Senior Thesis

Molecular Mechanisms of Aging and the
Potential of Extending Human Lifespan
Mitchell S. Kirby
A Thesis Presented to Princeton University
In Partial Fulfillment
For the Degree of
Bachelor of Arts
In
Molecular Biology
Princeton University, 2011
© Mitchell Kirby
This paper represents my own work in accordance with University regulations
__________________________________
I further authorize Princeton University to reproduce this thesis by photocopying or by
other means, in total or in part, at the request of other institutions or individuals for the
purpose of scholarly research
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ACKNOWLEDGEMENTS
The success of this thesis would have been impossible without the help and support of
many individuals. I would first like to thank Dr. Leon Rosenberg for his time, energy,
and constant encouragement. When I first walked into his office in November of my
junior year, I had no idea what I wanted to write my thesis about, and it was only through
discussions with him that a topic I loved arose. Zach Liebmann was also instrumental in
this thesis as he first got me interested in the subject of aging. I would also like to thank
Princeton University for providing me with the funding to meet with leaders in the field,
and Drs. Leonard Guarente and Colleen Murphy for taking the time to meet with me. I
am also indebted to the American Federation for Aging Research for allowing me to
attend one of their conferences on aging and lifespan extension. Additionally, Princeton’s
Department of Molecular Biology has given me the opportunity to learn from the best,
and has furthered my passion for science and I thank them for that. My friends,
particularly the members of the Pi Tau, have been significant influences during my time
here, and I am convinced that I have learned as much through them as through my
coursework. I am also especially grateful to my parents, Hyde and Bonnie, and to my
sister Samantha, for all their love and support throughout my thesis, my years at
Princeton, and my life.
For My Family
TABLE OF CONTENTS
LIST OF FIGURES ....................................................................................................... iii
ABSTRACT .................................................................................................................. iv
CHAPTER 1: INTRODUCTION TO AGING .................................................................1
Early Theories of Aging ..........................................................................................4
Thesis Goals and Outline ........................................................................................7
CHAPTER 2: THEORIES OF AGING AND DETERMINANTS OF LIFESPAN ...........9
Stochastic Damage Theories ................................................................................. 11
Free Radical Theory ..................................................................................... 11
Mitochondrial Theory................................................................................... 15
Genome Maintenance and Aging .................................................................. 18
Other Stochastic Theories ............................................................................. 21
Program Theories.................................................................................................. 24
Telomere Shortening Theory ........................................................................ 24
Genetic Control Theory ................................................................................ 28
Discussion ............................................................................................................ 29
Lessons from Comparative Biology.............................................................. 29
Comprehensive View of Aging .................................................................... 32
CHAPTER 3: THE MOLECULAR NETWORK OF LONGEVITY .............................. 36
Molecular Pathways of Longevity ......................................................................... 38
The Insulin/IGF1 Pathway............................................................................ 38
The Target of Rapamycin (TOR) Pathway.................................................... 42
The Sirtuins .................................................................................................. 48
Discussion ............................................................................................................ 53
CHAPTER 4: PROPOSED METHODS OF LIFESPAN EXTENSION ......................... 61
Methods of Lifespan Extension ............................................................................. 62
Dietary Restriction ....................................................................................... 62
Rapamycin ................................................................................................... 70
i
Resveratrol ................................................................................................... 73
Discussion ............................................................................................................ 78
CHAPTER 5: THE POTENTIAL OF EXTENDING HUMAN LIFESPAN THROUGH
PHARMACEUTICAL INTERVENTION ..................................................................... 81
Creating Drugs to Extend Lifespan ....................................................................... 82
Current Pipeline of Drugs for Age-Related Diseases..................................... 82
Rapamycin versus Resveratrol...................................................................... 84
Properties of an Ideal Lifespan Extension Drug ............................................ 86
The Ethics and Economics of Lifespan Extension ......................................... 88
Improving Our Perspective on Aging .................................................................... 89
Future Research............................................................................................ 89
A Network Approach to Aging ..................................................................... 91
Final Thoughts ............................................................................................. 92
REFERENCES .............................................................................................................. 95
Chapter 1 .............................................................................................................. 95
Chapter 2 .............................................................................................................. 96
Chapter 3 ............................................................................................................ 101
Chapter 4 ............................................................................................................ 106
Chapter 5 ............................................................................................................ 111
ii
LIST OF FIGURES
Figure 1.1: Average Life Expectancy in the United States……………………..….…....3
Figure 1.2: A Timeline of Early Aging Research………………………………..………5
Figure 2.1: Reactive Oxygen and Nitrogen Species……………………………...…......13
Figure 2.2: The Vicious Cycle of Mitochondrial Theory………………………...……..16
Figure 2.3: The End Replication Problem….………………...………………........…….25
Figure 2.4: Comprehensive View of the Aging Process………………………...….......34
Figure 3.1: Structure of the Insulin/IGF1 Signaling Pathway……………………..….....38
Figure 3.2: The TOR Signaling Pathway……………………………………...…...........44
Figure 3.3: Activities and Localization of the Mammalian Sirtuins…….…...….....…....49
Figure 3.4: A Network of Signaling Pathways Regulate Aging……………………...…54
Figure 3.5: Model for Longevity Signals in Proliferating vs. Non-Proliferating Cells....58
Figure 4.1: Activating SIRT1 Improves Many Age-Related Diseases……………..........76
Figure 4.2: A Network of Signaling Pathways Regulate the Response to CR,
Rapamycin, and Resveratrol………………………………………………………......…79
Figure 5.1: Current Sirtris Pipeline………………………………………………........…83
Table 3.1: Lifespan Regulation by Various Signaling Pathway Components…………...55
iii
ABSTRACT1
The mechanisms that regulate cellular senescence, organismal aging, and species-specific
lifespan depend on a synergy of pathways that are multifactorial and extremely complex,
though not yet completely understood. Recently, the development of new molecular
techniques has elucidated, at least in part, the primary pathways involved in aging. In
parallel with the search to uncover the factors that control aging is the endeavor to
discover methods of extending lifespan, in hopes of living both youthfully and longer.
Specifically, dietary restriction regimens, along with rapamycin and resveratrol feeding,
have been shown to increase lifespan in a variety of species. The illumination of the
molecular mechanisms of aging, coupled with the means of extending lifespan, provides
a foundation from which to determine a complete network of pathways that regulate
aging and the place of various lifespan extension methods within it. Furthermore, this
information acts as a stepping stone from which to evaluate the potential of extending
human lifespan safely and effectively through pharmaceutical intervention.
1
Parts of Abstract Adapted from my JP.
Kirby, M. Molecular Mechanisms of Aging and Proposed Methods of Lifespan Extension. Princeton
University, 2010.
iv
CHAPTER 1
INTRODUCTION TO AGING
The Fountain of Youth, a mystical spring that bestows youth and immortality on
those who drink from it, captured the attention of Spanish Conquistadors exploring the
American continent. Though many centuries have passed, the fascination with delaying
aging and living forever has never dwindled. Today, scientists serve as contemporary
conquistadors, searching for the real Fountain of Youth in the form of pharmaceuticals
and health regimens that increase lifespan, compress aging, and decrease the onset of
age-related diseases.
The complex scientific quest for eternal youth begins with a deceptively simple
question: What is aging? At its most basic level, aging is defined as the accumulation of
changes over time (Bowen and Atwood 2004). Although the concept is usually associated
with organisms, almost anything can age, sometimes even favorably. Wine, for instance,
develops changes in aroma, color, and texture that consumers enjoy. Unfortunately,
organismal aging isn’t so pleasant. Lenny Guarente, a leading researcher on longevity,
describes aging as “a multitude of factors on the cell and organismal level going wrong at
1
once, leading to disease, and ultimately, death” (Guarente Interview 2010). For the
purposes of this paper, aging will be defined as the accumulation of diverse deleterious
changes in cells and tissues that are responsible for the increased risk of disease and death
with advancing age (Harman 2003).
Aging can be classified in different ways. Chronological aging, for instance,
simply measures age by the amount of time an organism has been alive, and is the most
widely used definition. Interestingly, some define age as the time until an organism’s
death rather than from its birth (Birren and Cunningham 1985). On the other hand,
biological aging relies on the physical state of an organism to define its age. While some
forms of aging are universal, meaning that all people share their characteristics, others are
probabilistic, in which changes only affect a portion of the aging population, such as
Alzheimer’s or heart disease (Stuart-Hamilton 2006). Furthermore¸ the aging of
populations occurs when the average age of its individuals increases - many times
resulting from increases in average lifespan.
Aging can be thought of as the biggest killer worldwide, leading to the deaths of
100,000 people each day (de Grey 2007). Consequently, scientists, as well as lay people,
invest incredible amounts of time and money into finding substances that delay aging.
Consumers pay outrageous prices for the tiny possibility of living youthfully, longer, and
are expected to spend nearly 300 billion dollars annually on anti-aging products by 2013
(Stibich 2009).
Are society’s anti-aging efforts working? The short answer is yes, as evidenced
by a steady increase in average life expectancy over the last 50 years (Figure 1.1). In
2010, the average person is expected to live about 10 years longer than someone alive in
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1960. This remarkable lengthening of life expectancy can be attributed mainly to the
elimination of infectious diseases, improvements in hygiene, and the adoption of
antibiotics and vaccines (Tosato et al. 2007). If we progress at this same rate, someone
born in 2125 can expect to live to 100.
Presently, average lifespan has increased due to advances that allow people to
survive the aging process longer, not by delaying aging, or by increasing an individual’s
maximal lifespan. Current treatments are like playing medical whack-a-mole, repairing
one age-related defect at a time until a new one pops up. But, what if scientists could fix
the underlying cause of aging? Recently, researchers have uncovered mechanisms in a
variety of species that may make this idea a reality. The use of pharmacological agents
that act on biological targets crucial to the aging process could potentially slow, stop, or
even reverse aging. As such, this paper explores what is known about the underlying
mechanisms of aging to assess the potential of creating drugs to extend lifespan.
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Early Theories of Aging
Before embarking on the quest for lifespan extension, it is helpful to understand
the context in which modern aging research began. Though cultures have pondered the
significance of aging and death for millennia, it was not until 1532 that Muhammad ibn
Yusuf al-Harawi published the first document on the subject. Profound for its time, alHarawi noted the behavioral changes that occur with age, as well as substances “known”
to augment the aging process (Hayat 2007).
Three hundred years later, Alfred Russel Wallace developed a theory to explain
the presence of aging based on his own theory of evolution, which he published even
before Darwin’s. He postulated that evolution would promote individuals to die soon
after producing viable successors so as not to allow the old to steal resources from the
young (Stipp 2010). Because it speculates that death is encoded within an organism’s
heritable information, this idea became known as the death-program theory and was the
first of its kind to give an explanation for why aging exists. Despite its revolutionary
perspective, critics have refuted this theory, arguing that it is teleological in nature
because it specifies a purpose, but no mechanism (Stipp 2010).
Tragically, aging research of the late nineteenth and early twentieth century
devolved into grotesque experiments and techniques aimed at instilling youth and
longevity. This “gland madness”, as it was later termed, started around 1890 when
Charles-Edouard Brown-Sequard injected himself with dog and guinea pig testicles and
noted incredible rejuvenating effects (Stipp 2010). The 1920s and 1930’s bore witness to
thousands of implantations of animal testicles in men hoping to gain back the virility of
their youth, many of whom died due to infections, immune reactions, and other
4
complications. Additionally, many men, including Sigmund Freud, underwent the
Steinach Operation, which was essentially a vasectomy and was thought to restore male
vigor by increasing the secretion of testosterone from the gonads (Bullough 1995).
Though gland madness marked the first social craze stemming from modern science’s
attempt to “cure” aging, it cast a thick shroud over any serious research on aging,
condemning it as a front for charlatans (Stipp 2010).
It was not until Peter Medawar that a modern theory of aging was developed. Still
standing as one of the cornerstones of gerontology, Medawar hypothesized that after the
age of reproduction, the pressure of natural selection disappears and “abandons us to the
ravages of time” (Stipp 2010). Functionally, Medawar theorized that random, detrimental
mutations could accumulate if they exerted their effects only after the age of
reproduction, as such mutations would avoid being weeded out by natural selection
(Medawar 1952). Thus, Medawar believed that as a result of selection, organisms live
long enough to reproduce and effectively care for offspring, but no longer.
George C. Williams was puzzled by Medawar’s evolutionary theory of aging. He
found it strange that “aging” genes would enter the genome under this theory, especially
given the number of short-lived creatures (Stipp 2010). Taking evolutionary theory one
5
step further to solve this problem, Williams hypothesized that “aging” genes must
increase fitness if they are to accumulate in the genome over time (Williams 1957). Thus,
the same genes that cause age-related decline late in life must confer some reproductive
advantage if they are to be preserved. He coined this idea “the antagonistic pleiotropy
theory of aging” because such genes have multiple effects: beneficial when young and
detrimental when old (Williams 1957). Oddly, this theory implies that the same genes
that render the vigor of youth also cause the organismal decline associated with aging. An
example of this may lie in tumor suppressor genes, which prevent uncontrolled cell
growth during youth, but may reduce the ability of cellular self-renewal late in life (Stipp
2010).
A few years later, an English biologist named Thomas Kirkwood hypothesized
that because cells have a fixed amount of energy available to them, they must budget it
accordingly (Kirkwood 1977). For instance, cells must allocate energy to maintenance,
reproduction, repair, metabolism and many other functions. As a result of this scarcity,
each cellular function does not receive enough energy to operate perfectly. Kirkwood
argued that this imperfection sets the rate of aging because cells devote only enough
energy to quality control to ensure reaching the age of reproductive ability. Kirkwood’s
theory became known as the “disposable soma theory” because he highlighted that our
bodies are no more than “disposable gene packages”, unprotected from failure after
reproduction (Stipp 2010). Interestingly, this suggests that long-lived organisms devote
more energy to quality control, and that the earlier a species reproduces, the earlier it
ages.
6
Thesis Goals and Outline
Though the evolutionary theories of aging highlighted in the previous pages
present interesting hypotheses for why aging arose, as well as a strong context to begin
studying the subject, they say nothing about the molecular processes by which aging
actually operates. These hypotheses are merely ideas that logically explain why aging
should exist, but are founded more in logic than biology.
In reality, the mechanisms that regulate cellular senescence, organismal aging,
and species-specific lifespan depend on a synergy of pathways that are multifactorial and
extremely complex, though not yet completely understood (Kirby 2010). Over the past
few decades, the development of new molecular techniques has elucidated, at least in
part, the primary pathways involved in aging. In parallel with the search to uncover the
factors that control aging is the endeavor to discover methods of extending lifespan, in
hopes of living both youthfully and longer (Kirby 2010). The illumination of aging
mechanisms, side-by-side with means of extending lifespan, will provide a foundation
from which to determine a complete multidimensional network of aging pathways and
the place of various lifespan extension methods within it (Kirby 2010). Furthermore, this
information will act as a stepping stone from which to evaluate the therapeutic potential
of methods thought to extend lifespan in a variety of species.
