An undergraduate lecture on the biological aspect of senescence

1. Ageing: THE BIOLOGY
OF SENESCENCE
Aurnab Ghose 200218
200303

2. What is ageing?
Time-related deterioration of the physical functions
necessary for survival and fertility

3. Maximum Life Span
• Characteristic of a species
• It is the maximum number of years a member of a given species
has been known to survive
- humans ~ 120 years (Arking, 1998)
- tortoises and lake trout ~ >150 years
- domestic dog ~ 20 years
- laboratory mouse ~ 4.5 years
- fruit fly from eclosion ~ 3 months

4. Human Senescent Phenotype
senex (latin): old man or old age or advanced in age
- gray hair
- sagging and wrinkling skin
- stiff joints
- osteoporosis
- loss of muscle fibers and muscular
strength
- memory loss
- eyesight deterioration
- slowed sexual responsiveness

5. Ageing-associated pathologies
- cardiovascular disease
- cancer
- arthritis
- cataract
- osteoporosis
- type 2 diabetes
- hypertension, and
- Alzheimer's disease

6. 1. Occurs in all individuals of the population
2. Produces changes in function/ structure
3. Changes increase with age (progressive)

7. Biological Theories
Feature theory:
Genetically determined
Intrinsic to biological organization
(Hayflick limit)
Defect theory:
Accumulation of damages (wear and tear)

8. Causes of ageing
A. Wear and Tear
B. Genetic Instability
C. Oxidative damage
D. Mitochondrial genome damage
E. Telomere shortening
F. Genetically programmed ageing
G. Gene silencing
H. Caloric restriction
I. Hormonal control

9. A. Wear and Tear
(Weismann, 1891; Szilard, 1959)
• As one grows older, small traumas to the body and genome
build up
• At the molecular level
- the number of point mutations increases
- efficiency of the enzymes encoded by the genes decreases
- mutations occurring in a part of the protein synthetic
apparatus leads to the production of faulty proteins (Orgel,
1963)
- mutations in the DNA-synthesizing enzymes (DNA
polymerases) leads to an enhanced overall rate of mutation
in the whole organism (Murray and Holliday, 1981)

10. B. Genetic Instability
Mutational alterations such as point mutations in the DNA,
microsatellite expansions or contractions, amplification and
deletion of DNA sequences, gene rearrangements and
structural or numerical chromosomal aberrations
DNA damage
Genome Instability

11. Accelerated ageing
Segmental Progeroid Syndromes
- genetic disorder in which various tissues, organs or
systems of the human body age prematurely
• Bloom syndrome
• Cockayne syndrome
• Down s syndrome
• Progeria (Hutchinson-Gilford Progeria syndrome)
• Werner syndrome
• Xeroderma pigmentosum
Defective DNA repair is associated accelated ageing

12. Hutchinson-Gilford Progeria syndrome
- first identified in 1886 by Jonathan
Hutchinson and described independently
in 1897 by Hastings Gilford.
- rare genetic disease (1 in 8 million)
- Median age of death is 12 years
(mutation in Lamin A gene)

13. Causes of ageing
A. Wear and Tear
B. Genetic Instability
C. Oxidative damage
D. Mitochondrial genome damage
E. Telomere shortening
F. Genetically programmed ageing
A. Gene silencing
B. Caloric restriction
C. Hormonal control

14. C. Oxidative damage
Metabolism and its by-products result is reactive oxygen
species (ROS)
ROS poses a constant threat to macromolecules and creates a
state of oxidative stress (Martin et al., 1996)
- Cause lipid peroxidation, protein oxidation, damage to nucleic
acids
ROS species are detoxified by specific pathways that involves
the genes encoding superoxide dismutase (SOD) and
catalase

15. ROS are critical in the ageing process
• Drosophila melanogaster (fruit flies) overexpressing catalase and
SOD live 30 - 40% longer than control flies (Sun and Tower, 1999).
• In Saccharomyces cerevisiae (bakers yeast) deletion of copper-
zinc SOD (sod1- mutants) and manganese SOD (sod2- mutants)
leads to dramatic shortening of survival time. sod 1- sod2- double
mutants die within a few days (Longo et al., 1996).
sod2- mutants treated with agents that decrease superoxide
generation, the life span is extended (Longo et al., 1999).

