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Molecular Biology of Aging:
Genetics, Senescence, and Caloric
Restriction Models
What is aging?
Birth --> Aging --> Death
Aging is a process (developmental), not a stage
Does not cause death
Is it inevitable?
Does it just happen?
Or, is it programmed?
Is it a breakdown of normal function?
Think of it as programmed (genetically)
Aging: Cell --> organ --> organism --> population
Examples of programmed biological processes:
apoptosis, cell differentiation, aging
(1) apoptosis
 specific molecules and genes regulate it
it is a default pathway
proliferative signals must prevent it
(2) differentiation  actively maintained (cells are quiescent)
must counteract proliferative signals
also a default pathway
(3) aging

programmed (has a clock)
is a default pathway
must be counteracted to slow it down
inevitable (so far)
Topics of the Lecture
*Aging is characterized by changes in cellular
senescence.
*Caloric restriction experiments have given us valuable
clues into the aging process.
*Specific genes regulate functions common to aging and
cancer.
*Cancer and aging are intimately linked: assigned papers
1. Cellular senescence
Reddell (1998)
human fibroblasts
growth arrest (p53, p21)
telomerase and telomere shortening occurs in vivo too
Hayflick limit:
Somatic cells (human diploid fibroblasts e.g.) in vitro
lose their proliferative capacity after a certain number of
population doublings (PD).
Cells survive after this (post-mitotic).
But they cannot be stimulated to proliferate (unless
transformed).
Proliferative senescence is a well-defined cellular model
of aging.
Terminal proliferation arrest
Terminal proliferation arrest is a feature of senescence.
Mutations lead to cellular escape from senescence (p53 gene e.g.)
Cells with p53 gene mutations can also growth arrest.
Further suppressor gene inactivation or oncogenic changes allow continued
growth. (e.g p16 cyclin inhibitor)
These cells will also approach growth arrest unless additional genetic
changes occur (immortalization after crisis; SV40 T-antigen e.g.).
These are the three terminal proliferation states:
senescence
p53-independent arrest
crisis
also know as life-span checkpoints.
Is normal aging compatible with cancer?
How does p53 regulate terminal growth arrest?
DNA damage or a built-in “clock” (see below) may activate p53 and cells
become either growth arrested (DNA is repaired, healthy cell) or
undergo apoptosis (genetically damaged cell).
A third option is senescence which is distinct from temporary growth
arrest.
p53 > p21
Several genes are involved in life-span control:
Importantly, p53 increases expression of the
cdk inhibitor p21 (sdi1, waf1, cip1).
p21 mediates terminal differentiation or senescence in
G1/Go
Senescence
Proliferation
DNA synthesis
pRb (retinoblastoma) protein inhibits E2F (elongation factor) and inhibits
transcription. Hyper-phosphorylated pRb cannot bind E2F; transcription proceeds and
cells can enter the cell cycle.
Senescent cells fail to phosphorylate pRB. (why?…cyclinD declines and/or
p16 increases)
Normally, in cycling cells, cyclinD and cdk4 phosphorylate pRb. The p16 (Ink4)
protein can inhibit cyclinD and promote senescence.
The opposite is true in cancer: p16 allelic mutations and deletions lead to
immortalization and tumorigenicity.
Telomeres and DNA Replication
Telomeres
(the built-in clock)
The end-replication problem leads to shortened DNA “ends” (telomeres).
Possible mechanism of aging:
Telomeres shorten with each cell division because of the end-replication
problem.
Ends of chromosomes consist of repeats: 5'-(TTAGGG)-3’
A germ cell has 2.3X103 repeats (polymorphic): 30-200bp are lost per cell
division (5-30 repeats). Also occurs in vivo (possible clock).
Normally, cells express a reverse transcriptase (RT) that fills in the
gaps: Telomerase
Telomerase = RNA primer 5'-CCCTAA-3' + RT enzyme (elongates the Grich 3'end) + another protein component.
Telomerase is a ribonucleoprotein that uses its internal RNA component as
a template for the synthesis of DNA on the ends of chromosomes during
cell replication.
In mammals, telomerase is normally found only in embryonic cells, germ
cells and in low levels, in renewable tissue such as leukocytes.
Most somatic cells have no telomerase and thus can undergo only a limited
number of cell divisions before they senesce.
Telomerase is found at high levels in malignant cells allowing those cells to
divide indefinitely.
A correlation between high telomerase activity and increased proliferation
and tumor stage has been described.
Telomerase assays:
1. Telomere length-measured by hybridizing a radio-labeled telomeric (CCCTAA)3
probe to genomic DNA digested with HinfI and RsaI.
