1.   
Introduction

1.1  Healthspan

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The main purpose of aging research is to
identify signaling pathways and conserved genes that will extend the lifespan
of living organisms and improve the human experience of aging. However, it is
largely unspecified whether this will result in an extension of the healthy
time or just a period of frailty with increased occurrence of age-associated
issues (Bansal A., 2015). According to the
World Health Organization, an increased lifespan is accompanied by an increased
incidence of aging-associated diseases such as cancer and diabetes, which could
lead to an economic disaster of the healthcare system.  It is important thus, to consider that aging
is much more than a lifespan measurement and in future research, lifespan and
healthspan will always be intrinsically correlated.

1.2
C. elegans as model for aging

In order to answer important questions about
the biology of different organisms, a detailed study of model systems is
necessary and it is based on the idea that it is convenient to study
representative species at an extremely detailed level. In 1963, Sydney Brenner
introduced Caenorhabditis briggsae as
a potential model system to study the developmental processes in other
organisms. Later on, in 1974 Brenner would publish a milestone paper on the
genetics of a very related species, Caenorhabditis
elegans (Brenner, 1974).

C. elegans, with its short life cycle, can be
easily cultivated and is small enough to be handled in a larger scale and it shares
many fundamental cellular/molecular structures and biological characteristics
with more advanced organisms. In 1998, the complete genome (100-Mb) of this
model organism was published and a comparison of the human and C. elegans
genomes has confirmed that 60–80% of human genes are represented by a homolog
in C. elegans (Consortium, 1998). Today, we have a detailed knowledge
of C. elegans morphology, and together with its genetic map and complete
sequence of its genome it is easy to assume that this worm is now a major model
system in biology and can be used for genetic studies relevant to human biology
and disease.

Despite all the excellent advantages of C. elegans in aging research, there is
also an important disadvantage of this model for human aging research. C. elegans and nematodes in general, lack
many important organs/tissues including a brain, blood and internal organs.
This can easily lead to limited understanding of any tissue-specific signaling (Johnson,
2003).
However, many of the molecular pathways and genes involved in human are
conserved in C. elegans and thus this
model can serve as a template for understanding how to promote healthy aging in
humans.

The life cycle of C. elegans is extremely short, taking approximately 3 days under
optimal environmental conditions. Each hermaphrodite can lay about 300
self-fertilized eggs and when mated by males, hermaphrodites can produce up to
1000 fertilized eggs (L. Byerly, 1976). Under unfavorable
conditions the L1 larvae can molt to L2 larvae and if adverse conditions persist,
molts to a Dauer larvae. In this developmental decision, environmental
temperature and food availability are playing an important role. The Dauer larvae
exits when environmental conditions are favorable and then the worm molts to L4
stage.

 

Figure 1 C. elegans life cycle, adapted from www.wormatlas.org

 

 

As it is already mentioned, in adverse
environments (nutrient limitation, stress, shifts in temperature or
photoperiod), C. elegans forms a
resistant stage which is known as Dauer
stage. The Dauer larvae is an alternative L3 stage, a special diapause
stage in which the juvenile is resistant to a variety of stresses, shows a
special morphology and has an altered metabolism. The Dauer larvae has no mouth
or anus, does not feed and has a very thick cuticle. Figure 1 shows the
differences between a developing larva, such as L3 and a Dauer stage. In L3 the
nutritional intake is distributed from the intestine to different tissues,
while during Dauer formation, intestine and hypodermis are responsible to store
metabolic resources as lipids and glycocen (Antebi, 2008). These resources
will be used for the locomotion and nictation of the worm. Dauer are
non-feeding and they rely on their internal fat stores for survival. They are
known to be very long-lived, often living four to eight times longer than their
non-Dauer counterparts. When environmental conditions are favorable, worms molt
to L4 stage (Figure 2).

Figure2. Differences in L3 and Dauer larvae,
adapted from www.wormatlas.org

 

 

Apart from all the morphological and metabolic differences
between the long-lived larvae and the its non-Dauer counterparts, the Dauer
stage is also characterized by differential expression of a very large set of
genes (>2500 genes are non-Dauer and >2000 are Dauer specific).

 

1.3
Lifespan extension in C. elegans

The most
important genetic pathway that regulates life span in C. elegans is Insulin/IGF-1 signaling (IIS). The pathway starts
with the insulin/IGF-1-like ligands. These ligands will bind to the
INS/IGF-1-like receptor, which is known as DAF-2. The DAF-2 protein is
expressed all over the body of the worm and in 1993, Kenyon C. proved that
mutations in the daf-2 gene extend lifespan of C. elegans about 2-fold (Kenyon C, 1993).