16. Causes of ageing
A. Wear and Tear
B. Genetic Instability
C. Oxidative damage
D. Mitochondrial genome damage
E. Telomere shortening
F. Genetically programmed ageing
G. Gene silencing
H. Caloric restriction
I. Hormonal control

17. D. Mitochondrial genome damage
• Mutations in the mitochondria DNA could
- lead to defects in energy production
- lead to production of ROS by faulty electron transport
- induce apoptosis (programmed cell death)
• Longevity in mice could be lengthened by targeting catalase
to the mitochondria. Catalase removes H2O2. Mice with
targeted catalase had lower ROS, better heart function, better
eyesight as they aged. They also lived 20% longer than wild-
type mouse (Schriner et al., 1998).

18. Causes of ageing
A. Wear and Tear
B. Genetic Instability
C. Oxidative damage
D. Mitochondrial genome damage
E. Telomere shortening
F. Genetically programmed ageing
G. Gene silencing
H. Caloric restriction
I. Hormonal control

19. The end replication
problem
• Each DNA replication,
end of DNA loses 50~100
nt.
• Lose genes near the end
of chromosome
E. Telomere shortening
Telomeres and Telomerase

20. E. Telomere shortening
Telomeres and Telomerase
Telomeres are the ends of linear eukaryotic
chromosomes composed of non-coding tandemly
repeated sequences.
Eg. In humans, the sequence is TTAGGG
Function of Telomeres:
- serve as a buffer zone to protect coding genes
from end-replication problem
- serve as a gauge for mitotic age

21. Telomeric DNA provides a cushion of expendable non-
coding sequences to protect against the potentially
catastrophic attrition of important chromosomal material.
BUT,
In the absence of any compensatory mechanism, we
would be extinct due to the progressive shortening would
eventually exhaust the telomeric buffer zone!
E. Telomere shortening
Telomeres and Telomerase

22. The germ line and stem cells express telomerase that can
add telomeric repeats to the end of the chromosome to
compensate for the end replication problem
Telomerase is a ribonucleoprotein that has reverse
transcriptase activity.
The RNA component hTERC contains the complement
of the telomeric TTAGGG sequence
The protein component hTERT contains the reverse
transcription activity (Chen & Greier, 2006; Cristofari & Linger, 2006)
E. Telomere shortening
Telomeres and Telomerase

23. Telomerase activity
5 3
TERT (Reverse Transcriptase)
TERC (RNA template)
Okazaki and DNA Pol based synthesis

24. In humans, telomerase is turned off or down-regulated in
somatic tissues during development (Shay & Wright, 2006).
Therefore, telomeres shorten with each cell division.
This forms the basis of the counting mechanism that
limits the maximal number of divisions a cell can
undergo - replicative ageing.
A primary purpose of replicative ageing may be to serve
as a barrier for the formation of tumors.
E. Telomere shortening
Telomeres and Telomerase

25. Ageing of Normal Cells
Telomere
length
Doubling
1
Doubling
2
Doubling
80

26. Since telomeres are an essential key to cellular
replication, loss of telomeres is involved in the cell’s
inability to replicate.
Hayflick (1965) defined finite proliferative capacity or
replicative senescence as the cell s inability to divide.
Hayflick s limit: Number of divisions after which normal
cells stop proliferating
There are 2 pathways that limit the replication of
human cells (Patil et al., 2005)
- telomere dependent (telomere shortening and p53)
- induced by stress signals and dependent on
p16INK4A and pRb tumor suppressors (Patil et al., 2005)
E. Telomere shortening
Telomeres and Telomerase

27. Use of telomerase-immortalized cells in therapies for
age-related diseases
Shay and Wright groups (2005) have immortalized corneal epithelial
cells and keratinocytes with telomerase for the production of
engineered corneas for transplantation.
Telomerase-immortalized human bronchial epithelial cells retain
the ability to differentiate in organotypic culture with normal or
immortalized lung fibroblasts, indicating that normal cell
differentiation has been retained despite the expression of
telomerase (Vaughn et al., 2006).

28. Aging By Genetic Programme
Gene Theory
One or more ageing promoting genes active only
later in life
Evidence::
Microarray analysis
Death Genes or Gerontogenes

29. Microarray Analysis
C. elegans

30. Causes of ageing
A. Wear and Tear
B. Genetic Instability
C. Oxidative damage
D. Mitochondrial genome damage
E. Telomere shortening
F. Genetically programmed ageing
G. Gene silencing
H. Caloric restriction
I. Hormonal control

31. Gene Silencing as a Regulator of Lifespan
Sirtuins = Silent Information Regulator 2 (Sir2) proteins
Studies in S. cerevisiae have led to the conclusion that a gene (Sir2) involved in
promoting the silencing of chromatin may be a key regulator of ageing.
Sir2 proteins may repress the expression of key genes that promote ageing.
By increasing the expression levels, Sir2 would increase the lifespan of the
organism.
An increase in Sir2 expression is associated with lifespan extension in yeast
(Kaeberlein et al., 1999; Lin et al., 2000), C. elegans (Tissenbaum and Guarente,
2001) and Drosophila (Rogina and Helfand, 2004). Overexpression of another
Sirtuin (SIRT6) can extend the lifespan of mice (Kanfi et al., 2012)
Silencing is a process by which entire regions of the chromosomes
(encompassing blocks of genes) are rendered transcriptionally inactive.
Normal ageing is triggered by a gradual erosion in silencing.

