotic bacteria [62–65]. This is not surprising since SKN-1 has
been identified as an important protein in regulating several age-related pathways
(see Chaps. 9 and 17).
L. gasseri SBT2055 was found to extend lifespan, increase stress resistance and
improve several age-related declines [62]. The lifespan extension was dependent on
skn-1, and feeding with L. gasseri upregulated the expression of SKN-1, through
the phosphorylation and activation of the p38 MAPK protein PMK-1. Furthermore,
age-related and SKN-1 target genes, such as gst-4, sod-1, trx-1, clk-1, hsp-16.2 and
hsp-70 were also upregulated in response to feeding with L. gasseri. Reactive oxy-
gen species and the age-related mitochondria decline were also reduced, indicating
an overall activation of stress-responses. The probiotic bacteria L. rhamnosus
CNCM I-3690 similarly extends nematode lifespan and stress resistance dependent
on SKN-1 [64]. Contrary to the study with L. gasseri, which did not require the
insulin/IGF-1 receptor homolog DAF-2 and DAF-16 [62], L. rhamnosus requires
both DAF-2, DAF-16 and SKN-1 to extend lifespan [64]. This indicates that the two
bacteria activate different signalling pathways in the host as well as some common
ones. However, the downstream signalling from SKN-1 was not investigated in the
L. rhamnosus study. Instead, they demonstrated that L. rhamnosus had anti-
inflammatory properties in cell cultures and mouse models [64].
Bifidobacterium is another LAB genus that has been tested in C. elegans. Feeding
with B. infantis extends lifespan but not stress resistance [63]. The lifespan exten-
422 K.V. Christensen et al.
sion was abrogated in skn-1 and pmk-1 mutants, but was still induced in daf-16
mutants, demonstrating a requirement for SKN-1 and PMK-1, but not DAF-16.
A final example of communication between the bacteria and the host, is activa-
tion of the C. elegans mitochondrial stress response pathways induced by free oxy-
gen radicals generated by E. coli [66].
A part of the LABs classified as Group II can also belong in Group IV, as some
of these probiotic bacteria activate certain signalling pathways in the worm.
This group includes different bacterial species that have a positive or negative effect
on nematode lifespan for example B. soli, B. myoides, L.reuteri and L. salivarius
[32, 33, 45, 67], but where there is no current knowledge as to which bacterial or
host mechanisms cause the effect on lifespan. Further investigations of these bacte-
rial strains will eventually place them in some of the other four groups or perhaps
define new groups.
In conclusion, all of these studies demonstrate that the probiotic effects of differ-
ent bacteria and the host response pathways that are activated appear to be very
strain specific. Furthermore, not all LAB strains appear to be probiotic, as a couple
of studies have demonstrated that feeding with selected LAB strains can in fact have
negative effects on their host, such as decreased lifespan [51, 67]. Therefore, cau-
tion is required when handling probiotic bacteria and predicting their effects on the
host, as strains of the same genus and species might have widely different effects.
However, dealing with species differences is becoming much easier with advanced
DNA sequencing enabling better distinction between sub-species.
From this example it is clear that certain probiotics have effects on the host that are
conserved across species and that genetic analysis in C. elegans can inform on the
underlying biology.
Another study demonstrated that several LAB strains found to protect C. elegans
from Salmonella Typhimurium DT104-induced death also protected pigs from diar-
rhoea and improved their growth performance, whereas LAB strains found not to
protect C. elegans from pathogen-induced death did not protect the pigs either [68].
Again, this is an example that clearly illustrates that probiotic bacteria operate via a
conserved mechanism in different hosts. Therefore, it is also likely that additional
probiotic strains can be isolated using a similar approach.
There is further evidence that C. elegans can be used to identify probiotics which
are functional in other organisms. A study comparing C. elegans and a porcine
intestinal epithelial cell line as screening platforms for selecting probiotic bacteria
was to a large extent able to identify the same probiotic bacteria in the two systems
[58], although a few strains were only selected by one system. Furthermore, one
selected probiotic strain induced similar host defence responses in both models
[58].
