Molecular Biology of Aging: Genetics, Senescence, and Caloric Restriction Models

What is aging?

Birth --> Aging --> DeathAging is a process (developmental), not a stageDoes 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 fibroblastsgrowth 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:senescencep53-independent arrestcrisis

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.

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

p53 > p21

DNA synthesispRb (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.

Senescence Proliferation

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 G-rich 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.

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.

V D V TGV A I TG

Telomerase: Implications for cancer stem cells

1. Normal stem cells (like germ cells) have very long telomeres and high telomerase activity.

2. Cancer stem cells have shorter telomeres and lower telomerase activity.3. The therapeutic window for treating cancers of stem cell origin (majority of

cancers) is good.

}Therapeutic Window

2. Caloric restriction and organismic agingSohal 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 non-human 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 restrictionCalories are limited by 30-40%. Extends lifespan. Reduces pathology.

Gene silencingSilencing 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

AcAc

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Co-ActivatorP300/CBP

Cb

fß

RU

NX

2

AP

1

Gene regulation by acetylation / deacetylation

YA

P

TEF

MMP13gene

Gene Activation

CH3CH3

CH3CH3

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HDAC6

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Xp21 gene

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Gene Repression

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

Aldose reductaseToxic aldehydes

Increasedglucose

Inactive alcohols

Sorbitol Fructose

NADP+NADPH

GSHGSSG

NAD+ NADH

SDH

GRBrownlee, Nature 2001

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;107:137-48Negative control of p53 by Sir2alpha promotes cell survivalunder 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 pBabe-Sir2- 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;124:315-329Genomic 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 certainenvironmental stresses: starvation, high temperature, and freeradical generators. Methuselah encodes a G-protein coupledtransmembrane receptor.

MiceMutations 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 mutatedRegulates telomerase activity by mediating the unwinding of the DNA replication D-loopThis 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 Model Functionp21sdi-1 fibroblasts growth arrest; differentiationras fibroblasts growth arrest; p53 stabilization; p16

increases ROS

p53 fibroblasts transcriptional activator of p21sdi-1Sir2 yeast silencing of genome: prevents transcription

of rRNA (rDNA locus); prevents aginghistone deacetylase: represses genes

(acetylated histones: active gene)

SNF1 yeast kinase; regulation of non-glucose carbon sourcesdaf-2 C.elegans insulin/IGF1R homologue increases agingclk C.elegans increases cell division, feeding (metabolic rate)

clk mutants show increased life-span

methuselah Drosophila shortens lifespanSOD Drosophila free radical; ROSCatalase Drosophila 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 degenerationin aged telomerase-deficient mice . Nature Letter ePub November 28,

2010. 2. Liao Genetic variation in the murine lifespan response to dietaryrestriction: from life extension to life shortening Aging Cell (2010) 9:

pp92–953. Paul Hasty Rapamycin: The Cure for all that Ails Journal ofMolecular Cell Biology (2010) 2: 17–194. Gary Taubes Live Long and Prosper. Discover, Oct 2010, p. 80

OTHER:1. Telomere Web Sitehttp://resolution.colorado.edu/~nakamut/telomere/telomere.html2. 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. 282:8564. 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 408:255-2628. Sinclair DA, Guarente L. Unlocking the secrets of longevity genes. Sci. Am. 2006 Mar;294(3):48-51, 54-7.