As such, this paper will first survey what is known about the molecular
mechanisms of aging and the biological determinants of lifespan. From this, it will delve
into pathways implicated in longevity. The understanding of these mechanisms and
pathways will allow an informed evaluation of the potential of drugs and health regimens
to safely extend human lifespan. Finally, this information will be leveraged to speculate
7
about the possibility of successful lifespan extension in humans and to suggest direction
for future research.
8
CHAPTER 2
THEORIES OF AGING AND DETERMINANTS
OF LIFESPAN
Before delving into the feasibility of lifespan extension and the specific molecular
pathways thought to modulate organismal aging, it is important to understand what is
known about the aging process. Though researchers have not pinpointed a single cause of
aging, they have identified a few biological sources from which aging phenotypes can
arise. From this, they have developed theories to explain both the aging process as a
whole and the determinants of lifespan.
The modern theories of aging can be split into two categories: the programmed
and the stochastic. Proponents of programmed theories argue that aging arises from a set
biological timetable, possibly the same one that regulates childhood growth and
development (Kasabri and Bulatova 2010). Of these, the Gene Regulation and Telomere
Shortening theories provide the most comprehensive account of the aging process and are
supported by the most evidence. On the other hand, stochastic theories suggest that
damage to cellular macromolecular integrity accumulates over time, eventually leading to
the functional decline of the organism, and ultimately, its death (Wilson III et al. 2008).
9
Free radicals, DNA damage, mitochondrial dysfunction, protein damage, and
inflammation have all been implicated as mediators of stochastic aging.
Regardless of category, both theories implicate “senescent” cells as units of
organismal aging, and thus try to explain how cellular senescence arises. Senescent cells,
which do remain metabolically viable, are characterized by increased volume and a
flattened cytoplasm, as well as alterations in gene expression, nuclear structure, and
protein procession (Ben-Porath and Weinberg 2004). The exact mechanism by which
senescent cells influence organismal aging remains unknown, though some observations
shed light on how this might function. For instance, fibroblasts, the stromal support for
most renewable epithelial tissues, produce degradative and inflammatory enzymes upon
senescence. These changes can result in disturbed tissue structure and function, possibly
creating a favorable environment for preneoplastic cells (Krtolica et al. 2001).
Furthermore, senescent cells have been found to accumulate with age in the skin, retina,
and liver, as well as in hyperplastic prostate and atherosclerotic lesions, both of which are
associated with aging (Cerone et al. 2005; Itahana et al. 2004). Additional theories
suggest that senescence can deplete stem cell pools or disrupt their function (Shawi and
Autexier 2008). Interestingly, senescence serves as an example of antagonistic pleiotropy
(Ch. 1): it is beneficial early in life by preventing cancer development, but becomes
detrimental later in life as dysfunctional senescent cells accumulate (Campisi 2005).
Though the theories of aging are many times presented or considered to be
mutually exclusive, a better perspective views aging as a complementary process, relying
on many or all of the theories for an adequate explanation. Furthermore, the goal of
identifying a single cause of aging has recently been replaced by the view of aging as an
10
extremely complex, multifactorial process (Kowald and Kirkwood 1996). Thus, a global
view is needed when debating about the aging process as a whole (Holliday 2006).
In the following pages, the major theories of aging will be explained along with
experimental evidence supporting each of them. Once these theories have been
elucidated, it will be possible to develop a complementary picture of how the aging
process may realistically function. This will be accomplished by analyzing which theories
have the best experimental support and which factors of aging they best explain. In
addition, evidence from comparative biology will be leveraged to help elucidate the
mechanisms that regulate organismal aging and the factors that determine lifespan.
Stochastic Damage Theories
Free Radical Theory
Free radicals are atoms or molecules with unpaired electrons. Since electrons are
most stably found in twos, free radicals are highly reactive and seek to steal an electron
from other molecules to create a pair. Though the first electron becomes stably paired, the
electron donating molecule now harbors an unpaired electron, making it a free radical. It
now steals an electron from a nearby molecule, and the chain reaction continues. Though
radical reactions can be favorable, in the case of many biological reactions, they can
create widespread protein, lipid, and DNA damage within microseconds (Weinert and
Timiras 2010). Thus, it is easy to picture a mechanism by which free radicals can lead to
an accumulation of biological damage over time, and possibly the phenotypes associated
with aging.
11
The origins of the free radical theory date back to the 1950s when they were first
implicated in aging by Denham Harman. Harman, a reaction kinetics chemist at Shell Oil,
became familiar with free radicals after using them in several chemical processes for the
production of petroleum products (Colman 2009). At the same time, he became
fascinated with the causes and potential cures of aging, leading him to leave Shell for
Stanford Medical School. After completing his medical internship, he was hired as a
research associate at the Donner Laboratory of Medical Physics at UC Berkeley, where
he began a search for a basic cause of aging (Stipp 2010). Because of his previous work
at Shell, he could easily imagine how free radicals could react with biological molecules,
thereby wreaking havoc on living cells. Although he was met with widespread
skepticism, he was finally able to get his “free radical theory of aging” published in the
Journal of Gerontology in 1956 (Harman 1956).
In cells, free radicals are most commonly found as reactive oxygen and nitrogen
species (ROS/RNS). ROS include hydrogen peroxide and superoxide, mainly generated
as natural products of cellular metabolism, while RNS are found in the form of
peroxynitrate (Figure 2.1). Peroxynitrate, though not a free itself, is produced by a
reaction of two free radicals - superoxide and nitric oxide (Pacher 2007). ROS and RNS
are sometimes referred to as “pro-oxidants.” On the other hand, “antioxidants” are
chemicals that can donate electrons to free radicals without becoming free radicals
themselves, thereby stopping the damaging chain reaction (Stipp 2010).
12
To gather evidentiary support for his theory, Harman began to carry out
experiments in a short-lived strain of mice known as Laf1. Upon feeding these mice 2mercaptoethylamine (2-MEA), an antioxidant used to treat radiation sickness, he
observed a 30% increase in their average lifespan (Harman 1968). At the same time, a
study by Alex Comfort showed similarly promising results when another antioxidant
called ethoxyquin, greatly boosted the lifespan of the same strain of mice (Stipp 2010).
Even though it seemed that the free radical theory was gaining support, some of the data
posed limitations on the ability of free radical reduction to extend lifespan in humans.
Though on average the mice lived longer, many did not. Additionally, the doses of
antioxidants given to the rodents posed toxicity risks in humans (Stipp 2010). Finally,
despite the fact that these studies showed an increase in the average lifespan of the mice,
not one of them showed an increase in their maximum lifespan.
Notwithstanding the limitations of the evidence described above, the timely
discovery of superoxide dismutase in 1969 by Irwin Fridovich and Joe McCord launched
the free radical theory into the spotlight of biology and gerontology. Isolated from the red
13
blood cells of cattle, superoxide dismutase is a class of enzyme that catalyzes the
conversion of highly reactive superoxide into less reactive hydrogen peroxide and oxygen
(Fridovich and McCord 1969). Hydrogen peroxide can then be broken down into oxygen
and water by catalase, a prevalent cellular enzyme.
Gradually it became clear that superoxide dismutase serves as an evolved defense
against the harmful effects of free radicals generated by natural cellular metabolism. The
discovery of superoxide dismutase in all aerobic organisms suggests that oxidative free
radicals are universal and may play an integral role in the aging process (Finkel and
Holbrook 2000). In support of this idea, it was found that inserting extra copies of the
superoxide dismutase gene into Drosophila increased their average lifespan by 30% (Orr
and Sohal 1994). These data indicated that free radical-scavenging enzymes are sufficient
to delay aging (Tower 2000 and Tosato 2007). Additionally, increased levels of
superoxide dismutase were observed in flies selected for greater longevity (Arking et al
2000). Furthermore, studies in C. elegans have demonstrated that long lived worms show
an age-dependent increase in superoxide dismutase and catalase activity (Larsen 1993).
Unfortunately, given as a medicine to humans, superoxide dismutase was eliminated too
quickly, showed immunogenicity, and was formidably expensive (Stipp 2010).
According to De La Fuente, the free radical theory has received the widest
acceptance because it offers the most plausible explanation of the biological reactions
that mediate the aging process (2002). Consequently, interventions aimed at limiting or
inhibiting the production of free radicals may reduce the rate of aging and the onset of
age-related diseases (Harman 2003). As a result, 3 billion dollars worth of antioxidant
supplements, like vitamin C, are sold each year to those seeking such effects (Stipp
14
2010). Additionally, many other theories of aging have their roots in free radical theory.
These theories explore the results of oxidative damage to specific cellular components,
including mitochondria, DNA, and proteins.
Mitochondrial Theory
In 1972, Denham Harman published an extension to his free radical theory, which
he termed the mitochondrial theory of aging. Mitochondria are organelles that oxidize
sugar via an electron transport chain (ETC) to release energy in the form of ATP.
However, this respiratory electron transport chain can prematurely leak electrons to
oxygen, making it a key site for the production of superoxide radicals (Finkel and
Holbrook 2000). Though several biological reactions contribute to the steady state levels
of superoxide, mitochondria have been found to be the largest producers (Cadenas and
Davies 2000). Thus, it seemed natural to implicate them as mediators of aging.
Functionally, the theory suggests that mitochondria produce large quantities of
free radicals, which go on to damage the mitochondrial infrastructure, leading to less
efficient respiration and the production of even more free radicals. Thus, with age, this
“vicious cycle” (Figure 2.2) causes oxidative damage to accumulate exponentially,
leading to the decline of many cellular components, and eventually senescence
(Mandavilli et al. 2002).
15
Figure 2.2: The Vicious Cycle of Mitochondrial Theory
Innate
Mitochondrial
Free Radical
Production
Production of
More Free
Radicals
Less Efficient
Electron
Transport Chain
Damaged
Mitochondrial
Infrastructure
Interestingly, Harman even explained the limitations of free radical theory in the
context of mitochondrial theory. Citing the observed inability of antioxidants to increase
maximum lifespan, he explained that the mitochondrial membrane prevented the
administered antioxidants from reaching the main sites of free radical production and
damage (Harman 1972, Stipp 2010). Harman’s theory was supported when mice,
genetically altered to express extra catalase in their mitochondria, displayed an increase
in their average and maximum lifespan of 17% and 21%, respectively (Liu et al. 2003).
Furthermore, additional mitochondrial superoxide dismutase increased Drosophila
lifespan, though similar effects could not be duplicated in mice (Bayne and Sohal 2002).
Mitochondria are unique among organelles in that they contain their own DNA.
Consisting of a closed circular molecule, the mitochondrial DNA (mtDNA) encodes 13
electron transport chain enzyme proteins, 2 ribosomal RNAs, and 22 transfer RNAs, all
needed to form the electron transport chain protein synthesis system (Linnane et al.
16
1998). mtDNA is generally attached to the inner mitochondrial membrane where free
radicals from the electron transport chain are frequently released. Adding to the vicious
cycle described above, it has been hypothesized that mtDNA mutations introduce altered
enzymes into the electron transport chain, thereby increasing the rate of free radical
production (Tosato et al 2007).
While nuclear DNA (nDNA) is protected by histones and repair enzymes,
mtDNA lacks both of these protective measures and is left exposed to the oxidation of
free radicals (South 2003). As a result, the level of free radical damage to mtDNA is
already 16 times higher in 3 month old rats compared to nDNA damage (Richter 1995).
Even in studies of the human brain, mtDNA has been found to be ridden with 15 times as
many mutations as nuclear DNA by the age of 70, with damage increasing exponentially
with age (South 2003, Hayakawa et al 1996). By the age of 90, only 5% of muscle tissue
mtDNA was isolated at its full length, and many cells completely lacked cytochrome
oxidase, a major component of the electron transport chain (Linnane et al. 1998).
An important piece of mitochondrial theory is that it provides a mechanism by
which damage can increase exponentially with age, which may be a necessary component
of a successful theory of aging. If oxidative damage occurred linearly with age, one
would expect to see signs of aging appearing linearly as well. However, this is not the
case; most of the phenotypes of aging seem to appear more and more rapidly with
increasing age. Additionally, the fact that longevity is more strongly associated with the
age of maternal death than that of paternal death, suggests that mtDNA inheritance may
be a key factor in the determination of lifespan (Tosato et al. 2007). Some suggest that
species and organismal lifespan is determined by the rate of mitochondrial oxidative
17
damage (Stipp 2010). On an organismal scale, Linnane et al. offers an explanation for
how mitochondrial theory explains aging. He argues that if mtDNA mutations occur in
many cells in a tissue, the function of that tissue will be comprised and will consequently
contribute to age-associated pathologies such as skeletal muscular and neurological
degeneration, heart failure, stroke, and ultimately death (1998).
Genome Maintenance and Aging
At the same time Denham Harman was developing his free radical theory, another
scientist was formulating an explanation for aging at the Salk Institute for Biological
Studies. Leo Szilard, best known for his discovery of the nuclear chain reaction, left
nuclear physics for biology after the bombing of Hiroshima (Zetterberg et al 2009). Like
Harman, who applied his knowledge of free radicals to biology, Szilard applied his
knowledge of radiation. As such, Szilard hypothesized that the accumulation of radiation
induced DNA mutations causes aging. This became known as the somatic mutation
theory. Furthermore, he developed a mathematical “target-hit model”, which allowed for
the calculation of the average and maximum lifespan of a species based on mutation rate
(Szilard 1959). Though there are many flaws with Szilard’s model, he was the first to
implicate genetic damage as a cause of senescence (Zetterberg et al 2009).
Interestingly, reactive oxygen species can induce DNA damage, thus linking
somatic mutation theory to the free radical theory. However, somatic mutation can also
be caused by exogenous factors, such as food agents, industrial genotoxins, and UV
radiation (Lindahl 1993). Thus, somatic mutation theory is in one sense broader than free
18
radical theory, but at the same time narrower, since free radicals can damage more than
just DNA.
The development of molecular assays to quantify and characterize spontaneous
DNA damage allowed researchers to gather evidence supporting a role for somatic
mutation in aging. These assays first and foremost led to the realization that spontaneous
DNA damage is both plentiful and extremely diverse (Vijg 2008). Thus, there existed
sufficient genome damage to theoretically lead to senescent cells and possibly organismal
aging. Concordantly, Birney et al. argues that even a few mutational events could have
profound effects on the regulatory circuitry of the genome (2007). Specifically, apoptosis,
senescence, and cell cycle arrest, all responses to DNA damage, could cause agingrelated tissue degeneration, in part by impairing or depleting stem cell responses (Bell
and Van Zant 2004).
Once it became clear that DNA damage was widespread, scientists sought to link
this damage to aging. One of the earliest discoveries was that damage, such as double
stranded breaks, abasic lesions, base modification, and cross-linkaging accumulate with
age (Mullaart et al. 1990). Studies in fruit flies showed that average lifespan correlated
inversely with mutation rate (Vijg 2008). Furthermore, the lifespan of mice was reduced
after exposure to DNA-damaging agents (Bernstein and Bernstein 1991). Most
importantly, human studies found that chromosomal aberrations in peripheral blood
lymphocytes, as well as mutations in almost all tissues increase with age (Ramsey et al.
