Integrated Endocrinology and Metabolism

Lecture Notes
The use of nematode Caenorhabditis elegans in ageing research
Last time we spoke a lot about WHY it is useful to use model organisms
when investigating aging. Today, I build on that, and we will look a bit
closer at different technical approaches that we can use in these models,
and get more into the details about what we have learned about longevity
using these models and approaches.
It has been found that longevity mechanisms are maintained between
species.

why use other models? They are small usually

- Easy to handle and store
- Great genetics, mutants!
- First animal genome to be sequenced
- Many tools are developed
- Transparent
- Short life cycles and lifespands
- Human homologues
- Cheap!
Success of C.elegans in biological research
Sydney Brenner (b 1927), Berkeley, CA, USA, established C. elegans as
a novel experimental model organism. This provided a unique opportunity
to link genetic analysis to cell division, differentiation and organ
development and to follow these processes under the microscope.
Brenner's discoveries, carried out in Cambridge, UK, laid the foundation
for this year's Prize.
John Sulston (b 1942), Cambridge, England, mapped a cell lineage where
every cell division and differentiation could be followed in the
development of a tissue in C. elegans. He showed that specific cells
undergo programmed cell death as an integral part of the normal
differentiation process, and he identified the first mutation of a gene
participating in the cell death process.
Robert Horvitz (b 1947), Cambridge, MA, USA, has discovered and
characterized key genes controlling cell death in C. elegans. He has shown
how these genes interact with each other in the cell death process and
that corresponding genes exist in humans.

Integrated Endocrinology and Metabolism

Lecture Notes
Do worms really age behavioural senescence in C.elegans; decline in
memory and learning.
the classic genetic approach previously
mentioned has been found to be bias
free.

A c.elegans mutants daf-2 was found to live twice as long as the wild type.
Daf2 is an insulin like receptor. Lifespan is actively regulated by endocrine
signalling.

Remarkable longevity and stress resistance of nematode PI3K muatants,

mutation of age 1 results in a 10 fold increase in the lifespan. How does
insulin like signalling affect ageing?
The FOXO transcription factor DAF-16 is localised to the nucleus in daf-2
mutants. including antioxidant genes such as superoxide dismutase,
metallothionine, catalase, and glutathionine S-transferase
metabolic genes including apolipoprotein genes, glyoxylate-cycle genes,
and genes involved in amino acid turnover, and chaperones, particularly
small heat-shock protein genes, and antibacterial genes.
Daf2 receptor activity regylates the DAF-16/FOX0 transcription factor. In
terms of epistatic analysis, its a good idea to look at the interactions
between mutations and figure out genetic pathways.
so what are the transcription targets of
DAF-16? The secret of what ageing is
lies in discovering what biochemical
processes are regulated by daf-16/FOXO.
What are these biochemical processes?
Some might argue that we already know.

Integrated Endocrinology and Metabolism

Lecture Notes

How does DA-16/FOXO promote longevity?

1. Functional genomics use of high throughput techniques to exploit
the vast wealth of data produced by genome sequencing projects to
understand gene and protein function. A direct approach to
understand biological processes associated with ageing and
longevity.
Techniques to test different molecules in ageing on a large scale
- Transcriptomics: microarrays/RNA seq to determine differences in
gene expression levels.
- RNA interference, ie gene knock out
- Proteomics, differences ini protein levels and post translational
modifications.
- Metablomics, differences in small molecule metabolites.
Transcriptomics; microarrays
Genome wide approaches to identify DAF-16 target genes. Microarrays
and whole genome RNA sequencing. Used to identify the mRNA signature
of a sample or the differences in the mRNA expression between samples.

RNA mediated interference RNAi

Introduce double stranded RNA that
targets gene
X. mRNA of
gene X
degraded and
no more likely
less protein is
produced.
Which genes downstream of daf-2 are required for
longevity? Daf-2 mutants are long lived. Use of RNAi
to knock down candidate gene in daf-2 mutants? Is the gene required for
the longevity of daf-2?

Integrated Endocrinology and Metabolism

Lecture Notes
The search for longevity genes: stress resistance
DAF-16/FOXO upregylates the expression of many genes whose protein
products fight various forms of stress
1. Oxidative damae free radicals from mitocgondrial respiration
damage DNA and proteins. Protection by antioxidants.
2. Heat stress causes misfolding of proteins. Protection by
chaperones.
3. Pathogens causes infections. Protection by antimicrobial and
immunity proteins.
Stress resistance
Molecular chaperones are required for daf-2 longevity. Observations were
that not all genes regulated by daf-2 contribute to lifespan. The
contribution of each gene to daf-2 longevity is small.
All of the above defence systems contribute to daf-2 longevity.
Humans:
1. Oxidative damage increases with age.
2. Protein aggregation and misfolding increases with age.
3. Infections increase with age.
There is an increase in the expression of metabolic genes in daf-2 mutants
for fat storage and fat metabolism. Adiposity increases in ageing humans,
preferential storage of fat in abdomen and organs. Ames dwarf mice are
resistant to detrimental effects of high fat diet.
So do stress resistance and metabolism contribute to longevity?
CONCLUSIONS
1. Not all genes regulated by daf-2 contribute to life span.
2. The contribution of each gene to daf-2 longevity is small.
3. Several processes seem involved in longevity.
4. Daf-16 extends lifespan by increasing stress defences
5. Daf-16 extends lifespan by shifting metabolism.
The search for DAF-16/FOXO targets
1. Direct and indirect targets
Daf-16 increases the expression of gene X or direct binding to promoter of
gene Y results in expression of gene Y, protein Y alters expression of gene
X.
The DAM identification (DamID) procedure in C. elegans. (A) How DamID
works. A fusion protein consisting of DNA adenine methyltransferase
(DAM) and the protein of interest methylates GATC sites near binding
sites. Genomic DNA is digested with DpnI, which cuts only methylated
GATC sites. Adaptors are added, and DNA is digested with DpnII (which
cuts at unmethylated GATC sites) to assure selective amplification of
methylated DNA. A parallel DAMonly experiment is also performed to
control for nonspecific methylation. Samples are then labeled and
hybridized to arrays. (B) Schematic of plasmid constructs used for
preparation of transgenic strains. (C, D) Transgene expression. Nuclear

Integrated Endocrinology and Metabolism

Lecture Notes
localization of GFP was detected in UL1782 animals (expressing
DAMDAF16GFP) (marked with arrows in C) in body wall muscle and
anterior bulb of pharynx (circled) following heat shock. UL1787 animals
(expressing DAMGFP) (D) do not show nuclear localization. (E) DAF
16DAM methylation profile for ist1, one of several evolutionarily
conserved FoxO targets identified. (F) Average distribution of methylation
(DAF16DAM versus DAM) from peak center for 1135 peaks identified.

So
We

have identified pathways that

-

control longevity.
We have identified some processes that promote longevity.
How can we use this information to improve the way we age?