2. Enzymatic TRAP assays (PCR)-telomeric repeat amplification protocol assays
the activity of the human telomerase reverse transcriptase (hTERT).
Telomerase and Cancer:
Hahn et al. (1999) Nat Med. 5:1164-70.
Wild-type hTERT cDNA consists of 7 RT conserved motifs and
one unique TERT motif (T). Mutations in motif 3 which changes an
aspartate to alanine and a valine to isoleucine result in a catalytically
inactive dominant negative hTERT.
V D V TG
V A I TG
Expression of dominant negative hTERT inhibits telomerase activity in
several tumor cell lines, reduces the telomere length, and induces cell
death when the cells stop proliferating.
Tumorigenicity in vivo is completely abolished by dominant negative hTERT.
Therefore, telomerase extends cellular lifespan and inhibition of telomerase
effectively limits survival (even of tumor cells). Drugs that inhibit
telomerase should be useful to treat cancer.
Telomerase: Implications for cancer stem cells
1.
2.
3.
Normal stem cells (like germ cells) have very long telomeres and high
telomerase activity.
Cancer stem cells have shorter telomeres and lower telomerase activity.
The therapeutic window for treating cancers of stem cell origin (majority of
cancers) is good.
}
Therapeutic Window
2. Caloric restriction and organismic aging
Sohal and Weindruch (1996)
What is caloric restriction?
What does caloric restriction tell us about aging?
Reactive oxygen species (ROS)
Oxygen is used by aerobic organisms in many metabolic processes.
Reactive oxygen metabolites are generated from metabolism.
This results in a chronic state of oxidative stress leading to senescence
and loss of function.
The organism can neutralize these toxic metabolites with specific genes
(SOD; catalase; pyruvate) or reduce toxic by-products with liver
enzymes.
But this capacity declines with age. Solutions: produce less ROS (lower
caloric intake) or counteract (increased gene expression).
Evidence that oxidative damage is a major causal factor of senescence
and contributes to shorter life-span in Drosophila:
Overexpression of antioxidative enzymes in Drosophila increases lifespan and
reduces protein oxidative damage. Average lifespan increased from 60 days to
75 days in one line of fly.
Metabolic rate has an inverse relationship to maximum lifespan in several
species. That is, the daily calories consumed relative to body weight are
lower for long-lived species. However, total SOD or glutathione reductase
activity did not correlate with lifespan suggesting that a combination of
responses to oxidative damage is operative.
<-- 2-3 year old mice life-span
1/2 max increased from 30 to 48 mo
max increased from 36 to 55 mo
Reducing the amount of calories consumed
has a dramatic effect on body weight (A),
percent survival (B), and life-span (C) in
mice. Similar results have been obtained in
rats.
A combination of reduced oxidative damage
and improved repair capacity may be
responsible for these effects. Studies in nonhuman primates and in humans show
improvements in metabolic parameters, but
lifespan data is incomplete.
Caloric restriction
Bluher M, Kahn BB, Kahn CR (2003)
Extended longevity in mice lacking the insulin receptor in adipose tissue. Science 299:572-574.
CR increases longevity and is associated with reduced fat storage and
changes in insulin/IGF-1 pathways.
KO mice lacking fat-specific insulin receptor (FIRKO) have reduced fat
mass; do not show signs of age-related obesity. Food intake is normal.
Mean life-span increased 18% (about 134 days).
Reducing fat mass, without caloric restriction, increases longevity
through probable effects on insulin signaling.
Caloric restriction:
model systems to discover genes that regulate aging
Review article: Guarente and Kenyon 2000 Genetic pathways that regulate
ageing in model organisms. Nature 408:255-261.
Model systems to study aging:
Yeast; drosophila; fibroblasts; rodents; primates; human studies
Caloric restriction
Calories are limited by 30-40%. Extends lifespan. Reduces pathology.
Gene silencing
Silencing is transcriptional inactivation of genes. Yeast, like humans,
undergo aging and shut down expression of specific genes, which extends
the lifespan.
3. Specific genes and aging
Aging in budding yeast
Mortality curves
Gene regulation by acetylation / deacetylation
Co-Activator
P300/CBP
Ac
Ac
TEF
Ac
Ac
MMP13gene
Cbfß
RUNX2
YAP
Ac
Ac
AP1
Ac
Ac
Ac
Ac
Gene Activation
Ac
Ac
CH3
CH3
Gene Repression
MeCP
RUNX2
MeCP
HDAC6
CH3
CH3
X
p21 gene
Yeast silent information regulator (Sir) genes (Guarente) and the
control of aging.
Normally, the Sir proteins function to silence several loci in yeast including
telomeres and the mating type genes found at the HM loci.