1995; Dolle et al 1997). Interestingly, the rates of accumulation are tissue specific,
providing a possible mechanism by which many tissues fail before others.
19
Despite the observance of widespread DNA damage, cells from bacteria to
humans do have repair mechanisms in place to help ensure genome integrity. As such,
proponents of somatic mutation theory have implicated repair as a key site in the
modulation of the aging process. Genome maintenance is considered to include all
systems for sensing and signaling the presence of DNA damage, the repair of detected
damage, and the reconstruction of DNA higher order structure after repair is complete
(Vijg 2008).
In fact, genes encoding genome maintenance proteins belong to a category termed
longevity genes, a name given after the repair capacity of 7 species was directly linked to
their maximum lifespan (Sacher 1982; Hart and Setlow 1974). Repair capacity is
measured by assessing the ability of an organism to remove specific lesions produced by
treatment with low doses of genotoxic agents. A study of the repair capacity in rodents
indicated that older animals had lower levels of lesion removal, but the differences were
minor (Boerrigter et al. 1995). One specific repair pathway, known as base excision
repair, has received recent attention because of its importance in processing oxidative
DNA damage caused by ROS (Vijg 2008). In fact, most studies have shown that this type
of repair declines with age, possibly linking the somatic mutation theory with the free
radical theory.
With regards to DNA repair capacity and aging, a few limitations and
qualifications should be addressed. First, it should be noted that a decline in DNA repair
is not necessary for DNA damage to accumulate with age, and thus is not a required
component of somatic mutation theory. Furthermore, the assays that measure repair
capacity are hard to interpret and many other studies have found no link with aging (Stipp
20
2010). Finally, these studies say nothing about causality and are purely correlational. A
study to identify causality would need to generate defects in the genome maintenance
pathways and observe if this accelerates the aging process (Vijg 2008).
Despite the above limitations, studies of premature aging diseases, known as
progerias, implicate genome maintenance in aging. These diseases include Bloom
syndrome, Werner Syndrome, Rothmund Thompson syndrome, Cockayne Syndrome,
and Hutchinson-Gilford Progeria. Amazingly, all but one of these diseases involve
mutations in DNA RecQ helicases, proteins involved in DNA repair and recombination
(Kasabri and Bulatova 2010). Though these diseases cannot definitively say that defects
in genome repair cause aging, they do suggest that improper genome maintence can lead
to premature aging. It is a still unknown whether the causes of progerias are also
fundamental in the normal aging process (Kasabri and Bulatova 2010).
Other Stochastic Theories
While the free radical, mitochondrial, and DNA damage theories are backed by
the most evidence, some scientists propose other biological sources as mediators of the
aging process. Within the cell, proteins and membranes have been implicated as sites
where stochastic damage can accumulate. More broadly, some theorists cite the
populations of specific types of cells, such as immune cells, as important sites of damage.
Though these theories are presented as distinct, it is important to remember that the aging
process should be viewed as both complimentary and multifactorial. Thus, these
upcoming theories should be understood in the context of the theories already presented.
21
Error-Catastrophe theory argues that the production of proteins, in addition to
DNA, can be flawed (Wilson III et al. 2008). These damaged proteins accumulate, and
eventually lead to the phenotypes of aging. Somatic mutation, proteosome dysfunction,
pharmacological intervention and oxidative stress have all been suggested as causes of
protein dysfunction (Sander et al. 2008).
More specifically, cross-linkage theory argues that agents form covalent bridges
between macromoleucles, leading to cellular dysfunction (Mullaart 1990). Collagen, an
abundant protein in skin, cartilage, arteries, and tendons, has been found to accumulate
cross-links with age. Cross-links in this highly essential protein are thought to cause
numerous problems from high blood pressure to wrinkles (Stipp 2010).
Interestingly, free radicals have also been implicated in protein damage theories
by fostering cross-linking reactions. For instance, free radicals help form “Advanced
glycation end products”, or AGE’s, in which sugar molecules react with proteins, in
effect, gluing them together (Stipp 2010). These AGE’s are dysfunctional and are many
times targeted for degradation. Additionally, one study found that nearly half of the
protein taken from elderly people was scarred by free radical damage (Stadtman 1992).
Another site of damage implicated in aging is the plasma membrane. The plasma
membrane is a lipid bilayer, rich in protein, which serves as a selective barrier and a
regulator of cellular communication. Thus, defects in the membrane could seriously
impair the ability of cells to function efficiently. For example, lipofuscin, which are
pigment granules containing lysosomal digests, have been found to accumulate with age
near the plasma membrane, leading to membrane dysfunction (Amenta et al. 1989).
Additionally, lipofuscin accumulation is associated with many age-related diseases such
22
as Alzheimer’s and Parkinson’s (Amenta et al. 1989). Finally, membrane theory can also
be linked to the previous theories because such damage could originate from free radicals
or DNA mutations.
Immune theory, on the other hand, argues that a decline in immune system
functioning leads to an increased vulnerability to infectious disease and thus aging and
death (Kasabri and Bulatova 2010). Franceschi, who first proposed the theory, argued
that the immune system represents the most powerful mechanism to face stressors
(Franceschi et al. 2000). The theory was supported in 2006, when lymphocytes were
found to harbor increasing mutations with age (Suh and Vijg). Furthermore, the
functioning population of T cells and the levels of interleukin 2, which stimulates T cell
proliferation, have been found to decline with age (Kasabri and Bulatova 2010).
It should be noted that some scientists categorize immune theory as programmed;
however, it should be considered to be at least partially an extension of the stochastic
damage theories. For example, the study by Suh and Vijg indicates that damage
accumulates in immune cells, most likely leading to the observed decrease in immune
function. However, it is possible that immune system decline is regulated by programmed
theories as well.
Theories that implicate groups of cells or whole systems in aging, like immune
theory, should fall into a different class of theories. These theories should not be viewed
as the basis for aging, but as potential mechanisms by which fundamental cellular aging
can lead to aging on an organismal scale. For instance, stochastic damage accumulation, a
fundamental cause of aging, can lead to a decline in immune function, which in turn can
modulate organismal aging. Thus, immune theory should not be viewed as a fundamental
23
cause of aging, but more of a middle man in the process. Furthermore, a few other
stochastic theories exist, including neuroendocrine theory, but fall into this “middle-man”
category and will not be addressed here.
The free radical, mitochondrial, and genome maintenance theories offer the best
view of stochastic aging because other explanations are rooted in these theories. Finally,
these theories should be viewed as complementary. A realistic and comprehensive look at
the aging process will be presented at the end of this chapter to help foster a better and
more complete understanding.
Program Theories
Telomere Shortening Theory
Until the 1960s, it was believed that vertebrate cells had the ability to proliferate
indefinitely. However, this was overturned when Leonard Hayflick realized that normal
human cells stopped replicating in culture. From this, he hypothesized that the cells
possessed some “molecular clock” to keep track of how many times they had replicated.
Intrigued, Hayflick mixed male fibroblasts that had divided many times with female
fibroblasts that had only divided a few times. His hypothesis was confirmed when only
female cells were observed after a certain number of cell divisions (Hayflick and
Moorehead 1961). Thus, the cells had some way of recording how many cell divisions
they had been through, and at some point (around 40 divisions), now termed the
“Hayflick Limit”, stopped replicating.
This mechanism, which prevented normal cells in culture from replicating after a
certain number of divisions, was termed cellular senescence (Hayflick 1965).
24
Furthermore, not only were these cells terminally arrested, they also had altered
physiologies and decreased function (Campisi 2003). Naturally, Hayflick proposed that
this proliferative limit could be at the heart of the aging process, and in 1965 published
the cellular senescence theory of aging (Hayflick 1965). This theory hypothesized that
senescent cells, produced via a replicative limit, accumulated and caused functional
decline, eventually leading to death.
In 1971, Alexei Olovnikov identified a mechanism to partially explain the
existence of replicative senescence – the end-replication problem (Figure 2.3). It was
known that DNA polymerase can synthesize only in the 5’ to 3’ direction. Furthermore,
since RNA primers are removed, the lagging strand is never fully replicated and loses
base pairs equal to the length of the primer with each cell division (Olovnikov 1971).
Thus, genes at the ends of chromosomes lose portions of their code with each cell
division.
Figure 2.3: The End-Replication Problem
Leading Strand Synthesis
5’
3’
3’
5’
5’
3’
3’
5’
Parental DNA
Leading Strand
Lagging Strand
Lagging Strand Synthesis
5’
3’
3’
5’
5’
3’
3’
5’
5’
3’
3’
5’
Unreplicated
Region
Adapted from Senescence.info, Integrative Genomics of Ageing Group
25
RNA Primer
However, the presence of telomeres, identified almost five years beforehand,
reconciled how cells maintain appropriate functioning even with the end-replication
problem (Berendes and Meyer 1968). Telomeres, located at chromosome ends, are highly
specialized DNA sequences consisting of repeated 5’-TTAAGGG-3’ sequences in
humans (Ahmed and Tollefsbol 2001). The ends of telomeres are composed of single
stranded 3’ G-rich overhangs bound by telomere binding proteins. These proteins help
form a T-loop structure, which protects the telomere ends from recognition as double
stranded breaks by DNA repair machinery (Smogorzewska and de Lange 2004). Thus,
telomeres serve as buffer regions, allowing the loss of DNA due to the end-replication
problem without the degradation of genetic information.
Additionally, the shortening of telomeres with each cell division provides a
mechanism to explain the presence of the Hayflick Limit. Telomeres shorten with
successive cell divisions until they reach a critically short length, at which time they
trigger permanent growth arrest and the phenotypes of cellular senescence (Kasabri and
Bulatova 2010). This occurs because, at this length, chromosome ends are sensed as
double stranded breaks which activate the DNA damage response machinery (Verdun
and Karlseder 2007). This process initiates the G1 DNA damage checkpoint and
upregulates p21/p16, leading to senescence (Verdun and Karlseder 2007). When this
checkpoint is deactivated, translocations, fusions, or rearrangements within these DNA
regions can occur, creating a favorable neoplastic environment (de Lange 1994).
Though telomeres were known to exist since the late 1960s, it was not until 1985
that their mechanism of maintenance was uncovered. Working in the ciliate
Tetrahymena, Carol Greider and Elizabeth Blackburn identified the enzyme responsible
26
for synthesizing telomeres (1985). This enzyme, now called telomerase, is a reverse
transcriptase that uses an RNA template to add the characteristic telomeric repeats to the
ends of chromosomes (Shawi and Autexier 2008). It should be noted that recently, a
telomerase-independent method of telomere synthesis has been identified. Furthermore, a
recent study found that telomerase may serve additional cellular roles apart from
maintaining telomeres (Choi et al. 2008).
Though not expressed in most human tissues, telomerase expression may be
necessary for cellular immortalization (Rhyu 1995). In fact, 85% of cells from human
tumors have been found to express telomerase, suggesting it may be required for cells to
escape replicative senescence (Shay 1998). Conversely, the attrition of telomeres due to
the lack of expression of telomerase in normal human cells may constitute a fundamental
basis for cellular aging (Artandi 2006). As a result, it has been hypothesized that
increased telomere expression may slow or reverse aging. In support of this, one study
found that cells given an exogenous source of telomerase maintained a youthful state,
proliferated indefinitely, and showed a reversal of senescent characteristics (Funk et al.
2000).
Interestingly, progerias have been found to be associated with telomere
dysfunctions, supporting a role for telomeres in the aging process. For instance, the
mutant DNA helicase protein in Werner’s syndrome is essential for telomere replication
and stability (Tosato 2007). Furthermore, twin studies have shown that longer telomeres
correlate inversely with the risk of mortality, and that greater differences in length are
linked to greater differences in the twins’ ages at death (Sander et al. 2008). Another
study, which analyzed cerebral samples, found that longevity is associated with longer
27
telomeric DNA (Nakamura et al. 2007). Though it is clear that telomeres are at the heart
of replicative senescence, there is not enough evidence to say if or how much telomeres
affect aging on an organismal scale.
Genetic Control Theory
Genetic control theory argues that the programmed upregulation and
downregulation of certain genes controls the aging process. Furthermore, the theory
defines senescence as the time when age-associated phenotypes are manifested (Kasabri
and Bulatova 2010). Some proponents of genetic control theory see aging as a
continuation of the genetic program that modulates growth and development earlier in
life.
Support for genetic control theory comes mainly from genetic manipulations in
laboratory animals that increase lifespan. In fact, numerous genes in organisms from
yeast to mice have been found to significantly extend maximum lifespan. For instance,
altering the C. elegans gene, Daf-2, to reduce its expression, more than doubles the
lifespan of adult worms (Sprott and Pereira-Smith 2000). Additionally, the gene
humorously named “I’m Not Dead Yet” can be mutated to produce Drosophila with
double their normal life span (Rose 1984). In yeast, overexpression of the “longevity
assurance gene-1” (LAG-1), which encodes a membrane protein, increases the replicative
lifespan of yeast by 30% (Jazwinski 1993). Though dozens of other genes have been
found to modulate lifespan, listing each one here would not be useful.
In support of genetic control theory, many of the genes identified that modulate
lifespan have been found to exist in only a few different molecular pathways. These
28
pathways tend to be extremely complex and generally play roles in cellular growth,
development, and nutrient sensing. The Target of Rapamycin (TOR), Sirtuin, and Insulinlike signaling pathways have been shown to be crucial in regulating the lifespan of a
diverse array of species. These pathways will form the basis for the next chapter because
of their close ties to the aging process and because they offer the greatest targets for
pharmaceutical intervention in aging.
Other studies in support of genetic control theory, especially ones performed in
humans, use correlation to assess the effect of genetic differences on lifespan. One study
found a locus on chromosome 4 that may promote extreme longevity, as found in studies
of human centenarians and their relatives (Puca et al. 2001). Additionally, twin studies
attribute 30% of the variation in longevity to genetic factors, with the remaining 70%
arising from environmental and behavioral factors (Perls et al. 2000). Amazingly, studies
in C. elegans have shown that genotype differences can confer up to a 1000% increase in
life expectancy (Sander et al. 2008). Because of their potential to extend lifespan, the
specific pathways implicated in longevity will be the focus of the next chapter.
Discussion
Lessons from Comparative Biology
A central question of aging research is why maximum lifespan varies from one
species to the next. As a result, understanding the determinants of species-specific
lifespan via comparative biology helps to elucidate the fundamental causes of human
aging. Correlational studies, which compare lifespan in multiple species with differences
in their biological infrastructure, are especially helpful in this pursuit. When applied to
29
the aging theories presented in the previous pages, these studies can help evaluate which
theories are actually at play and which are most powerful in the determination of lifespan.
One of the most well documented correlations compares a species’ size with its
lifespan. Across the animal kingdom, generally speaking, the larger the physical size of
an average member of the species, the longer its lifespan (Buffenstein and Jarvis 2002).
For instance, mice, rabbits, lions, and elephants have lifespans of 4, 10, 30, and 60 years,
respectively (Stipp 2010). Along the same lines, Steve Austad developed a measure
called the Longevity Quotient (LQ). The LQ is a ratio of the maximum lifespan of a
species to its average weight. Short-lived animals for their body mass have LQs less than
1 and long-lived animals have LQs greater than 1 (Buffenstein and Jarvis 2002). Thus,
finding animals with extreme longevity (LQs much greater than 1) provides opportunities
for uncovering mechanisms that delay or slow the aging process.