Figure 3 shows that when the ligand binds to
the DAF-2 receptor, a chain of signaling proteins and other messengers is
transduced inside the cell. The longevity of daf-2 is known to be dependent on daf-16. DAF-16 is a transcription factor from the FOXO-family and
plays a key role in the life span extension mediated by reduced
Ins/IGF-signaling. PI(3)K (phosphatidylinositol-3OK-kinase) consists of two
subunits: AGE-1 and AAP-1 and plays an important role in the activation of
DAF-2 signal. The PI(3)K activity of the AGE-1/AAP-1 complex can be
counteracted by the triphosphate PIP3 phosphate DAF-18. The PIP3 intracellular
messenger molecule can bind on PDK-1, which is known that when mutated results
in increased longevity. For this reason, PDK-1 acts downstream of DAF-2, AAP-1
and AGE-1 in this pathway. PDK-1 phosphorylates the kinases AKT-1 and AKT-2
that also act downstream in this cascade. DAF-16 can be phosphorylated then by
AKT-1 and AKT-2 and the phosphorylated DAF-16 will be remained in the cytosol
where it is inactive. This means that active Ins/IGF-1-like signaling leads to
inactivation of the DAF-16 transcription factor.

 

Figure 3. C. elegans insulin/IGF-1 (IIS) like
signaling pathway, adapted from: An Overview of Stress Response and
Hypometabolic Strategies in Caenorhabditis elegans: Conserved and Contrasting
Signals with the Mammalian System  (Lant B, 2010)

 

Dietary restriction is also known to increase
life span in different species. In C.
elegans there a few techniques where lifespan can be improved such as
reduction of food availability (bacteria), growth in axenic media and mutation
of specific nutrient transporters in the gut.

Other very important regulators of lifespan in C. elegans are mitochondria. clk-1 was the very first mitochondrial mutant
and it was proved that life extension can also be due to disruption of
mitochondrial activity (S Felkai, 1999). However, the
increased lifespan provided by mitochondrial mutants can cause a slowing down
in other processes such as development, movement and eating behavior. In this
case, an increase in lifespan doesn’t translate into improved health span.

Recently, it became clear that an extensive use
of chemical and natural compounds can also increase the lifespan of model
organisms such as worms, flies and even mice. One of the first compounds to be
tested for their ability to improve the lifespan were the antioxidants and it
was based on Harman’s theory of oxidative stress (Fang Wang,
2011) (Wang X.,
2015).
Rosmanic acid is one of the antioxidant compounds; a chemical compound found in
a variety of plants with antioxidant properties and is been using for aging
studies.

 

 

 

1.4
Biological techniques developed and used in C.
elegans

One of the major trends in biological research
in order to investigate the function of genes is RNAi (RNA interference) and
its first application was in 1998 by Andrew Fire and Craig Mello for the
nematode C. elegans (Andrew Fire, 1998). Few years
later, (2006) they will receive the Nobel Prize for Physiology and Medicine for
their discovery.

RNAi refers to gene silencing by introducing a
double-stranded RNA (dsRNA) which leads to an efficient loss of the target
mRNA. Synthetic dsRNA introduced into cells can induce suppression of specific
genes of interest. During a functional genetics study, researchers can knock
down the genes of interest in order to inactivate the corresponding mRNAs by
introducing a double stranded RNA (dsRNA, a mixture of both sense and antisense
strands). In C. elegans there are
three ways of delivering dsRNA for an official gene knockdown: injection,
soaking and feeding. Injection of just a few molecules of dsRNA per cell is
known to be sufficient to completely silence the homologous gene’s expression.
RNAi can also be used for large-scale screens that systematically shut down
each gene in the cell in order to identify the components necessary for a
particular process. Detailed RNAi studies have been carried out in C. elegans and for each knock-down the
resulting phenotypes are available in Wormbase.

However, in order to discover the function of a
certain gene it is crucial to know where the gene is active inside the worm’s
body and when it is expressed during its development. One of the most popular
technique for the localization of gene expression at a protein level is via
reporter strains expressing green fluorescent protein (GFP), isolated from the
jellyfish Aequoria victoria. This
protein is encoded in the normal way by a single gene that can be cloned and
introduced into cells of other species. The primary advantage of C. elegans is
the transparency of its body which makes it ideal model for the localization of
the gene expression. The freshly translated protein is not fluorescent, but
within a few hours it undergoes a self-catalyzed post-translational
modification in order to generate an efficient and bright fluorescent color.
The transparency and the relative thinness of C. elegans allows for microscopic analysis in vivo without animal dissection. GFP tagging is considered as the
clearest and easiest method of showing the distribution and dynamics of a
protein in a living organism and thus it is a valuable tool in C. elegans research.

A consisting set of genetic C. elegans strains expressing
GFP-reporters to monitor the activation of cytoprotective signaling pathways
can be used for healthspan experiments (table below). More specifically, in
this project we will focus on DAF-16/FOXO a well-known key regulator increasing
longevity and likely healthspan. However, we can’t use GFP::daf-16 reporter strain, because it is
impossible to measure its activation, since DAF-16 is always present.
Therefore, we will study the activation of its target SOD-3 in an GFP::sod-3 reporter strain, because SOD-3 is
activated when DAF-16 is in the nucleus.

 

 

Reporter

Signaling pathway

HSP-6::GFP

HSP-16.2 is a heat shock-related stress
response protein in the cytoplasm of the cells. It is a direct mediator of
heat shock transcription factor (HSF-1) signaling, known to play an important
role in lifespan signaling.