Sir proteins are histone deacetylases + NAD+ cofactor = gene repression
As cells age, silencing is lost at telomeres and HM loci and is gained at the rDNA
locus (consists of 100-200 random repeats of genes of the large and small
rRNAs).
The amount of Sir2 at the rDNA locus is predictive of lifespan: sir2 deletions
have a short lifespan while strains with an extra copy of sir2 have extended
lifespan.
Therefore, an increase in rDNA silencing by Sir2 increases lifespan.
Remember: you need more rRNA to synthesize proteins and grow
This may be related to metabolic state (caloric restriction) since rRNA synthesis
occurs when abundant carbon sources are available to yeast (shorter lifespan)
while under starvation conditions less rRNA is necessary (longer lifespan).
Other yeast genes may also regulate aging (see review and Table).
ROS
Toxic
aldehydes
Aldose reductase
Increased
glucose
Inactive alcohols
Sorbitol
NADPH
NADP+
GR
GSSG
NAD+
SDH
Fructose
NADH
Brownlee, Nature 2001
GSH
Too much glucose increases Aldose Reductase, which depletes NADPH.
NADPH is needed by GR to generate GSH to reduce ROS.
Low GSH means increased ROS and oxidative stress.
SDH (Sorbitol Dehydrogenase) converts sorbitol to fructose, thus depleting NAD+.
Sir2 is a histone deacetylase that is
activated by NAD
High glucose --> PKA activated
---> less NAD…Sir2 less active
---> shorter life-span
Low glucose --> less signaling
--> more NAD…active Sir2
--> increased life-span
Luo et al
Cell. 2001 Oct 19;107:137-48
Negative control of p53 by Sir2alpha promotes cell survival
under stress
•
•
•
•
Sirt2 is the mammalian NAD-dependent histone deacetylase
Mediates gene silencing
Mammalian Sir2alpha physically interacts with p53
Sir2alpha represses p53-dependent apoptosis in response to DNA damage and
promotes cell survival under stress
• Relevance for cancer therapy: Inhibit Sir2 deacetylase activity
Inhibition of the apoptotic response to
oxidative stress by mammalian Sir2 .
Both mock-infected cells and pBabeSir2- infected cells were either not
treated (I and III) or treated with
200uM H2O2 (II and IV). 24 hr later,
the cells were photographed under a
microscope.
Mostoslavsky et al.
Cell. 2006 Jan 27;124:315-329
Genomic instability and aging-like phenotype in the absence of
mammalian SIRT6
• Sirt6 is an NAD-dependent histone deacetylase; found in nucleus and is
chromatin-associated
• Promotes resistance to DNA damage and suppresses genomic instability
• SIRT6-deficient mice develop lymphopenia, lower body fat, severe metabolic
defects; die at 4 weeks of age
• Function of SIRT6 is to promote normal DNA repair
• Aging-associated degenerative disease is a result of SIRT6 deletion
Osteopenia and lordokyphosis in SIRT6-deficient mice. X-irradiation and bone mineral density
analysis were performed on 3.5-week-old mice of the indicated genotypes.
Ageing in C. elegans: life-span and cell division
Short Life Span
Ageing in C. elegans: life-span and cell division
Daf-2 encodes an insulin/IGF1 receptor homologue which, when mutated,
leads to extended lifespan (Kenyon). This signalling pathway also contains
other genes which affect aging: age-1, a PI-3kinase and pdk-1, a
phosphoinositide-dependent kinase. Mutations in these genes also retard
aging.
The ligand for Daf-2 in C. elegans is Ceinsulin-1 which, when its expression is
inhibited by dominant negative methods, extends the lifespan.
Therefore, it is hormones that control aging in C. elegans. In fact, hormonal
control of SOD expression actually extends the lifespan of motor neurons by
40-50% without affecting metabolic rate.
Another locus, clk-1, appears to modulate the growth of C. elegans at
different temperatures. That is, the lower the temperature, the slower the
metabolism and the longer the lifespan.
Drosophila
The methuselah gene shortens lifespan. Mutations in this gene
extend lifespan by 35% and the fly are resistant to certain
environmental stresses: starvation, high temperature, and free
radical generators. Methuselah encodes a G-protein coupled
transmembrane receptor.
Mice
Mutations in the p66shc extend lifespan by about 30% and may
alter the cellular response to oxidative damage stress. However, this is
probably an oversimplification.
Premature aging in humans
•Aging is a highly conserved evolutionary process.
•Hormonal regulation in humans may turn out to be true.
•Reduced caloric intake may be related to hormone control.
•Delay in the aging process may also delay some of the
pathologies (cancer).