Two such animals with extreme longevity are the little brown bat and the naked
mole rat, with LQs of 5.8 and 10, respectively (Buffenstein and Jarvis 2002). One study
compared free radical production in short-tail shrews, which have a low LQ, with that of
the long lived bats. In support of the free radical and mitochondrial theories, this study
found that the bats’ mitochondria were at least twice as efficient as those of the shrews.
Another study compared the levels of lipids prone to free radical damage in bats, naked
mole rats, and mice which also have low LQs (Hulbert 2007). Again in support of free
radical theory, the mice had nearly 9 times more of this lipid. This protein in turn makes
them more susceptible to free radical damage, possibly explaining their shorter lifespans
compared to the bats and mole rats.
30
Thus, both the rate of free radical production, and the defenses against such
radicals, provide an explanation for much of the variance in species-specific lifespan.
Furthermore, free radical theory, which links energy expenditure with aging, helps
explain why small animals with fast metabolic rates are short-lived (Stipp 2010).
However, one study found that lower antioxidant levels are correlated with longer
lifespan, which does not make sense upon first glance (Barja 2000). Scientists have
reconciled this finding by arguing that increased lifespan can be achieved one of two
ways: by decreasing radical production or by increasing the defense system against these
radicals (Stipp 2010).
Thus, the species in the study with lower antioxidant levels
probably produced fewer radicals to begin with, and thus needed fewer antioxidants.
In support of the genome maintenance theories, it has been observed that longerlived species have more efficient mechanisms in place to ensure genome integrity. One
example is found in the “hominid slowdown”, or the observed decrease in the rate of
mutation with increasing species lifespan during primate evolution (Hoyer et al. 1972).
Protein integrity has also been linked to longevity. One study found that the proteins of
naked mole rats were nearly 10-fold more resistant to unfolding than proteins from mice
(Perez et al. 2009).
Thus, comparative biology indicates that different species have evolved different
mechanisms for extending longevity. However, these mechanisms all seem to fall into the
realm of the stochastic theories. Thus, it seems that these theories most likely play some
role in regulating the aging process, even in humans. However, this is not to say that
programmed theories are uninvolved in a realistic model of aging. These theories are
harder to test using comparative biology, especially because it is still unclear what
31
specific genes are the modulators of the aging. On the other hand, a highly conserved set
of pathways have been linked to aging and are found in nearly all animals. These
pathways form the basis for the next chapter and are at the forefront of current aging
research.
Comprehensive View of Aging
Armed with an understanding of the major theories of aging, it is now possible to
form a realistic view of how the aging process may operate on an organismal scale.
Throughout this chapter it has been stressed that the theories of aging should be seen as
complimentary and not mutually exclusive. As such, though each theory is supported by
a plethora of evidence, it is clear that no one theory alone explains the entire phenomenon
of aging.
The best perspective views aging as the result of stochastic damage accumulation,
with the rate of such accumulation determined by both genetics and environment.
Niedernhofer et al. supports this view, arguing that aging is determined both by stochastic
damage, which causes functional decline, and genetics, which determines the rate of
damage accumulation (2006). Thus, this suggests that the stochastic theories form the
basis for the existence of aging. The programmed theories, on the other hand, serve a
regulatory role by augmenting the mechanisms by which stochastic damage arises,
leading to differences in lifespan.
An explanation such as this seems to make sense when considering even things
that aren’t alive, since any complex system can age. Take a personal computer, for
instance. When first purchased, the computer boots up quickly, runs smoothly, and rarely
32
crashes. Over time, however, damage accumulates to its various components from natural
wear-and-tear and internet viruses. Slowly, as this damage builds, the computer begins to
run sluggishly, and after a certain point, the computer crashes. Luckily, hardier
components and antivirus software have been developed to help increase the lifespan of
personal computers.
Though the mechanisms are far more complex, organismal aging occurs via a
process not so different from that of the computer. The complex cellular components
accumulate damage with time, leading to functional decline, and eventually the failure of
entire systems. Like the computer, organisms have developed mechanisms to help reduce
the accumulation of this damage. Superoxide dismutase, the free radical scavenger, and
DNA repair proteins, which improve genome integrity, are two of dozens of examples of
evolved mechanisms for curbing damage accumulation. Like the antivirus software, these
safeguards contribute to increased lifespan.
Thus, organismal aging results from the stochastic accumulation of damage.
Furthermore, the rate of accumulation is partially governed by genes encoding the most
susceptible proteins (like those of the mitochondria) or those whose function is to reduce
this damage in the first place (like DNA repair proteins).
Although the discussion above provides a simple way to understand aging
holistically, it says nothing about the specific biological mechanisms that regulate aging
on a cellular level. The answer to this question comes from the specific stochastic and
programmed theories, which implicate certain cellular components in aging.
33
Figure 2.4: Comprehensive View of the Aging Process
Neuroendocrine
Imbalance
Cell
Membrane
Damage
Mitochondrial
Damage
Cellular
Senescence
Exogenous
Toxins
Damaged
Proteins
ROS
Accumulation of
Senescent Cells
DNA
Damage
Telomere
Shortening
Decreased Tissue
Function
Impaired
Immune
System
Altered
Gene
Expression
System Failure
Death
Factors Affecting Aging on an
Organismal Level
Factors Affecting Aging on a
Cellular Level
Figure 2.4 presents a model for how these theories work together to cause both
cellular senescence and organismal aging. The large blue box on the left of the figure
contains the key cellular mechanisms that lead to senescence. For instance, innate
mitochondrial inefficiency leaks ROS, which create more mitochondrial damage and
more radicals. These ROS also damage other cellular components, such as DNA, proteins
and the cell membrane. Damaged proteins lead to more DNA damage through inefficient
repair. Damaged DNA leads to further impaired protein production and altered gene
expression, perpetuating a cycle of exponential damage accumulation. Though ROS offer
one source for this perpetuated damage, exogenous toxins and telomeres also lead to a
similar vicious cycle.
34
Thus, the theories of aging are intertwined and act concertedly to bring about
cellular senescence. These processes occur in all cells, causing an accumulation of
senescent cells (small box). Since these cells are less efficient than younger healthy cells,
their accumulation leads to decreased tissue function. Eventually, stem cells lose their
proliferative capacity, immune cells respond inadequately to threats and expel
inflammatory chemicals, and endocrine cells begin secreting an imbalance of hormones.
These factors, along with many others, increase the rate of damage accumulation in cells,
and thus, increase the production of senescent cells. This process accelerates with age
until entire systems fail, and the organism dies.
In the future, this morbid picture of aging may be replaced. The development of
pharmaceutical agents with various cellular targets may not only slow the aging process,
but make it more tolerable. As such, the next chapter explores the key molecular
longevity pathways implicated as targets for pharmaceutical intervention.
35
CHAPTER 3
THE MOLECULAR NETWORK OF
LONGEVITY
Equipped with a theoretical understanding of the aging process, it is now useful to
explore the specific molecular pathways thought to regulate lifespan. The fact that
species-specific lifespan varies from days to decades suggests that mutations acquired
over evolutionary time are sufficient to extend lifespan thousands of times over (Kenyon
2010). This extreme order of variation begs the question, which genes regulate the
lifespan of an organism? Though the previous chapter argued that aging arises from the
stochastic accumulation of damage, it also implicated genetic components as master
regulators of the rates of such accumulation. Here we explore these genes and their
associated molecular pathways.
Most of the mutations found to extend lifespan affect genes involved in stressresponse or nutrient sensing (Kenyon 2010). Theoretically, the specific involvement of
these types of pathways is appropriate. When nutrients are abundant, organisms are able
to develop, grow, reproduce, and as a result, age quickly. On the other hand, when
36
nutrients are scarce, growth and development is halted, stress-responses are activated, and
the organisms live longer (Stanfel et al. 2010).
From an evolutionary perspective, it is clear why this type of molecular
organization would be conserved. When external conditions are harsh, these pathways
direct a molecular shift towards protection and maintenance, allowing the organism to
live longer so that it may reproduce at a more favorable time (Kenyon 2010). Once in
place, these pathways gained mutations that altered their basal activity leading to the
wide variety of lifespans found in the animal kingdom (Kenyon 2010). In support of this
theory is caloric restriction, which shows that limiting available nutrients extends the
lifespan of species from yeast to primates (Colman et al. 2009). As such, caloric
restriction will be extensively covered in the next chapter.
So, which pathways have been found to regulate organismal lifespan?
The
insulin/IGF1 pathway, which is responsive to glucose levels, a signal of nutrient
availability, was the first to be discovered and is highly conserved (Kenyon 2005).
Additionally, the target of rapamycin (TOR) pathway responds to signals from nutrients
and growth factors and has been found to affect lifespan from yeast to man (Stanfel et al.
2010). Finally, the sirtuins, a class of nutrient sensors that regulate gene expression, have
also been found to modulate aging (Donmez and Guarente 2010).
Though other proposed molecular aging regulators exist, these three pathways are
the most promising in terms of their breadth and their potential to be augmented by
pharmacological intervention. In this chapter, the structure of these pathways, along with
evidence supporting their role in regulating aging will be explored.
37
Molecular Pathways of Longevity
The Insulin/IGF1 Pathway
The insulin/insulin-like growth factor-1 (insulin/IGF1) pathway was the first to be
implicated in animal aging, and as a result, is the best characterized (Houtkooper et al.
2010). Furthermore, the relationship between insulin signaling and longevity, suggests an
important function for hormones in the aging process (Kenyon 2010).
Figure 3.1: Structure of the Insulin/IGF1 Signaling Pathway
Worms (C. elegans)
Flies (D. melanogaster)
Insulin-like Peptides
Insulin-like Peptides
DAF-2
Mammals (M. musculus)
Insulin
IGF1
IR
IGF1R
InR
IRS
CHICO
AGE-1
P13K
PTEN
P13K
PTEN
PTEN
PDK1
PDK1
PDK1
AKT/PKB and SGK
AKT/PKB and SGK
AKT/PKB and SGK
DAF-16
FOXO
Pro-aging
Genes
Sir-2.1
Longevity
Genes
Pro-aging
Genes
SIR2
Longevity
Genes
FOXO
Pro-aging
Genes
SIRT1
Longevity
Genes
Source: Adapted from Russell and Kahn, 2007
Before delving into the insulin/IGF1 pathway’s role in aging, it is important to
understand its structure (Figure 3.1). In lower organisms like nematodes, the insulin and
IGF1 pathways converge on a single receptor, a tyrosine kinase, called DAF-2
(Houtkooper et al. 2010, Kimura et al. 1997, Russell and Kahn 2007). Similarly, flies
contain a single receptor called the insulin receptor (InR) (Tatar et al. 2001). Mammals,
however, have separate insulin and IGF1 receptors whose activities are linked by the
insulin receptor substrate (IRS) proteins which promote interactions between the
38
receptors and the phosphoatidylinositol 3-kinase (P13K) (AGE-1 in C. elegans) (Russell
and Kahn 2007).
The binding of an appropriate insulin-like ligand leads to the phosphorylation of
P13K via the kinase activities of the receptors (Russell and Kahn 2007). Now in its active
form, phosphorylated P13K leads to the synthesis of two proteins which consequently
activate 3-phosphoinositide-dependent protein kinase-1 (PDK1) (Russell and Kahn
2007). Activated PDK1 then phosphorylates AKT/protein kinase B (PKB) and
serum/glucocorticoid-regulated kinase (SGK), thereby activating them (Russell and Kahn
2007). Once active, these proteins phosphorylate the class O forkhead box transcription
factors (FOXO), also called DAF-16 in C. elegans (Russell and Kahn 2007). This
phosphorylation event leads to the inactivation of FOXO by nuclear exclusion (Kaletsky
and Murphy 2010; Lin et al. 2001). As a result, the relegation of FOXO to the cytoplasm
renders it incapable of activating its longevity enhancing transcriptional targets
(Houtkooper et al. 2010).
Thus, upregulation of the insulin/IGF1 pathway leads to FOXO inactivation and
an increase in the transcription of pro-aging genes. Conversely, downregulation of the
pathway prevents FOXO phosphorylation, allowing it to stay in the nucleus to aid in the
transcription of genes that promote longevity. Microarray analysis has found that these
genes are involved in cellular stress-response, antimicrobial activities, and metabolism
(Murphy et al. 2003). Thus, FOXO seems to be the key intermediate in the insulin/IGF1
pathway’s regulation of lifespan.
Furthermore, this pathway is activated by insulin-like peptides, which are
produced in response to high blood glucose levels, a signal that the organism has access
39
to nutrients. Thus, the insulin/IGF1 pathway can be linked to the lifespan extending
effects of dietary restriction. When nutrients are scarce, the pathway is downregulated
due to a lack of stimulating insulin peptides. As a result, FOXO remains
unphosphorylated and is able to promote the transcription of longevity genes, thereby
extending lifespan.
The insulin/IGF1 pathway was first linked to lifespan regulation when, nearly two
decades ago, mutations in daf-2 and age-1 doubled the lifespan of nematodes (Freedman
and Johnson 1988; Kenyon et al. 1993; Kaletsky and Murphy 2010). In support of the
idea that daf-16 (FOXO) serves as the insulin/IGF1 pathway’s crucial lifespan regulator,
daf16-/daf2- worms showed no lifespan extension (Kenyon 2010). This finding was
confirmed in flies when lifespan extension was achieved through FOXO overexpression
alone (Hwangbo et al. 2004). Thus, lifespan extension through the insulin/IGF1 pathway
requires a FOXO homolog.
The insulin/IGF1 pathway has been found to exert its influence on lifespan in
mammals, in addition to lower organisms, indicating that its role in aging has been highly
conserved. For instance, the pathway has been found to regulate the lifespans of several
genetically engineered mice. Fat-specific Insulin Receptor KnockOut (FIRKO) mice have
increased lifespans of nearly 20% (Bluher et al. 2003). Furthermore, the Ames dwarf
mouse, which harbors a mutation in a transcription factor necessary for proper pituitary
development, has reduced circulating IGF1 levels and as a result an increased lifespan
(Brown-Borg et al. 1996, Russell and Kahn 2007). Finally, small dogs, which have
decreased IGF1 signaling, tend to outlive larger dogs (Kenyon 2010; Kaletsky and
Murphy 2010). It should be noted, however, that organism-wide deletions of insulin
40
signaling in mammals are usually lethal, whereas downregulation or tissue-specific
deletion sometimes lead to lifespan extension.
The insulin/IGF1 pathway has even been found to influence human longevity. For
instance, Ashkanazi Jewish centenarians have a higher incidence of impaired IGF-1
receptor signaling (Kenyon 2010; Suh et al. 2008). Furthermore, a study published in
early 2011 showed that Ecuadorians with the dwarfism known as Laron Syndrome, a
disease caused by decreased IGF-1 levels, had remarkably lower rates of cancer and
diabetes (Naik 2011). It is still unclear, however, whether these dwarves exhibit increases
in lifespan, since they tend to die early from substance abuse and accidents (Naik 2011).