Taken together, these studies demonstrate the relevance of C. elegans as a screen-
ing model organism when identifying novel probiotics for applications in livestock
and humans. In this context, the ability to inexpensively generate germ free indi-
viduals as well as maintaining larges cultures are strong benefits of the C. elegans
model. However, there are also some limitations that should be kept in mind.
A concern when using C. elegans in host-microbe interactions is that bacteria
have never been observed to infect the intestinal cells of the worm (see Chap. 2).
Rather it seems that bacteria only colonize the intestinal lumen. This is in contrast
to the human intestine, where pathogenic bacteria can transverse the intestinal bar-
rier and colonize the intestinal cells. Especially for studying the antimicrobial effect
of a probiotic strain, the worm response might be different from that seen in humans,
due to the difference in intestinal colonization. Furthermore, human studies have
found probiotic bacteria to have an effect on several different tissues in the human
body that are not found in the worm. For obvious reasons these tissue cannot be
studied directly in C. elegans.
The main reasons for using C. elegans to study probiotics are the easily accessible
genetic and biochemical methods combined with the fact that effects on organismal
lifespan can be determined. Furthermore, as the worms eat bacteria as natural food
sources, and since bacterial mutagenesis can be done fairly simply, C. elegans pres-
ents a system where both host and food can be mutagenized to identify which genes
are required for the probiotic effect in both species, and within a relatively short
time frame. For example, the effect of bacterially synthesized folate on C. elegans
lifespan was identified by using a mutagenized E. coli strain [59] and the effect of
424 K.V. Christensen et al.
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Chapter 19
The Future of Worm Ageing
Gordon J. Lithgow
Abstract The history of the C. elegans model in ageing research is a glittering suc-
cess story. Since Tom Johnson’s realization that the longevity displayed by Mike
Klass’s mutants, at the University of Colorado in the late 1980s, resulted from muta-
tion of a single gene (age-1), we have witnessed the rapid development of this sub-
field of ageing research. The chapters in this book attest to the dedicated work of
scores of labs that utilize the worm in an effort to understand ageing. Hundreds of
researchers gather together every year at various C. elegans meetings to consider the
molecular pathways and physiological consequences of the myriad of mutations
that determine lifespan in this organism. The worm meeting ranks as one of the larg-
est ageing meetings on the calendar. During the last 25 years, increasing lifespan
has been the goal and also the gold standard of genetic interventions in ageing. The
focus on the lifespan phenotype and its manipulation has allowed ageing research to
go well beyond the worm model and enter the mainstream. But what is the future for
our beloved worm in ageing research? Will we continue to see new labs established
working on ageing or is the ageing field about to move to more complex, and (per-
haps) more human-relevant models?
The history of the C. elegans model in ageing research is a glittering success story.
Since Tom Johnson’s realization that the longevity displayed by Mike Klass’s
mutants, at the University of Colorado in the late 1980s, resulted from mutation of
a single gene (age-1), we have witnessed the rapid development of this subfield of
ageing research. The chapters in this book attest to the dedicated work of scores of
labs that utilize the worm in an effort to understand ageing. Hundreds of researchers
gather together every year at various C. elegans meetings to consider the molecular
pathways and physiological consequences of the myriad of mutations that deter-
mine lifespan in this organism. The worm meeting ranks as one of the largest ageing
meetings on the calendar. During the last 25 years, increasing lifespan has been the
goal and also the gold standard of genetic interventions in ageing. The focus on the
lifespan phenotype and its manipulation has allowed ageing research to go well
beyond the worm model and enter the mainstream. But what is the future for our
beloved worm in ageing research? Will we continue to see new labs established
working on ageing or is the ageing field about to move to more complex, and (per-
haps) more human-relevant models?
It seems likely that the worm will continue to be an effective model for discovery of
genetic and chemical interventions that extend lifespan. Worm labs are still able to
compete for grants around the world and discoveries being made are increasingly
staggering. Despite the challenges in funding, postdoctoral trainees should be
encouraged to build their careers on the worm model. But for the ageing field as a
whole there will be increasing pressure to translate the discoveries of the last
quarter-century into preclinical and clinical research. How do we respond to this
pressure as individual researchers and as a community?