HSP-16.2::GFP

HSP-6 is a heat shock-related stress response
protein, a robust marker of mitochondrial stress. Its potential interactions
with healthspan-promoting signaling cascades makes this protein an extremely
interesting candidate to reveal healthspan promoting mechanisms.

PHA-4::GFP

PHA-4 is a direct target of TOR-mediated
signaling, an important regulator of diet-induced changes in life- and healthspan.

NHR-57::GFP

NHR-57 is involved in the response to hypoxia
and activation of the NHR-57::GFP reporter is a reliable readout for HIF-1
activity (which is known to modulate lifespan in C. elegans).

GCS-1::GFP

GCS-1 (?-glutamine cysteine synthase heavy chain) is
predicted to function in a conserved oxidative stress response pathway, and
is under the control of the SKN-1/Nrf transcription factor.

GST-4::GFP

GST-4 (gluthathione S-transferase) is
involved in oxidative stress response, under control of SKN-1.

SOD-3::GFP

The activation of SOD-3 is monitored, which
is a target of DAF-16/FOXO
(well-known key regulator in longevity).

HSP-4::GFP

HSP-4 (Hsp70-related protein) is a
stress-induced regulator of aging related signaling in the endoplasmic
reticulum (UPR in ER).

 

 

 

 

2.
Methodology

2.1
Nematode and bacteria culturing

In its natural habitats, C. elegans feeds on a wide range of microorganisms such as Escherichia coli, Serratia marcescens etc. In this project we will use E. coli as a standard food source for C. elegans culture. This bacterium can
be easily grown on an agar surface. The agar mixture contains (NGM) commercially available agar,
peptone, NaCl, CaCl2, MgSO?, and is buffered at ph 6 with 25mM phosphate
buffer. Finally, sterol is added because C.
elegans cannot synthesize and E. coli
does not contain this essential molecule. NGM mixture will be sterilized in an
autoclave and the hot fluid agar will be poured into petri dishes. Then small
amount of C. elegans worms will be
spread over the agar surface. The hermaphrodites will use the bacteria as a
food source and will rapidly produce offspring by self-fertilization. Age-synchronized
worms will be used in all assays.

 

2.2 RNA interference (RNAi)

There are a few ways to carry out RNAi in C. elegans and in this project, we will
use the method of feeding. Bacteria producing the desired dsRNA will be given
to worms and either they or their progeny will be scored. This method is the
least labor intensive and most inexpensive method. For feeding, it is important
to allow the worms to feed for 2-3 days before they or their progeny are
assayed. Here, we will study the effect of 3
candidate polymorphisms.

2.3
Fluorimetry

Reporter
strains expressing Green Fluorescent protein (GFP) are available in the lab and
will be used in order to quantify the activation of the stress pathways. The
primary advantage of using GFP is its ability to visualize reporter gene expression
in live animals, in this case worms.

2.4
Compounds

To
determine the effects of the different compounds, wild type worms with the
chemical compounds at different concentrations will be treated in NGM to 20°C. To
prepare the treatment plates, rosmanic acid, trehalose and ampelopsin will
first be dissolved in sterilized distilled water and then each stock solution
will be added into autoclaved NGM plates at 55°C to the final
concentrations (Figure 4). In all assays, age-synchronized worms will be used.

 

Figure 4.
Graphical representation of compounds to culture media

 

3.    Research
proposal

 

WP number:

1

Start month:

February

duration:
(month)

4

Person months:

1.5

Title: The effect of healthspan promoting genes and compounds on
stress response pathways in C. elegans

Objective: The conduction of healthspan experiments using a reporter panel to
study promising healthspan-promoting candidates

Task(s):

Task 1.1: C. elegans and
bacterial culturing

Escherichia coli is chosen as a
standard food for C. elegans culture. This bacterium will be grown on a agar
surface. The agar mixture (NGM) will contain
commercially available agar, peptone, Nacl, CaCl2, MgSO?, and this mixture then is
buffered at Ph 6 with 25mM phosphate buffer. Finally, sterrol is added.

Task 1.2: Suppression of specific genes of interest- RNAi

In the RNAi experiment, we will study the effect of
candidate polymorphisms  favoring
healthy aging discovered in cohorts of healthy aged persons (available from
the Genome Wide Association Study or GWAS) on specific signaling pathways (HSP-6::GFP,  SOD-3::GFP and GST-4::GFP).
In order to knock-down the genes of interest RNAi
will be performed in the reporter strains by feeding: bacteria producing the
desired dsRNA will be fed to worms.

Task 1.3: Compounds

Rosmanic acid, ampelopsin (also known as dihydromyricetin) and trehalose will be tested in C. elegans for their ability to extend
the lifespan of C. elegans. These
compounds will be synthesized, stored to water solutions and added to culture
medium of wild type strains.

Task 1.4: Fluorimetry

Reporter strain expressing Green Fluorescent
Protein (GFP) will be used in order to quantify the activation of the stress
pathways. In order to evaluate the degree of activation during RNAi
treatments, a positive control (using a treatment that evokes the stress
signal) and a negative control (empty vector) will be used.