Thus, it seems that insulin signaling may play a significant role in human aging and
disease, and may serve as a potential target for lifespan extension.
The downregulation of the insulin/IGF1 pathway in specific tissues has proven to
be a valuable experimental technique in understanding the pathway’s role in aging. The
FIRKO mice, for instance, which present a model of tissue-specific insulin-signaling
downregulation, have shown that hormone signals produced in adipose tissue may be
sufficient to signal the desired rate of aging to other tissues (Russell and Kahn 2007).
Likewise, studies that overexpress daf-16 in C. elegans intestinal cells, the main sites of
fat storage, support the existence of a feed-forward loop. It is hypothesized that in this
feed-forward loop, overexpressed daf-16 decreases the expression of daf-2, which in turn
increases the activity of daf-16 in other tissues (Russell and Kahn 2007). Klotho, a
transmembrane protein that circulates in the blood, has been implicated as the messenger
that delivers the anti-aging signal to other tissues, since its tissue-specific overexpression
is sufficient to extend lifespan (Kuroso et al. 2005; Russell and Kahn 2007).
41
Thus, decreases in insulin-like signaling in one tissue, which decreases the rate of
aging, can be spread to other tissues. The fact that fat tissue is conserved as the site of
signaling in multiple organisms suggests a potential site for intervention in the lifespan
extension of humans (Murphy, Personal Communication, 2011). Downregulating the
insulin/IGF1 pathway, specifically in human adipose tissue, could spread an appropriate
anti-aging signal to other tissues without disturbing the signals required for proper
growth, development, and metabolism.
The understanding of the downstream effectors of insulin/IGF1 signaling is
important in determining how the pathway augments longevity. The discovery that no
daf-16 target on its own can mimic the lifespan extending effects of daf-2- implies that
the extended longevity phenotype is regulated by the upregulation and downregulation of
many genes (Murphy et al. 2003). Regardless, these findings support a role for the
insulin/IGF1 pathway in mediating the stochastic accumulation of damage associated
with aging.
The Target of Rapamycin (TOR) Pathway
Rapamycin, a macrolide secreted by bacteria to compete with fungi for nutrients,
was first isolated from the soil of Rapa Nui, the island from which its name is derived
(Sharp 2010). Initially, rapamycin was used medicinally as an antifungal agent, but later
was found to have better value as a potent immunosuppressant (Vezina et al. 1975). The
drug’s role in lifespan extension and aging began when Michael Hall identified that
rapamycin was acting on a protein complex, which he termed the Target of Rapamycin
(TOR) (Hall 2010).
42
Like the insulin/IGF1 pathway, studies of TOR have shown that it acts as a sensor
by which nutrient availability is tied to cellular growth (Stanfel et al. 2009). Furthermore,
downregulating TOR, through genetic deletion or rapamycin feeding, led to significant
lifespan extension in a variety of species (Houtkooper et al. 2010).
An understanding of the structure of the TOR pathway is necessary to
contextualize its role in the aging process. The TOR proteins are highly conserved
protein kinases, which consist of two paralogs in yeast, termed TOR1 and TOR2, while
mammals possess only one, called mTOR (Stanfel et al. 2009).
mTOR participates as the catalytic subunit in two protein complexes, mTORC1
and mTORC2. These complexes have distinct functions, and only TORC1 is sensitive to
rapamycin (Hall 2010; Stanfel et al. 2009). Complex one consists of mTOR, the
regulatory associated protein of mTOR (Raptor) and the mammalian LST8/G-protein βsubunit like protein (mLST8/GβL) (Lamming 2010). When nutrients are abundant,
interactions between mTOR and Raptor are weakened, enabling the mTOR kinase
domain to phosphorylate its targets (Kim et al. 2002). mTORC2, on the other hand,
consists of mTOR, rapamycin-insensitive companion of mTOR (Rictor), mammalian
stress-activated protein kinase interacting protein 1 (mSIN1), and potentially other
components (Lamming 2010; Frias et al. 2006).
The TOR complexes control two separate branches of a signaling pathway:
mTORC1 is thought to regulate cell growth by sensing nutrients and growth factors, and
mTORC2 is thought to control cell division and survival (Sparks and Guertin 2010)
(Figure 3.2). The upstream regulators of TORC1 are better understood than those of
TORC2 (Stanfel et al. 2009). Upstream, Akt signals for the activation of TORC1 in
43
response to insulin and other growth factors by inhibiting tuberous sclerosis complex 2
(TSC2) (Avruch et al. 2006). TCS2 is an inhibitor of Rheb, which directly activates
TORC1 (Inoki et al. 2003).
In summary, Akt activation inhibits TCS2, which
consequently prevents inhibition of Rheb, which then goes on to directly activate
TORC1. Additionally, though not depicted in the figure below, AMP-activated protein
kinase (AMPK) has been identified as a repressor of TORC1 activity (Bolster et al.
2002).
Figure 3.2: The TOR Signaling Pathway
Taken from Sparks and Guertin 2010
mTORC1’s effects on mRNA translation and ribosome biogenesis play important
roles in regulating cell growth and maintenance signals. Two downstream effectors, the
ribosomal S6 protein kinase (S6K) and the eukaryotic translation initiation factor 4E
binding protein (4E-BP), are essential for TOR’s control of these functions (Bjedov et al.
44
2010). When nutrients are abundant, TOR stimulates protein production through these
downstream effectors. Autophagy, or the directed degradation of cellular components, is
inhibited by TORC1 and is suggested to play a crucial role in longevity (Houtkooper et
al. 2010). This makes sense as upregulating the recycling of cellular parts would help
keep damage from accumulating. Furthermore, the fact that TOR-induced autophagy
occurs in nutrient replete conditions ties this pathway to caloric restriction.
mTORC2, on the other hand, responds to growth factors, such as the insulin and
insulin-like growth factors described above, and functions to regulate stress responses
essential for cell survival (Stanfel et al. 2009). Though many features of TORC2’s
upstream regulators remain poorly understood, a new study found that NIP7, a protein
that controls ribosome assembly, acts as an upstream activator of TORC2 by promoting
its association with the ribosomal 60S subunit (Hall 2010). At a conference hosted by the
American Federation for Aging Research (AFAR), Michael Hall proposed that this form
of TORC2 activation ensures that TORC2 will only be active in cells with many
ribosomes, i.e. growing cells (Hall 2010).
mTORC2’s activation of Akt identifies a site of cross-talk between the two
complexes and highlights the potential of feedback loops (Stanfel et al. 2009). Because
Akt inactivates FOXO in the insulin signaling pathway, it seems that it is an extremely
important protein that may integrate the regulatory activities of both TOR and insulin
signaling. Additionally, Akt inactivation, which downregulates both the TOR and insulin
pathways, suggests that Akt may be a master regulator of longevity. At the very least, in
both pathways, Akt inactivation leads to a cellular switch towards the expression of
longevity assurance genes.
45
What is the evidence that supports TOR as a mediator of lifespan? Studies of
TOR inhibition, whether through genetic deletions or pharmacological intervention, have
shown increased environmental stress resistance, leading to increased lifespan in species
from yeast to mice (Kenyon 2010). The fact that TOR serves as an integrator of
information from amino acids, insulin, ATP, stress, and growth factors indicates, at least
theoretically, that it would be a prime molecular site to mediate aging and lifespan
(Kapahi 2010).
Studies in three invertebrate model organisms - yeast, nematodes, and fruit flies have provided the firmest evidence for TOR’s role in aging. In yeast, TOR1 deletion,
rapamycin inhibition, S6K homolog deletion, and mutations in downstream targets of
TOR involved in protein synthesis, have all been found to increase replicative lifespan
(Stanfel et al. 2009; Fabrizio et al. 2001; Powers et al. 2006; Steffen et al. 2008).
Similarly, lifespan extension in C. elegans can be achieved by inhibiting or
downregulating the TOR homolog (let-363), the raptor homolog (daf-15), the S6K
homolog (rsks-1), certain ribosomal proteins, and a variety of mRNA translation
initiation factors (Stanfel et al. 2009; Hansen et al. 2007; Jia et al. 2004; Pan et al. 2007).
Studies in flies confirmed these findings and also showed that overexpressing the TSC1
and TSC2 homologs, as well as inhibiting TOR, exclusively in fat bodies, was sufficient
to extend lifespan (Stanfel et al. 2009; Kapahi et al. 2004; Luong et al. 2006).
Importantly, TOR controls lifespan through a pathway distinct from the
insulin/IGF-1 pathway, as studies in C. elegans indicate that increased lifespan is
achieved independently of daf-16 (FOXO) (Kenyon 2010). However, TOR lifespan
46
mediation is similar to that of the insulin/IGF1 pathway in that it requires specific
transcriptional changes to extend lifespan (Kenyon 2010).
The fact that TOR regulates downstream effectors involved in mRNA translation
suggests that protein synthesis plays a critical role in the aging process. Understanding
how downregulating protein production leads to lifespan changes will be crucial in
determining TOR’s role in aging. Additionally, TOR inhibition leads to at least 3 other
cellular changes including autophagy, stress response, and mitochondrial-associated
metabolic changes (Stanfel et al. 2009). Elucidating the role of these changes will also
prove fruitful in the understanding of TOR’s role in aging and the aging process as a
whole.
Currently, evidence is lacking to definitively conclude whether TOR signaling
affects aging in mammals; however, a few recent studies suggest this might be the case.
In 2009, a study led by Dave Sharp demonstrated that feeding mice a specific formulation
of rapamycin increased their lifespans by nearly 20 percent even when administration
started late in life (Sharp 2010). Furthermore, Sharp has started to explore rapamycin
induced gene expression changes in mice and has isolated 40 targets in fat, 50 in the liver,
and 3 in both (Sharp 2010). These genes will serve as prime candidates for research into
how rapamycin and the TOR pathway regulate mammalian lifespan. Brian Kennedy and
colleagues are exploring the potential of mouse models that lack certain ribosomal
proteins as potential models of age-related diseases (Kennedy 2010). In particular, mice
with a deletion of the gene Rpl22, which encodes a portion of the ribosomal 60S subunit,
display a cardioprotective phenotype and female mice with deletions of a similar gene,
Rpl29, are protected from obesity when fed a high-fat diet (Kennedy 2010). Dudley
47
Lamming, a postdoctoral researcher in the Cambridge based Sabatini lab, described the
lab’s development of a mouse strain with partially depleted levels of mTOR and mLST8
(Lamming 2010). Female mice in this strain have increased longevity and display
decreased mTORC1 activity, and hence bear the name DEnACO mice (Decreased
Endogenous Activation of mTOR complex One) (Lamming 2010). These mice
demonstrate that even minor inhibition of the mTOR signaling pathway can have
dramatic effects on lifespan.
Finally, human studies, though few and far between, have linked TOR signaling
to a variety of age-related diseases. For instance, mTOR has been implicated in diabetes
as TOR activation has been found to lead to insulin resistance (Stanfel et al. 2009). TOR
has also been linked to cardiac hypertrophy, inflammatory diseases, and many different
cancers (Hall 2010). The studies in mice described above should help to better understand
mTOR’s role in human aging. Specifically, reconciling gender differences, changes in
gene expression, the function of TOR signaling in different tissues, mTORC2 signaling,
and the roles of autophagy and protein synthesis, will be critical in understanding how
TOR regulates aging and lifespan.
The Sirtuins
The sirtuins refer to the seven human homologues of the yeast transcriptional
repressor Sir2, and are appropriately termed SIRT1-7 (Verdin 2010). Functionally, the
sirtuins are a group of NAD+-dependent deacetylases, which remove acetyl groups from
histones as well as a variety of other proteins (Houtkooper et al. 2010). The seven sirtuins
vary widely in substrate specificity, function, and cellular distribution with sirtuins 1, 2,
48
6, and 7 occupying the nucleus, 1 and 2 occupying the cytoplasm, and 3, 4, and 5
occupying the mitochondria (Verdin 2010) (Figure 3.3).
Like the other molecular pathways implicated in longevity, the sirtuins act as
nutrient sensors. Furthermore, the fact that their activity is modulated by the
NAD+/NADH ratio, a measurement of both the metabolic state and health of cells,
suggests why they may be prime targets for longevity control (Russell and Kahn 2007;
Schafer and Buettner 2001).
SIRT1, the closest homologue of Sir2, has received the most attention due to the
discovery that it, at least in part, mediates the lifespan extending effects of caloric
restriction in yeast, flies, and worms (Houtkooper et al. 2010; Russell and Kahn 2007).
Interestingly, SIRT1 seems to be tightly linked to the insulin/IGF1 pathway, as its
overexpression achieves lifespan extension in C. elegans by activating daf-16, likely
directly by deacetylation (Berdichevsky 2006; Kenyon 2010). Furthermore, SIRT1 has
49
been shown to deacetylate FOXO in mammals, which leads to a cellular shift towards
stress resistance (Kenyon 2010).
The fact that insulin/IGF1 mutants do not require Sir2 for lifespan extension
implies that the sirtuins may act on daf-16 and thus lifespan independently, at least in
nematodes (Kenyon 2010). This difference may arise from differing downstream effects
when daf-16 is deacetlyated versus phosphorylated, the latter of which occurs during
insulin/IGF1 signaling. In addition to daf-16/FOXO, SIRT1 substrates include a variety
of proteins that regulate transcription, including p53 and NF-kB (Verdin 2010).
Another suggested member of the sirtuin pathway is AMP-activated protein
kinase (AMPK), an enzyme responsible for cellular energy homeostasis, which may
boost NAD+ levels, thereby activating SIRT1, and leading to deacetylation of its
downstream targets (Canto et al. 2009; Houtkooper et al. 2010). One such target, PGC1α, leads to improvements in mitochondrial activity, respiratory metabolism, and
oxidative stress responses (Canto et al. 2009). AMPK’s role in longevity was confirmed
when metformin, a drug that increases the activity of AMPK was found to increase the
lifespan of mice (Anisimov et al. 2008).
Unlike the TOR and insulin signaling pathways, the sirtuin pathway is less clearly
tied to lifespan. In lower organisms, such as yeast, flies, and worms, Sir2 has been shown
to regulate lifespan, though mammalian studies have provided less concrete evidence
(Kenyon 2010). For instance, nematodes harboring a duplication of the Sir2 ortholog,
sir-2.1, exhibit significant increases in lifespan (Guarente 2010). Furthermore, those with
sir-2.1 deletions have shortened lifespans (Guarente 2010). However, it seems that sir-2.1
overexpression may induce lifespan extension through modulation of the insulin/IGF1
50
pathway as sir-2.1 activates daf-16 directly by deacetylation (Russell and Kahn 2007). In
flies, SIR2 overexpression is also sufficient to extend lifespan, though it is still unclear
whether FOXO is required (Russell and Kahn 2007).
In mammals, the sirtuins have not yet been found to increase lifespan, though they
do seem to play a significant role in diseases of aging, such as diabetes, cancer, and
inflammation (Donmez and Guarente 2010). Recently, however, feeding mice
resveratrol, a compound reported to activate the sirtuins, did extend the lifespan of mice
on high-fat diets, but did not affect the lifespans of normally fed mice (Pearson et al.