For many years, worm geneticists claimed, usually at the opening of the grant
applications, that their research was highly significant for the future of human
health. Indeed, it is commonplace to read claims that understanding the basic biol-
ogy of ageing will result in meaningful new therapeutic avenues for age-related
disease in humans. This stems from the view that we are all studying “conserved”
mechanisms, and that what is good for a worm is likely to be good for humans as
well. Many of us have gone as far as to say that ageing itself is the root “cause” of
most chronic human disease in developed countries and consequently manipulation
of the ageing phenotype could lead to the eradication of age-related diseases. If age-
ing causes multiple diseases, but can be slowed, then surely ageing becomes the
major target for therapeutics. The origins of this idea are the observations made on
long-lived caloric restricted mice that appear to have postponed or even no obvious
specific disease with increasing age. Moreover, simply looking down the micro-
scope at worms rendered long-lived with a chemical compound has a profound
psychological effect. Frequently these worms, that should be dead, appear vibrant
and healthy. The researcher seeing this cannot help but dream that the same, simple
interventions must be possible in humans. But is this all true? Are we at the begin-
ning of a radically different outcome of ageing in humans? The pressure on ageing
researchers to deliver on our collective promise can only increase.
But, I can hear you say “That’s not my problem. I do the basic research. I dis-
cover the genes. I discover the compounds. It’s up to the clinicians to take this and
do something important with it”. Of course, there is truth to that. Very few scientists
working on model organisms follow through on their discoveries to identify a new
drug or initiate a clinical trial. Of those that do, some find themselves totally out of
their depth and flounder. But collectively, we have to take some responsibility for
the translation of the discoveries described in this volume. Such action is needed not
19 The Future of Worm Ageing 433
only because of the promises made in grant applications but because if we truly
believe that ageing is so important to the origins of disease then morally we have to
make others believe this to be the case. This is particularly important for those bio-
medical scientists who are in a position to take these discoveries towards the clinic.
What are the challenges that prevent the worm from playing a major role in the
future of biomedical breakthroughs? The first challenge is the one faced by all
model organisms; they are not humans. If the truth be told, they are not even normal
organisms. The vast majority of research in ageing is conducted on lab adapted
animals living in less than ideal environments on unnatural and probably subopti-
mal diets. Wild-type N2 worms on E. coli OP50 is a wholly artificial system that is
great for the study of many biological processes, such as development, but does it
makes sense for ageing studies? Many of the genes that modulate ageing are
involved in mechanisms that respond to environmental and nutritional changes. It’s
possible that these genes would have radically different effects on ageing under
more realistic conditions. We need to carefully consider how that affects our ability
to predict what ageing mechanisms we discover are relevant to humans.
Another major challenge is that of reproducibility. Every few weeks, major jour-
nals publish doom and gloom articles that claim that an extremely large fraction of
the biomedical literature appears to irreproducible junk. Depending on your per-
spective this is a storm in a teacup or a serious problem that could undermine scien-
tific progress, not to mention future Federal funding. Of course there are very robust
and reproducible effects on longevity; has anyone ever failed to observe the
increased lifespan of a daf-2 hypomorph? This result, first made by Cynthia
Kenyon’s lab, is perhaps the most reproduced observation in ageing research and in
a pathway that is surely relevant for human ageing and disease. That said, ageing
research may have a particular problem with reproducibility. Take the simple notion
that feeding a specific chemical compound can extend lifespan of C. elegans grown
in standard lab conditions. On the face of it, this should be highly reproducible; it’s
a simple task of making up the media correctly, dissolving an appropriate amount of
compound, applying it to the agar plates, adding synchronously ageing worms: then
watching them die over the course of a month. Surely, such a straightforward exper-
iment should be highly reproducible. However, there are many contrary examples.
In the interest of full disclosure, the main findings of one of my own most-cited
manuscripts failed to be reproduced by another respected lab (although we could
always reproduce our own data). This pattern has been repeated down the years in
other labs. Not only does this reduce the confidence in the particular compounds
being studied, but while the experiments appear deceptively simple, in reality there
may be many unreported or undetected differences in protocols, lab strains and
bacteria between labs that can have profound effects on the results obtained. This
suggests a need to increase our documentation of how we go about such studies.