•It appears that the genetic diseases of aging (progeria,
Werner’s) are reminiscent of normal aging but cause
abnormalities that are extremely pleiotropic, severe, and
progressive.
•These diseases may be linked with normal aging at the level of
DNA damage and cellular damage by ROS.
lamin A mutation in a 15-year old girl
Premature aging in humans: Werner’s syndrome
The WRN gene is mutated
Regulates telomerase activity by mediating the unwinding of
the DNA replication D-loop
This may occur by activation of WRN helicase activity through
interaction with the POT1 protein:
Werner syndrome protein: functions in the response to DNA damage and replication
stress in S-phase. Cheng WH, Muftuoglu M, Bohr VA.Exp Gerontol. 2007
Sep;42(9):871-8. Epub 2007 May 10. Review.
Centenarian Studies
Geesaman BJ, Benson E, Brewster SJ, Kunkel LM, Blanche H, Thomas G, Perls TT, Daly MJ,
Puca AA. Haplotype-based identification of a microsomal transfer protein marker associated
with the human lifespan. Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):14115-20.
(Elixir Pharmaceuticals, Cambridge, MA 02139, USA)
•Identified linkage within chromosome 4 and identified a haplotype marker
within microsomal transfer protein as a modifier of human lifespan.
•Microsomal transfer protein is the rate-limiting step in lipoprotein
synthesis. May affect longevity by subtly modulating this pathway.
•This study provides proof of concept for the feasibility of using the
genomes of LLI (long-lived individuals) to identify genes impacting
longevity.
TABLE of some aging genes
Gene
p21sdi-1
ras
Model
fibroblasts
fibroblasts
Function
growth arrest; differentiation
growth arrest; p53 stabilization; p16
increases ROS
p53
Sir2
fibroblasts
yeast
transcriptional activator of p21sdi-1
silencing of genome: prevents transcription
of rRNA (rDNA locus); prevents aging
histone deacetylase: represses genes
(acetylated histones: active gene)
SNF1
daf-2
clk
yeast
C.elegans
C.elegans
kinase; regulation of non-glucose carbon sources
insulin/IGF1R homologue increases aging
increases cell division, feeding (metabolic rate)
clk mutants show increased life-span
methuselah
SOD
Catalase
Drosophila
Drosophila
Drosophila
shortens lifespan
free radical; ROS
free radical; ROS
Articles
C. p53 and aging article on premature aging (Student 1)
Assigned article:
•Tyner et al. 2002 p53 mutant mice that display early ageing-associated
phenotypes. Nature 415:45-53.
Background reading:
•Ferbeyre and Lowe 2002 The price of tumour suppression? Nature 415:26-27.
D. SIRT1 and aging (Student 2)
Assigned article:
•Sinclair SIRT1 Cell 135, 907–918, November 28, 2008
Background reading:
•The genetics of ageing Cynthia J. Kenyon NATURE Vol 464; 25 March 2010
E. Additional Reading (Bibliography)
RECENT:
1. Jaskelioff et al Telomerase reactivation reverses tissue degeneration
in aged telomerase-deficient mice . Nature Letter ePub November 28,
2010.
2. Liao Genetic variation in the murine lifespan response to dietary
restriction: from life extension to life shortening Aging Cell (2010) 9:
pp92–95
3. Paul Hasty Rapamycin: The Cure for all that Ails Journal of
Molecular Cell Biology (2010) 2: 17–19
4. Gary Taubes Live Long and Prosper. Discover, Oct 2010, p. 80
OTHER:
1. Telomere Web Site
http://resolution.colorado.edu/~nakamut/telomere/telomere.html
2. Sohal and Weindruch 1996 Oxidative stress, caloric restriction, and aging. Science 273:59-63.
3. Hayflick 1999 Aging and the genome. Science 283:3019 Ibid. 285:838; ibid. 282:856
4. Greider and Blackburn 1996 Telomeres, telomerase, and cancer. Sci. Amer. 274:92-7.
5. Reddel 1998 Genes involved in the control of cellular proliferative potential. Ann NY Acad Sci
854:8-19.
6. Hahn, Stewart, Brooks, York, Eaton, Kurachi, Beijersbergen, Knoll, Meyerson, and Weinberg
1999 Inhibition of telomerase limits the growth of human cancer cells. Nat Med. 5:1164-70.
7. Guarente, L and Kenyon, C. 2000 Genetic pathways that regulate ageing in model organisms.
Nature 408:255-262
8. Sinclair DA, Guarente L. Unlocking the secrets of longevity genes. Sci. Am. 2006
Mar;294(3):48-51, 54-7.