2008; Kenyon 2010).
In terms of diseases of aging, research has provided more conclusive evidence
supporting a role for the sirtuins. For instance, low to moderate SIRT1 overexpression in
the hearts of mice rendered significant improvements in age-dependent cardiac
hypertrophy, apoptosis, fibrosis, cardiac dysfunction, and the expression of senescent
markers (Alcendor et al. 2009; Donmez and Guarente 2010). Furthermore, mice
overexpressing SIRT1 had lower incidences of diseases of aging, including diabetes and
cancer, though they did not show improvements in lifespan (Herranz et al. 2010;
Houtkooper et al. 2010).
Even in humans, the sirtuins have been implicated as regulators of healthspan. In
support of this, studies have linked energy expenditure to single nucleotide
polymorphisms in SIRT1, and have found a strong correlation between SIRT1 expression
and insulin sensitivity (Lagouge et al. 2006; Rutanen et al. 2010). Additionally, a small
human study demonstrated that variation in SIRT3, a sirtuin found in the mitochondria, is
linked to longevity (Bellizi et al. 2005; Houtkooper et al. 2010).
51
Thus, evidence is lacking to conclude whether the sirtuins regulate mammalian
lifespan and it is still unclear how they influence lifespan in lower organisms, though
several ideas exist. In yeast, for instance, it was proposed that Sir2 extends lifespan by
decreasing the production of extrachromosomal ribosomal DNA circles (Kenyon 2010).
Recently, however, it has been suggested that Sir2 regulates aging by silencing genes at
telomeres (Dang et al. 2008). The sirtuin’s effects on mitochondrial respiration and
oxidative stress have also been implicated as mechanisms by which these proteins
influence aging (Canto and Auwerx 2009). Finally, sirtuin-mediated regulation of
inflammation has been proposed as a method of controlling aging, as inflammation plays
a large role in the pathogenesis of many age-related diseases (Donmez and Guarente
2010). In support of this, SIRT1 and SIRT6 have been found to inhibit the activities of
NF-kB, a key activator of inflammatory responses to stress that has been implicated in
aging (Adler et al. 2007; Donmez and Guarente 2010).
Thus, not much is known about how the sirtuins affect age-related diseases in
mammals or the lifespan of lower organisms. Elucidating these mechanisms, as well as
the interactions between the sirtuins and other signaling pathways implicated in aging,
will be extremely important in determining how the molecular activities of the sirtuins
may or may not influence aging. This will also serve to better evaluate them as candidates
for augmentation in the pursuit of lifespan extension. As of now it seems that SIRT1 is
the best classified regulator of aging; however, it will also be important to determine the
role of the other sirtuins, as they may also significantly affect senescence in their
respective cellular compartments. In the following chapters, the lifespan extending
52
potential of resveratrol, which has been shown to activate SIRT1, will be assessed and the
current pipeline of pharmaceuticals that act on the sirtuins will be evaluated.
Discussion
In the previous pages, the pathways implicated in aging were presented as more or
less distinct. However, just as the theories of aging are complementary, the signaling
pathways shown here work together to synergistically regulate growth and development
in response to nutrients and other signals. As such, the achievement of safe and effective
lifespan extension should consider an entire network of pathways and not any one
pathway alone. Understanding the components of this complex network and how they
interact will be the critical next step in determining how the aging process operates. As
such, a model for the network of longevity pathways and their effects on aging is
presented below (Figure 3.4).
Thus far, a few key intermediates have been discovered that link these three
pathways together. AKT, for instance, functions in the insulin signaling pathway to
inhibit FOXO, but also regulates the TOR pathway by indirectly activating mTORC1.
Additionally, FOXO ties the sirtuin and insulin signaling pathways together, and seems to
be an extremely important regulator of longevity. Finally, AMPK regulates both TOR
and the sirtuins.
53
Longevity Pathways
Figure 3.4: A Network of Signaling Pathways Regulates Aging
Nutrients
Lack of Nutrients
Autophagy
mTORC1
Insulin Receptor
AMPK
S6K
AKT/PKB
?
mTORC2
4EBP
?
Protein Synthesis
Sirtuins
Daf-16/FOXO
Telomere
Maintenance
Effects on Aging
Longevity Genes
Protein
Integrity
Aging and
Senescence
Genome
Integrity
p53
PGC-1α
NF-kB
Mitochondrial
Function
Inflammation
ROS
Portions adapted from Houtkooper et al. 2010
Importantly, all of these shared intermediates contribute synergistically to
improvements in lifespan. For example, AMPK both activates the sirtuins and inhibits
mTORC1. Because upregulation of the sirtuins and downregulation of TOR are both
linked to increased lifespan, AMPK simultaneously renders the same effects on lifespan
in two separate pathways (Table 3.1). This same effect can be seen with the actions of
AKT and FOXO.
54
The linkage of these important signaling pathways provides prime sites for further
research into aging and lifespan extension. It will be important to distinguish which
branches of this network are most important to lifespan regulation and by what
mechanisms. Uncovering this information could be accomplished by knocking out the
ability of these intermediates to interact with one branch. For instance, the acetylation site
on FOXO could be augmented to prevent regulation by SIRT1, which would isolate its
activities to within the insulin signaling pathway. This would also prove fruitful in
identifying changes in gene expression when FOXO is acetylated versus phosphorylated.
It should be noted that these pathways are extremely complex and intertwined, so studies
of this sort may not be straightforward. As such, identifying other interactions within
these signaling branches will be important in understanding further how they are
intertwined and how the regulation of other proteins are involved.
Modifying multiple parts of this signaling network may yield insights into
achieving safer and more effective lifespan extension. For instance, a recent study found
that the combined deletion of daf-2 (Insulin Receptor) and rsks-1 (S6K) acted
55
synergistically to extend the lifespan of C. elegans nearly 5-fold (Kapahi 2010). Thus,
regulating multiple components of this complex network may provide a method for
achieving a remarkable lengthening of life. Furthermore, the ability to control multiple
components may allow each piece to be controlled more mildly. Consider the situation
where inhibiting one component 95% is required to increase lifespan; however, inhibiting
this component 20% and another component 20% has the same effect. Thus, the ability to
mildly regulate multiple components may make lifespan extension safer by reducing
side-effects.
Understanding how these pathways render their effects on aging and lifespan will
also be crucial. It seems that they must, in some way, regulate the accumulation of
damage to various cellular components, as this is what causes aging (Ch. 2). Figure 3.4
provides a model for how these pathways may control such damage accumulation. For
instance, the sirtuins’ role in telomere silencing and its activation of p53 may serve to
improve genome integrity through stricter control of cell cycle checkpoints and tumor
suppression. The TOR pathway’s role in regulating autophagy and protein synthesis may
serve to regulate protein integrity in response to changing environmental conditions.
Activation of PGC-1α and inhibition of NF-kB by the sirtuins may play a role in reducing
free radical levels by improving the functioning of mitochondria and limiting the
presence of inflammation. These are just a few mechanisms by which these signaling
pathways may alter the accumulation of damage associated with aging. Elucidating the
changes in gene expression rendered by these pathways is necessary to understand how
these pathways regulate aging.
56
A recently published study concerning the insulin/IGF1 pathway’s role in
reproductive span and lifespan may shed light on how these pathways augment the aging
process. This study investigated the role of two TGF-ß (transforming growth factor-beta)
signaling pathways, one which alters longevity through insulin/IGF1 signaling and
another which regulates reproductive span through the (Small body/Male tail abnormal)
Sma/Mab pathway (Luo et al. 2009). The Sma/Mab pathway was found to greatly
increase reproductive span without proportional effects on lifespan and acted
independently of known modulators of somatic aging (Luo et al. 2009). As such, this
pathway was the first to be found to regulate reproductive span independently of somatic
aging (Luo et al. 2009).
The ability to split the extension of reproductive span and somatic aging indicates
that the two are regulated by distinct molecular mechanisms (Luo et al. 2009). Likewise,
in a subsequent paper, the researchers showed that regulating reproductive span involved
improving chromosome segregation, DNA damage response, and other activities related
to genome maintenance (Luo et al. 2010).
57
Figure 3.5: Model for Longevity Signals in
Proliferating vs. Non-Proliferating Cells
Non-Proliferating Cells
Proliferating Cells
Insulin Signaling Receptor
(DAF-2, IR, IGF-1R)
Sma/Mab Signaling
DAF-16/FOXO
Pro-Longevity
Genes Promoting
Pro-Aging,
Genomic
Integrity
Short Reproductive
Span
Anti-Longevity
Pro-Longevity
Long Reproductive Span
Genes Promoting
Protein Integrity
Somatic Longevity
Created from Talk with Colleen Murphy (Murphy 2011)
The fact that the germ line cells of C. elegans are analogous to the proliferating
cells of mammalian tissues led Colleen Murphy to propose a model for the signaling
involved in human aging (Figure 3.5). She argues that in proliferating tissues, where
DNA is rapidly transcribed and copied, the upregulation of a homologous Sma/Mab
pathway plays an important role in improving genome integrity, and thus lifespan
(Murphy, Personal Communication, 2011). On the other hand, in non-dividing cells, the
maintenance of proteins, through the insulin signaling pathway, becomes more important
because proteins are turned over more slowly and the DNA is less dynamic. In support of
this, genes upregulated by the insulin/IGF1 pathway have been found to be involved
exclusively in protein integrity (Murphy, Personal Communication, 2011). This
differentiation in needs, she argues, is the evolutionary reason behind the separation of
these two signaling pathways (Murphy, Personal Communication, 2011). In summary,
58
this model hypothesizes that proliferating cells require improvements in genome integrity
for lifespan extension, whereas in non-proliferating cells, maintaining protein integrity
becomes more important. This has important implications for aging and lifespan
extension in humans. If this model is correct, researchers should focus on improving
genome integrity in proliferating cells, such as stem cells. In non-proliferating cells, like
neurons, the focus should shift towards maintaining the integrity of proteins.
If this model holds, it would be interesting to determine if different signaling
pathways operate in dividing or non-dividing tissues. For instance, the sirtuins may
regulate genome integrity through p53 mediated activities and telomere silencing, so is
this pathway the dominant lifespan regulator in proliferating tissues? On the other hand,
the insulin/IGF1 pathway exclusively regulates protein integrity, so does its activity
dominate aging and lifespan determination in non-proliferating tissues? Further research
into aging and lifespan extension in proliferating versus non-proliferating tissues will be
important in confirming or denying this hypothesis.
The tissue specificity of these pathways in general is another important piece that
needs to be uncovered before the mechanisms of aging in mammals can be understood
and lifespan extension made feasible. Studies suggest that these signaling pathways have
a particularly important role in fat tissue. For instance, knocking out a particular receptor
exclusively in fat tissue has been enough to increase lifespan in the whole organism. Thus
it seems that there is a mechanism to spread an “anti-aging” signal to the rest of the
tissues of the organism. The klotho protein, described previously, may be important in
this signaling though it is likely that other components are involved.
59
The ability of one tissue to affect the aging of an entire organism may be a key
insight into the safe modulation of healthspan and lifespan. These pathways are involved
in the complex regulation of growth and development throughout the body, and thus
globally changing their activities may have unwanted side-effects in certain tissues.
Therefore, if it is possible to regulate the activity of a specific lifespan regulator in one
tissue, it may send the appropriate signals to the other tissues to safely delay aging and
extend lifespan without unwanted effects. The fact that fat seems to be conserved as a
tissue whose receptors can mediate lifespan throughout the body makes sense as it is an
ideal site for nutrient sensing.
Equipped with an understanding of the molecular pathways implicated in
longevity determination, it is now possible to begin evaluating the feasibility of lifespan
extension in humans. In hopes of achieving this goal, this paper will first explore known
interventions that mediate lifespan in a variety of species. After assessing the efficacy and
limitations of these interventions, as well as their context in the network of longevity
pathways, the actual potential of developing safe and effective drugs to extend lifespan in
humans will be explored.
60
CHAPTER 4
PROPOSED METHODS OF LIFESPAN
EXTENSION
An understanding of the theories of aging and the network of molecular pathways
that regulate longevity provides a meaningful context in which to evaluate the proposed
methods of lifespan extension. Though there are no known methods that definitively slow
the aging process or extend lifespan in humans, a few interventions have shown promise
in this regard. Dietary restriction (DR), or simply the sustained reduction of caloric
intake, has been shown to increase lifespan and reduce age-related disease in organisms
from yeast to primates, making it the best-characterized anti-aging intervention (Stanfel
et al. 2009; Minor et al. 2010).
Recently, studies in a variety of organisms identified the insulin/IGF1, TOR, and
sirtuin signaling pathways as potential mediators of DR-induced lifespan extension. As
such, chemicals that augment the activity of these pathways have been identified and
implicated as potential lifespan extenders. Rapamycin, a TOR inhibitor, and resveratrol, a
potential sirtuin activator, have received the most attention in this realm due to their
ability to extend lifespan and ameliorate age-related diseases in many species.
61
Additionally, these substances have provided a framework through which to study the
aging process as a whole and a starting point for the development of more potent
modulators of lifespan.
In the current chapter, these proposed methods of lifespan extension will be
evaluated with regard to their effects in many species, the signaling pathways by which
they act, and their advantages and limitations in the regulation of human aging. This
understanding will enable the proposal of future research to clarify the roles of these
methods in aging and lifespan extension. In the final chapter, this information coupled
with the conclusions of previous chapters, will be leveraged to evaluate the potential of
safely and effectively extending human lifespan.
Methods of Lifespan Extension
Dietary Restriction
Dietary restriction (DR), sometimes referred to as caloric restriction (CR), has
been found to increase lifespan and delay the onset of age-related diseases in many
species and is currently the best-characterized anti-aging intervention (Chatzidaki 2010).
In fact, the effectiveness of DR was first documented in studies as early as the 1930’s
when it was observed that rats fed a reduced calorie diet lived nearly twice as long as
normally fed rats (McCay and Crowell 1934; Minor et al. 2010).
The term dietary restriction refers to any intervention that reduces food intake,
with caloric restriction the intervention most commonly studied. Generally, CR entails a
20% to 40% daily reduction in caloric intake, though in some lower species complete
fasting has been tested as well (Minor et al. 2010). In humans, CR would be undesirable
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and extremely difficult to maintain. As a result, many scientists have evaluated other
dietary paradigms that may prove more feasible and that may shed light on the
mechanisms by which CR renders its effects (Minor et al. 2010). For instance,
intermittent fasting (IF), which commonly uses 24 hour gaps in feeding, has been shown
to increase lifespan as well (Chatzidaki 2010). Some scientists argue that the effects of
CR are due to a reduction in protein intake and have thus set out to study the effects of
protein restriction (PR) with varied results (Minor et al. 2010). Additionally, it is possible
that the dietary absence of specific amino acids, most commonly methionine, may
mediate the effects of CR (Miller et al. 2005; Houtkooper et al. 2010). Though the current
chapter will mainly focus on studies of CR, the effects of IF and PR will be explored
when applicable to shed light on the biological mechanisms of CR.