434 G.J. Lithgow
Another hurdle to translation is that the kinds of things we assay are largely dis-
connected from the day-to-day measures that clinical science cares about. Efforts to
humanize the worm are therefore very valuable. For example, going back to Chris
Link’s original strains expressing human amyloid beta in the early 1990s, the worm
has made an enormous contribution to our understanding of proteostasis, as it relates
to both age-related disease and ageing. However, even the most dramatic worm
discoveries don’t have clinicians cancelling their golf round to try to get a call into
the worm lab. Our science usually is at the bio- end of the biomedical spectrum,
even when we go out of our way to introduce some features of a human disease. In
fact, the phenotype that is the fundamental basis of the field, lifespan, rarely reso-
nates at a practical level with anyone deeply concerned with developing therapies
for people.
Of course some researchers have questioned the lifespan phenotype as the best
measure of healthy ageing. Some have made good progress in developing “healths-
pan” measures that may indeed reflect measures made in people, such as the famous
frailty index. Along the way, there will be considerable debate about what assays are
best or how they are interpreted. A parallel debate is happening amongst clinical
scientists interested in human clinical trials in ageing. Recently, there has been con-
siderable interest in using a large panel of equally weighted measures of health and
age-related changes in mouse ageing experiments. A similar approach may become
standard for the worm and help us convey the degree to which some modulations of
ageing appear to increase healthspan. One thing is clear, increasingly sophisticated
ways of looking at tissue, cellular and organelle function will be providing more
accurate and meaningful ways to assess healthspan. The ongoing development of
automated handling and high content analysis for C. elegans is spectacular. Whether
automated lifespan machines become commonplace remains to be seen, but adop-
tion of automation for various healthspan measures is a priority for the field. It’s
probably too soon to sell all the dissection scopes, but clearly microfluidic devices
will be generating a lot of the important data in years to come.
Perhaps the major hurdle for translation is that we do not run into people with the
skills to make it happen on a daily basis. Of course there are large translational cen-
tres everywhere, but I would speculate that not many of them contain worm labs.
There are exceptions, but most worm labs are based in molecular, cellular biology
departments. Likewise, many of us have given “Grand Rounds” presentation at
local hospitals but rarely does this result in a clinical or preclinical collaboration.
Why are we not more connected? Only a handful of worm ageing people attend
conferences like the Gerontological Society of America’s annual meeting; thou-
sands of healthcare providers and social gerontologists mingle at such meetings in
almost total ignorance of the progress that has been made in understanding the
underlying process at the heart of healthcare. Most of us prioritize the annual worm
meeting but we also have to think about who else needs to understand the discover-
ies we are making. We need to think about reaching the people who can actually
translate our science. While most scientists like to collaborate, translation requires
a truly interdisciplinary approach. Very different kinds of scientists and clinicians
need to work closely with each other and this generally needs the incentive of
19 The Future of Worm Ageing 435
increased access to funding and other resources. Whether we believe that this kind
of team science really works, it seems likely that ageing research is increasingly
going to require such organization. We have seen over the last 10 years an increase
in multi-model publications, and reviewers more frequently ask a worm lab to pro-
vide evidence of conservation in a mammalian cell system. This may morph into
requests to see clinical relevance of discoveries made in worm ageing.
Will worm ageing labs exist in 10 years? Of course, but the overall picture may be
quite different. The extent of Federal funding of academic institutions across the
world will be a major factor but equally important will be the attitudes of Deans and
Department Chairs who do the recruiting; if there is grant money for ageing research,
what kind of researchers are likely to attract it? That’s who the Deans and Chairs go
after. The worm remains competitive today because of the fast pace and low cost of
doing very significant science. If, however, there is a perception that the worm has
given up most of its ageing secrets it may not be seen as investment worthy.
How do we know if we have made all the major discoveries already? If we had
one or a series of interventions that rendered worms immortal then we might say
that we were close to understanding ageing. But what if that is impossible? We can
probably already say that it is not possible from the manipulation of a single gene
nor by the many tens of thousands of chemical that have been tested for lifespan
extension. So for now, there is plenty of scope for trying to understand all this. We
do know that ageing is complex. The complexity is challenging and possibly the
major immediate challenge. We have not ventured far into understanding how so
many genes modulate lifespan or how they interact. We also haven’t explored the
natural variation in ageing in truly wild strains. It has been suggested that many
interventions that increase lab lifespan are merely “fixing” problems of lab adapted
animals being housed under less than ideal conditions. The use of wild strains may
be helpful in this regard.