32. Catalytic function of SIR2
SIR2 acts as a histone deacetylase and promotes silencing (Imai and Armstrong).
It is a novel kind of histone deacetylase in that it requires NAD (Nicotinamide
adenine dinucleotide) to function.
The histone deacetylase activity of SIR2 is required for all of its in vivo functions,
including promoting longevity.

33. Causes of ageing
A. Wear and Tear
B. Genetic Instability
C. Oxidative damage
D. Mitochondrial genome damage
E. Telomere shortening
F. Genetically programmed ageing
G. Gene silencing
H. Caloric restriction
I. Hormonal control

34. Caloric restriction
Caloric restriction (CR) typically refers to a diet in which calories are
limited by 30-40% compared with animals fed ad libitum.
Has been shown to extend lifespan in yeast, worm, flies, spiders and
mammals (Lin et al., 2000; Masoro, 2005; Tatar, 2007).
Exact mechanism by which calorie restriction extends life is unknown.
One hypothesis is that calorie restriction slows metabolism, thereby
slowing the production of toxic ROS, and in turn slowing ageing.
Indy (I’m not dead yet) was the first gene in the fly to be speculated to be
involved in CR. Reduction of Indy gene activity leads to lifespan
extension (Rogina et al., 2000).
INDY is a transporter of Kreb s cycle intermediates localized in the
plasma membrane of the fly s fat body cells (Knauf et al., 2002).

35. Age (months)
Caloric restriction

37. Caloric restriction
It has been shown that life-span-extending effect of CR is
blocked in flies mutant for dSir2 (Rogina and Helfand, 2004).
Citric acid cycle
Oxidative phosphorylation

38. Causes of ageing
A. Wear and Tear
B. Genetic Instability
C. Oxidative damage
D. Mitochondrial genome damage
E. Telomere shortening
F. Genetically programmed ageing
G. Gene silencing
H. Caloric restriction
I. Hormonal control

39. Hormonal control
There is a conserved genetic pathway, involving the response to insulin or
insulin-like growth factors, in mice, C. elegans, flies that regulates ageing.
Mutations that damage a gene called daf-2 doubles the worm’s lifespan
Kenyon et al., (1993) Nature

40. daf-2 gene encodes a hormone receptor
DAF-2 receptor
Cell
ageing
Hormonal regulation of ageing

41. The DAF-2 hormone receptor is similar to two human hormone
receptors: the receptors for insulin and IGF-1
Insulin or
IGF-1 receptor
CellFood Uptake
Growth
ageing?

42. Insulin receptor in Model Organisms
C. elegans Fruit Flies Mice
Insulin/IGF1 Insulin/IGF1 InsulinIGF1
DAF-2
(Insulin/IGF-1
Receptor)
Insulin/IGF-1
Receptor
IGF-1
Receptor
Insulin
Receptor
Longevity Longevity Longevity Longevity

43. AGING
GENETIC FACTORS
Apolipoprotein E2/3
DIET and LIFESTYLE
Low Calorie Intake
Physical and Mental Exercise
Dietary antioxidants
Dietary FOLATE
Oxidative stress
Impaired energy metabolism
Protein aggregation
DNA damage
Neuronal Dysfunction/Death
Behavioural alteration
Adaptation
GENETIC FACTORS
Apolipoprotein E4
ENVIRONMENT
High Calorie Intake
Physical and Mental Inactivity
Infectious Agents
Disease
Impaired Neurogenesis
and Regeneration
Glial Cell
Abnormalities
Neuronal dysfunction and Behavioural alteration

44. Summary
The ageing process like most biological process is subject to
regulation.
Several interacting agents may promote longevity - caloric
restriction, protection against oxidative stress and the
factors activated by a suppressed insulin pathway.
Single gene change also has been shown to lead to
longevity
As human life expectancy increases due to advances in our ability
to prevent and cure diseases, we are still left with a general
ageing syndrome that is characteristic of our species.