Though the mechanisms of CR are not well understood, it is generally believed
that nutrient-sensing pathways play a crucial role. In theory, the ability to reduce the rate
of aging in response to food shortage or poor environmental conditions should be
evolutionarily conserved as it would allow an organism to prolong reproductive viability
until conditions improve. As such, it makes logical sense that nutrient-sensing plays an
integral role in controlling the rate of aging. Thus, CR and the nutrient-sensing pathways
described in the previous chapter likely slow aging through an overlapping and highly
conserved mechanism.
In order to determine if a specific pathway component is involved in the
mechanism of CR, scientists determine whether the addition of a CR regimen increases
lifespan beyond what is observed when altering the pathway component alone (Kenyon
2010). If no change in lifespan is observed, the component is likely involved in mediating
63
the effects of CR. For instance, the lifespan extension rendered by TOR inhibition has
been shown to be unaffected by the addition of CR in many species and is thus the most
consistently linked pathway to CR (Kenyon 2010). Additionally, even in mammals, TOR
signaling was found to be inhibited by calorie restriction (Wu et al. 2009). Other
scientists argue that CR is mediated, at least in part by the sirtuins. Proponents of the
sirtuins cite the fact that the proteins are NAD+-dependent deacetylases that act as
sensors of cellular energy status, making them prime candidates for setting the rate of
aging (Donmez and Guarente 2010). Consistent with the conclusions of the previous
chapters, it is likely that a network of signaling pathways regulates the biological
response to CR rather than a single, linear pathway (Fontana et al. 2010).
Studies in lower organisms have helped elucidate the molecular mechanisms of
CR. In yeast, lifespan extension can be achieved by complete starvation, as moving cells
from a nutrient-rich medium to pure water has been observed to double chronological
lifespan (Fontana et al. 2010). Furthermore, a 2007 study in yeast showed that TOR and
CR-induced lifespan extension is achieved by the nuclear localization of the transcription
factors Msn2 and Msn4, both of which boost the expression of Pnc1, a Sir2 regulator
(Medvedik et al. 2007). Thus, it is likely that TOR and the sirtuins are part of an
interwoven longevity pathway that responds to CR and is conserved even in higher
organisms (Medvedik et al. 2007).
In C. elegans, some methods of DR require Daf-16 and AMPK, suggesting a role
for the insulin/IGF1 signaling pathway and the sirtuins (Greer and Brunet 2009).
Additionally, the TOR pathway seems to be involved as autophagy and the oxygensensing transcription factor, HIF-1, both downstream effectors of TOR, aid in the worms’
64
response to DR (Fontana et al. 2010). Though mild DR has been shown to increase worm
lifespan through a sir2.1 dependent mechanism, many other DR regimens have increased
lifespan in the absence of sir2.1 (Wang and Tissenbaum 2006; Greer and Brunet 2009;
Kenyon 2010).
Thus, it seems that different methods of dietary restriction induce lifespan
extension through overlapping and sometimes independent mechanisms. The fact that
lifespan can still be extended in the absence of a particular pathway component does not
provide definitive evidence that the component is not involved in the DR response. In the
absence of a specific component, the complex signaling network may provide a
compensatory mechanism for still achieving increased lifespan. Understanding when and
how these pathways contribute to the effects of DR is an ongoing pursuit and should
yield valuable insights.
Studies in Drosophila further confirm the involvement of multiple, overlapping
pathways in the DR response. For instance, insulin/IGF1 mutants with increased
longevity only partially respond to DR as if their genetically increased lifespan was
achieved by an overlapping mechanism (Clancy et al. 2002; Kenyon 2010). Additionally,
in flies, both the TOR and sirtuin pathways are involved, as Sir2 deletion prevented DRinduced longevity, and the TOR target, 4E-BP, was upregulated during DR (Rogina and
Helfand 2004; Zid et al. 2009). In support of protein restriction, fly lifespan was
increased solely by a reduction in amino acid intake, with methionine and a few other
essential amino acids playing a key role (Grandison et al. 2009).
Studies in mice are the most practical for establishing the mechanisms by which
DR extends healthy lifespan in mammals (Fontana et al. 2010). Even rodent lifespan can
65
be increased by up to 60% through DR (Anderson et al. 2009). It is thought that this is
achieved, at least in part, by postponing the occurrence of chronic diseases because DR
mice are nearly 5 times less likely to die of severe organ pathology compared to controls
(Shimokawa et al. 1993; Fontana et al. 2010). In opposition to these findings, though a
few mice responded to the regimen, one study found that the effects of DR were greatly
reduced in wild mice (Harper et al. 2006). These findings highlight the limitations of
using mice raised in the lab and suggest that lifespan studies should use genetically
heterogeneous mice for the greatest relevance and applicability.
Research into amino acid restriction in rodents has also proven fruitful. Rats fed a
diet lacking tryptophan exhibit increased lifespan, as well as improved hair growth and
delayed tumor formation, likely due to a decrease in the synthesis of CNS proteins,
specifically serotonin (Segall and Timiras 1976). Unfortunately, such serotonin depletion
would most likely lead to an increased risk of suicide and psychosis in humans,
diminishing the prospect of this form of amino acid restriction (Minor et al. 2010).
Methionine restriction has also been found to increase lifespan and decrease oxidative
stress, insulin signaling, and age-related pathologies in mice (Minor et al. 2010).
Rodent studies also support the involvement of a network of pathways in the
mediation of CR-induced longevity. For instance, the absence of the TOR component,
S6K1, led to increases in lifespan and healthspan in female mice through a gene
expression pattern similar to that of CR (Selman et al. 2009). Additionally, at the
American Federation for Aging Research Conference on mTOR, Dave Sharp described a
currently unpublished study that found significant decreases in downstream mTOR
effectors during CR in mice, with the most profound differences found in fat (Sharp
66
2010). The insulin/IGF1 pathway is likely involved, as CR fails to extend the already
increased lifespans of mice with growth hormone receptor mutations (Arum et al. 2009;
Kenyon 2010). The sirtuins have also been implicated in DR-induced longevity in mice,
as SIRT1 seems to be required for the effects of dietary restriction and its overexpression
generates a phenotypic profile similar to that of DR mice (Chen et al. 2005; Bordone et
al. 2007). Additionally, the mRNA levels of PGC-1α, a sirtuin target, are increased
during CR and are thought to influence the metabolic balance between carbohydrates and
fats (Anderson et al. 2008).
Recently, a study in primates suggested that the effects of DR may apply to
humans as well. In 2009, the eagerly awaited results of a long-term monkey DR study
were published providing the first evidence of the longevity effects of DR in a species
closely related to humans (Colman et al. 2009; Austad 2009). This study, performed in
rhesus monkeys undergoing 30% DR for 20-years, found a significant reduction in agerelated deaths and lower incidences of tumors, cardiovascular disease, and diabetes
(Colman et al. 2009). Furthermore, at the time the results were reported, half of the
control animals had died compared to only 20% of DR animals, suggesting that DR
effectively extends lifespan in primates as well (Colman et al. 2009).
Steve Austad argues that some aspects of the study diminish the strength of the
findings on lifespan (2009). For example, though the difference in survival was
statistically significant (P = 0.03), it relied on the exclusion of many deaths from
accidents or factors not related to aging (Austad 2009; Colman 2009). Austad argues that
if all of the deaths are included, the results are no longer statistically significant (P=.16).
His confidence in the data is dependent on ensuring that the excluded deaths did not
67
result from some aspect of the DR regimen (2009). Regardless, he concludes that the
study clearly shows that DR produces many health benefits in the monkeys, particularly
in the realm of glucose regulation, though he believes the effects on longevity are less
definitive and dramatic than those found in rodent studies (2009).
Along the same lines, human studies of dietary restriction have shown clear health
benefits, while effects on lifespan have yet to be conclusively studied. As in rodents and
monkeys, voluntary CR produced significant cardiovascular, glucose control, and
hormonal improvements in humans, particularly involving insulin/IGF1 signaling (Smith
et al, 2010; Fontana et al. 2008). Unfortunately, with few exceptions, human DR studies
have been primarily performed in the obese, with many of the observed benefits derived
purely from weight loss (Smith et al. 2010). One such exception, which monitored the
health of 8 non-obese people undergoing CR in a biosphere, found marked improvements
in glucoregulation and cardiovascular health (Walford et al. 2002). The CALERIE study,
funded by the National Institute on Aging (NIA), is currently investigating the effects of
2 years of CR in non-obese and healthy individuals, with preliminary results paralleling
the benefits in metabolism and physiology observed previously (Smith et al. 2010).
Though conclusive and well-controlled data is lacking, it seems DR may lengthen
lifespan in humans as well. In support of this, the people of Okinawa, who practice a diet
similar to calorie restriction, have remarkably long life expectancies and one of the
highest incidences of centenarians in the world (Kagawa 1978; Chatzidaki 2009). Thus, it
seems that even short-term DR provides health benefits in humans, while long-term DR
likely delays the onset of age-related diseases and extends lifespan. Definitive answers to
these questions are expected within the decade (Smith et al. 2010).
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Despite the potential of DR to increase the lifespan and healthspan of humans, a
few barriers need to be addressed to assess the safety and feasibility of such a regimen.
For one, the side-effects of DR need to be evaluated, especially with regard to reduced
immune function and wound healing, as animals in these studies have been kept in
pathogen free environments and deficiencies in these processes have been observed
during DR (Fontana et al. 2010; Reed et al. 1996).
Additionally, gerontologists believe that most people would choose not to adhere
to DR even if obvious health benefits are demonstrated (Minor et al. 2010). The
hypothesis that protein restriction, or the restriction of specific amino acids, mediates the
benefits of CR should be studied in hopes of developing a more practical and compliable
regimen for humans. Analyzing the health and longevity of people with Phenylketonuria
could prove fruitful as many people with this disease restrict their protein intake
throughout their entire lives. Though a study such as this would be difficult to control, it
would at least potentially provide evidence supporting the benefits of a PR regimen.
Realistically, any method of lifespan extension that requires dietary changes is not
ideal. As a result, researchers are currently searching for substances that mimic the
effects of CR; as such a substance would improve lifespan and healthspan without
requiring any changes in lifestyle. Understanding the network of pathways that regulates
CR is crucial to developing such a drug. Unsurprisingly, studies of the molecular
mechanisms of CR have implicated the TOR, insulin signaling, and sirtuin pathways as
integral components of the network of CR-induced longevity. Consequently, the two
most promising lifespan extending drugs are rapamycin and resveratrol, which are
thought to mediate their effects by modulating the TOR and sirtuin pathways,
69
respectively. These proposed methods of lifespan extension will thus be the focus of the
following sections.
Rapamycin
As described in the previous chapter, TOR inhibition is sufficient to extend the
lifespan of a wide variety of species. Thus, a substance that effectively downregulates
TOR signaling may serve as a prime lifespan extension candidate. Remarkably, such a
TOR inhibitor has been in use for decades. Rapamycin, a selective mTOR inhibitor, was
first isolated from soil bacteria on Easter Island in the 1960s, and received FDA approval
in 1999 for its immunosuppressive properties, which prevent organ transplant rejection
(Minor et al. 2010; Rapamune Prescribing Information).
TOR inhibition is achieved by rapamycin binding to FKBP12, its intracellular
receptor, which then binds to the FKBP12-Rapamycin Binding (FRB) domain of mTOR,
disabling its kinase activity (Huang et al. 2003). This bioactivity was confirmed when
reduced phosphorylation of the mTORC1 target S6K was observed upon treatment with
rapamycin (Harrison et al. 2009; Austad 2009). Originally, it was thought that rapamycin
selectively inhibits mTORC1, though new evidence suggests that mTORC2 may be
rapamycin-sensitive as well, at least in certain circumstances (Stanfel et al. 2009).
Understanding when and where rapamycin is able to inhibit mTORC2 in vivo, as well as
the role of mTORC2 in modulating lifespan will be crucial in evaluating rapamycin as an
anti-aging drug candidate.
Rapamycin’s ability to inhibit mTOR suggests that it may be able to increase
lifespan as well. This hypothesis was first supported in 2006, when rapamycin extended
the chronological lifespan of yeast (Powers et al. 2006). Additionally, rapamycin
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increased the lifespan of Drosophila specifically through the TORC1 branch of the TOR
pathway and required changes in autophagy and protein synthesis (Bjedov 2010).
Interestingly, these effects were also achieved in DR flies with already maximized
lifespans, suggesting that the rapamycin-induced longevity operated, at least partially,
through a distinct mechanism (Bjedov 2010).
These results prompted the study of rapamycin’s effects in mammals. The first
research of this kind was published in 2009 and boosted rapamycin further into the antiaging spotlight as it extended the lifespan of male and female mice by 9% and 14%,
respectively (Harrison et al. 2009). Though these numbers are not quite as dramatic as the
lifespan extension of certain genetic alterations in other species, this study was
remarkable for a few reasons. Firstly, rapamycin feeding did not begin until 600 days of
age, the equivalent of 60 years old in humans (Harrison et al. 2009; Austad 2009). This is
the latest any intervention has been successful in extending rodent lifespan (Austad
2009).
Furthermore, the regimen extended both median and maximum lifespan, a
generally accepted requirement for demonstrating that aging was in fact slowed (Austad
2009). Maximum lifespan in this study, as well as in many others, is defined as the age of
death at the 90th percentile of survivability (Harrison et al 2009). Adding credibility to the
study, two independent testing sites replicated the results (Austad 2009). Finally, as
explained earlier in the chapter, it is important to perform lifespan studies in subjects with
varying genetic backgrounds to maximize the applicability of the results. Importantly,
this study used genetically heterogeneous mice to avoid genotype-specific effects
(Harrison et al. 2009).
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The results of the above study definitively show that rapamycin is sufficient to
extend the lifespan of mice. In light of this discovery, new doors are opened for
understanding the roles of rapamycin and TOR in aging. For one, the function of TOR
inhibition in the mechanism of dietary restriction needs to be uncovered. Understanding
this will help determine the extent and limitations of rapamycin in mediating lifespan and
healthspan. Additionally, with efficacy clearly demonstrated in mice, it is time to assess
the benefits of rapamycin in primates, and potentially humans.
Apart from lifespan, it is also important to understand rapamycin’s role in
ameliorating age-related diseases. Since mTOR functions in the clearance of toxic protein
aggregates, rapamycin may serve to prevent or treat diseases caused by such aggregation,
such as Alzheimer’s, Huntington’s, or Parkinson’s (Zemke et al. 2007). Research by Eric
Klann, of New York University, has proposed that mTOR may contribute to the
phenotypes of obsessive-compulsive and autism spectrum disorders, and hopes to explore
rapamycin as a treatment for these debilitating diseases (Klann 2010). Furthermore,
rapamycin is thought to inhibit inflammatory feedback loops associated with cellular
senescence, tumorigenesis, and other age-related pathologies (Kapahi 2010). The ability
of rapamycin to affect glucoregulation and cardiovascular health should also be explored
as these diseases of aging are commonly improved by the augmentation of pathways
involved in longevity.