The future? Scientists are not particularly good at predicting the future. There are
clearly major challenges in growing and maintaining labs and expanding their num-
ber across the world. Sometimes it seems like we know a lot about worm ageing but
understand very little. The next generation have the challenge of integrating the ever
growing body of information into much clearer understanding of the relationship
between ageing and disease (sometimes referred to as geroscience). This will
require different and broader skillsets and collaborations. In the end though, I think
that a bright, young graduate student will still want to join a worm ageing lab 10, 20,
50 years from now. They might be entering an interdisciplinary team. They might be
working directly with clinicians. They will likely conduct experiments in multiple
models. But the main reason they will want to join a C. elegans ageing lab is that
this field was, is, and still will be cool!
Index
A B
Ageing, 2–5, 42–44, 46–49, 51, 52, 54–56, 63, Behavior, 10, 19, 21, 22, 36, 85, 87, 88, 164,
65, 67–72, 85, 87, 91–100, 109–112, 173–176, 194, 195, 198, 199, 376, 395
118, 119, 121, 124, 126–128, 164, 167,
168, 170–181, 192–194, 198, 202–204,
225–233, 246, 250, 252–261, 266–278, C
286, 290, 291, 296, 298, 299, 307, 308, Caenorhabditis elegans (C. elegans), 2–5,
311, 312, 316–319, 321, 322, 332, 10–17, 20, 22–30, 32, 35, 36, 41, 42,
335–337, 339–347, 356, 367, 369–378, 45, 46, 48, 51, 53–56, 63, 64, 67–72,
380, 381, 412, 413, 416–424, 432–435 84–89, 92–97, 99, 100, 110, 111, 113,
Age-related diseases, disease models, 63, 114, 118, 121, 123–125, 127–128,
69–71, 85, 90, 178, 179, 181, 207, 138–146, 148–156, 164, 165, 169–181,
233, 255, 260, 266, 267, 270, 272, 192, 194, 196–200, 202, 203, 207,
292, 311, 346, 347, 367, 371, 374, 221–229, 231–233, 246–255, 257, 259,
377, 432, 434 260, 266, 267, 269, 271–278, 286–292,
Aging, 10, 12–25, 27, 29–36, 138–150, 294–299, 308, 309, 312, 313, 315–322,
152–156, 164, 167, 168, 171, 172, 332, 334, 336–347, 356, 357, 363–381,
174–178, 202–204, 251, 254, 255, 258, 394, 395, 399–402, 413, 417, 418,
395, 397 420–424, 433–435
ALH-6, 396, 399 Cap-dependent, 289
AMP-regulated protein kinase (AMPK), Cuticle, 12–19, 22, 26, 30, 31, 225, 233
194, 195, 336, 339, 366, 368–371,
375, 378, 399
Anatomy, 10, 12, 14, 16, 20, 23, 25–28, 30, D
32, 35, 36 daf-2, 2, 42, 46–48, 64, 96, 140, 168, 193,
Antioxidants, 65, 85, 87, 89, 90, 94–95, 253, 272, 291, 308, 337, 363, 421, 433
99, 176, 203, 219, 221–222, 226–230, Dauer, 2, 12, 41, 64, 145, 194, 221, 273, 319,
254, 259 373
Atg8/LGG-1/2, 332–333, 335, 345–347 Decline, 10, 12, 18–20, 23, 26, 29, 32, 33, 35,
Autophagy, 4, 5, 19, 27, 70, 87, 89, 93, 96–97, 87, 96, 100, 124, 126, 138, 140–144,
99, 113, 115, 116, 118, 123–124, 127, 149, 150, 152, 155, 156, 163, 165, 167,
152, 164, 266, 268, 271–272, 277, 312, 170–178, 181, 192, 206, 226, 254, 255,
313, 317, 332–348, 365, 370–372, 374, 257, 259, 270–273, 346, 376, 377, 421
378, 395, 399 Diet, 143, 144, 148, 313, 318, 366–368,
Axon regeneration, 171–172 393–402, 413–418