Before successful lifespan extension can be achieved with rapamycin, a few sideeffects and limitations need to be addressed. For one, rapamycin is a potent
immunosuppressant. Thus, the negative effects caused by the impairment of the immune
system need to be evaluated to ensure they outweigh the benefits of lifespan extension.
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Furthermore, research needs to focus on the benefits of rapamycin in non-pathogen free
environments, as to properly evaluate its immunogenic effects. This will also help to
determine the optimum dosage for maximizing lifespan extension while minimizing the
effects of immunosuppression. Additionally, rapamycin may impair some cellular
functions, particularly growth and protein synthesis (Blagosklonny 2010). Assessing how
these functions impact wound healing and other aspects of repair and growth will be
important as well.
Promising results of lifespan extension in mice and the clear molecular
mechanism of rapamycin make it an extremely compelling candidate for improving
health and increasing lifespan. It may be the case that rapamycin is not the ideal
compound for this task. Fortunately, it may serve as a starting point for the development
of more effective and tolerable substances for ideal lifespan extension. Regardless,
rapamycin will be an important tool for understanding many aspects of aging and will
surely be a high priority during the next decade of aging research.
Resveratrol
The breadth of research linking the sirtuins to caloric restriction led scientists to
screen compounds for their ability to activate the sirtuins in hopes of finding a CR
mimetic (Minor et al. 2010). The discovery of SIRT1’s enzymatic activity made
screening easier and led to the identification of resveratrol as a potential SIRT1 activator
(Howitz et al. 2003).
Interestingly, resveratrol was found in its highest concentrations in plants
associated with health benefits. For instance, Polygonum cuspidatum, a plant root extract
73
used in oriental folk medicine, contains the highest concentrations of resveratrol of any
natural source (Szkudelska and Szkudelski 2010). Furthermore, the abundance of
resveratrol in red wine has been used as an explanation for the French Paradox. The
French Paradox describes the observation that, despite consuming a diet high in fat and
enjoying regular cigarette smoking, the French have lower incidences of coronary heart
disease, potentially from the cardioprotective effects of drinking relatively large
quantities of red wine (Minor et al. 2010; Criqui and Ringel 1994).
Since its discovery, resveratrol has been linked to CR. For instance, one study,
which examined genome-wide transcriptional profiles of mice on a CR diet or a diet
supplemented with resveratrol, observed a remarkable overlap in transcriptional changes
in both groups (Barger et al. 2008). Specifically, resveratrol mimicked aspects of CR in
terms of insulin-mediated glucose uptake, cardioprotectivity, and alterations in chromatin
structure and transcription (Barger et al. 2008). On the other hand, in yeast, CR has been
observed to extend lifespan in the absence of the sirtuins (Kaeberlein et al. 2004). Thus,
consistent with previous findings in this paper, it is likely that the sirtuins do play a role
in CR-mediated longevity, but are merely one element of a large and intertwined network
of signaling pathways.
Studies have also investigated reveratrol’s effects on longevity. In lower
organisms, such as yeast, worms, and flies, reveratrol has been shown to increase lifespan
(Howitz et al. 2003; Wood et al. 2004). Furthermore, resveratrol was able to increase the
maximum lifespan of N. furzeri, a short-lived seasonal fish, by up to 59% (Terzibasi et al.
2009; Camins et al. 2010)
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However, in mammals, the effects of resveratrol on lifespan are less clear. For
instance, resveratrol was able to increase the median lifespan of mice fed high-fat diets,
but did not affect the longevity of normally fed mice (Baur et al. 2006). Additionally,
resveratrol feeding was shown to ameliorate many aspects of aging, including
inflammation, heart disease, decreased motor function, and weakening bones, without
extending lifespan (Pearson et al. 2008). Thus, although resveratrol may not be able to
extend lifespan in normal individuals, it may be able to improve some aspects of aging in
the overweight and elderly. Further research exploring the effects of resveratrol on many
measures of health, particularly those associated with aging, is warranted.
Though it is clear that resveratrol interacts with the sirtuins, the mechanism by
which this occurs and leads to changes in healthspan has yet to be completely understood.
Previously, it was thought that resveratrol activates SIRT1 directly; however, a recent in
vitro study reports that this may be the result of an experimental artifact, as resveratrol
was unable to activate SIRT1 under the conditions measured (Beher et al. 2009). In
support of a different mechanism, Sir2.1 extends lifespan through Daf-16/FOXO in C.
elegans, whereas resveratrol does not (Kenyon 2010).
It is likely that the mechanism underlying the link between resveratrol and the
sirtuins is more complex than previously thought. One model proposes that resveratrol
mildly inhibits mitochondrial respiration, leading to an increase in the AMP/ATP ratio,
thereby activating AMPK, and thus SIRT1 (Canto et al. 2009; Houtkooper et al. 2010).
Another idea suggests that resveratrol might alter the substrate specificity of SIRT1
(Camins et al. 2010). Further research that uncovers the mechanism by which resveratrol
75
activates the sirtuins is necessary to properly evaluate the drug as a potential calorie
restriction mimetic and a benefiter of health.
Figure 4.1: Activating SIRT1 Improves Many Age-Related Diseases
Source: Sirtris Pharmaceuticals Website
Despite the lack of concrete evidence supporting resveratrol’s ability to increase
lifespan, the drug has real potential in the mitigation of age-related diseases (Figure 4.1).
For instance, resveratrol has been found to improve glucose homeostasis and insulin
sensitivity in a variety of tissues, and thus may have potential as a treatment or preventer
of diabetes (Camins et al. 2010). The fact that resveratrol was able to improve many
measures of health in obese mice suggests it may have beneficial effects on
glucoregulation, a common problem that develops in the overweight. Initial studies of the
French Paradox and recent research suggest that resveratrol possess cardioprotective
76
properties as well. At first, resveratrol was thought to mediate its beneficial effects on the
heart through its antioxidant properties, however, it is now also thought that the
compound attenuates mitochondrial ROS production and increases the expression of
superoxide dismutase (Orallo 2006; Camins et al. 2010).
Resveratrol has also received a lot of attention for its potential to improve
neurodegenerative diseases associated with aging.
For one, the drug decreases
neuroinflammation and improves memory loss in aging mice with infections, suggesting
that it may attenuate acute cognitive disorders in elderly individuals (Abraham and
Johnson 2009). Additionally, reports have suggested that resveratrol may serve to protect
against Alzheimer’s Disease, as it was found to be an inhibitor of acetylcholinesterase
and it reduced the signaling of the inflammatory cytokine NF-kB when stimulated by βamyloid (Vingtdeux et al. 2008; Moon et al. 2008; Camins et al. 2010). Additionally,
moderate red wine consumption has been linked to a lower incidence of Alzheimer’s
disease and dementia in general suggesting that resveratrol may indeed possess
neuroprotective properties (Minor et al. 2010). As such, resveratrol has been suggested to
show beneficial effects in Parkinson’s disease models as well (Camins et al. 2010).
Though resveratrol was identified as a potential SIRT1 activator less than a
decade ago, it has received widespread attention for its possible effects on longevity and
its clearer role in mediating age-related disease. Future studies need to determine the
mechanism by which resveratrol activates the sirtuins, its role as a CR mimetic, and other
potential targets through which the compound acts. As research progresses and this
knowledge surfaces, it may also be beneficial to synthesize compounds that directly and
77
more potently activate the sirtuins. Regardless, resveratrol will be a prime starting place
and tool for such synthesis and understanding.
Discussion
The past decade of aging research has witnessed significant advances in
uncovering the molecular mechanisms of aging and potential interventions to extend
lifespan. Though dietary restriction is the best characterized lifespan extending
intervention, it is also the least practical. As such, the discovery that resveratrol and
rapamycin influence aging provides promising leads for feasible lifespan extension in
humans. Though these compounds may not be marketable, they will surely help to
uncover the mechanisms underlying DR and the aging process in general. Additionally,
the chemical structures of these compounds may serve as frameworks for the
development of safer and more effective regulators of longevity.
The first step forward will be to more clearly determine the signaling changes that
occur as the result of DR, rapamycin, and resveratrol. Evidence in this chapter
definitively shows that these interventions render their effects on lifespan and healthspan
through a mechanism that overlaps highly with the molecular signaling pathways
implicated in longevity (Ch. 3). Unfortunately, the current perspective tends to isolate
the mechanisms of such interventions to specific signaling pathways. Consistent with the
conclusions of previous chapters, this view needs to be replaced by a network of
intertwined pathways (Figure 4.2).
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Figure 4.2: A Network of Signaling Pathways Regulate the Response
to CR, Rapamycin, and Resveratrol
?
mTORC2
AKT/PKB
Daf-16/FOXO
PGC-1α
Insulin Receptor
p53
S6K
4EBP
mTORC1
Caloric
Restriction
AMPK
NF-kB
Sirtuins
PNC1
Rapamycin
Msn2 + Msn4
Resveratrol
Changes in Metabolism and Protein Synthesis
Increases in Lifepsan and Healthspan
Such a model of interconnected signaling pathways should provide future
direction and help to explain discrepancies in the data. For instance, as described earlier
in the chapter, activation of the sirtuins produced a transcriptional profile similar to that
of DR, however, in some cases, DR-induced lifespan extension was achieved in the
absence of the sirtuins. This seems contradictory when these pathways are viewed in
isolation. However, a network model can help explain these findings. The fact that the
sirtuins are not required for the effects of DR does not mean that they are uninvolved. It
is likely that in the absence of the sirtuins a feedback mechanism enhances DR’s effects
on the other pathways, and thus compensates for the deficiency of this signaling
component.
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Each branch of this network is tweaked by the activities of the other branches,
leading to a fine-tuned system of nutrient sensing, metabolism, and longevity regulation.
The dramatic and conserved effects of DR are likely due to its impact on multiple
branches of this signaling network. The fact that these pathways are interdependent
greatly complicates the ability to determine the precise mechanism of DR. Future studies
should seek to quantify changes in the activity of various components of this network
during effective DR. This quantification will serve as a benchmark for the testing of
potential lifespan extending compounds. For instance, if DR decreases mTORC1 2-fold,
the dose of rapamycin can be altered to achieve equal inhibition. However, DR may also
increase the activity of SIRT1 by a factor of 5, while the same rapamycin dose only
increases it 2-fold via feedback mechanisms in the signaling network. Thus, either higher
doses of rapamycin or the administration of multiple compounds acting on different
components will be required to mimic DR. In this pursuit, maximizing efficacy while
maintaining safety will be the preferred protocol. As such, lower doses, and thus the coadministration of multiple drugs that act on various network components is the likely
outcome.
Though recent advances have propelled safe and effective lifespan extension into
the realm of possibility, there is still much to learn. In the final chapter, the feasibility of
this remarkable task will be evaluated by leveraging the information of previous chapters.
Furthermore, the chapter will suggest future direction, predict likely outcomes, and
briefly explore the ethical factors surrounding the quest for prolonging youth.
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CHAPTER 5
THE POTENTIAL OF EXTENDING HUMAN
LIFESPAN THROUGH PHARMACEUTICAL
INTERVENTION
An understanding of the molecular mechanisms of aging, coupled with the known
methods of lifespan extension, provides a firm foundation from which to develop drugs
that extend human lifespan safely and effectively. The discovery that CR accomplishes
just this in a few closely related organisms, suggests that such a goal may be attainable.
Unfortunately, strict adherence to a CR regimen is undesirable for the majority of the
population, even if it is effective. Thus, developing pharmaceutical agents that produce
the same benefits as CR, without requiring changes in lifestyle, provides an ideal strategy
for realistic lifespan extension. The discovery that the modulation of a few intertwined
nutrient-signaling pathways produces changes in healthspan and lifespan in many
organisms suggests that the development of such CR mimetics may be possible.
Additionally, the recent identification of rapamycin and resveratrol, two natural
substances that augment these signaling pathways and improve many measures of agerelated health, greatly supports such a strategy for realistic lifespan extension.
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In this final chapter, the potential of safe and effective lifespan extension in
humans will be evaluated with regard to a variety of factors. The current pipeline of drugs
for age-related diseases, the properties of an ideal lifespan extension drug, and some
ethical considerations will be explored. From this, future research needed to achieve such
a goal will be proposed. Finally, the paper will conclude with a discussion of an
appropriate perspective with which to view aging and predictions of what is likely to
come.
Creating Drugs to Extend Lifespan
Current Pipeline of Drugs for Age-Related Diseases
The discovery that resveratrol ameliorates many aspects of aging through the
modulation of SIRT1 activity, led to the formation of Sirtris Pharmaceuticals, a company
dedicated to “developing proprietary, orally available, small molecule drugs with the
potential to treat diseases associated with aging, including metabolic, inflammatory,
neurodegenerative and cardiovascular diseases” (Sirtris Pharma. 2010). Sirtris’ pipeline
includes its own resveratrol formulation along with more potent SIRT1 activators that it
developed in-house (Sirtris Pharma. 2010). The anti-aging potential of these drugs led to
the purchase of Sirtris for 720 million dollars in 2008 by GlaxoSmithKline (Caroll 2010).
Since natural sirtuin activators displayed low potency, Sirtris, through the use of
high-throughput screening, was able to identify novel, selective SIRT1 activators with
much higher potency (Camins et al. 2010). Though other compounds have been
developed, there are currently only 3 compounds in the Sirtuin pipeline: SRT501,
SRT2104, and SRT2379 (Figure 5.1) (Sirtris Pharma. 2010).
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Unfortunately, at the end of 2010, Sirtris abandoned development of its special
formulation of resveratrol, SRT501, because clinical trials for multiple myeloma showed
that it “offers minimal efficacy while having a potential to indirectly exacerbate a renal
complication common in this patient population" (Caroll 2010). As a result,
GlaxoSmithKline executives have chosen to focus “on more selective SIRT1 activator
compounds that have no chemical relationship to SRT501 and more favorable drug-like
properties" (Caroll 2010).
Thus, the compounds SRT2104 and SRT2379 currently have the greatest
potential. These new chemical entities (NCEs) are said to activate SIRT1 1000-times
more potently than resveratrol (McBride 2008). Preclinical studies of SRT2104 in animal
models demonstrated improvements in glucoregulation, indicating potential for the
treatment of type 2 diabetes (Sirtris Pharma. 2010). As such, SRT2104 was Sirtris’ first
compound to enter human trials, and phase I trials in healthy individuals demonstrated
that the compound was both safe and well tolerated (Camins 2010). These promising
results pushed SRT2104 into phase II trials in human participants with type 2 diabetes,
and results are eagerly anticipated (Clinicaltrials.gov 2011). According to Sirtris,
83
SRT2104 is also being evaluated in patients with metabolic, inflammatory, and
cardiovascular diseases (Sirtris Pharma. 2010). The compound SRT2379 is also under
evaluation in clinical trials, with phase I trials currently underway that investigate the
safety and pharmacokinetics of the drug in male volunteers (Camins et al. 2010). Though
these drugs are the most promising of their kind, their long-term effects on lifespan and
healthspan have yet to be determined (Minor et al. 2010).
Rapamycin versus Resveratrol
Currently, rapamycin and resveratrol are the best-characterized substances that
mimic some aspects of CR. So, which substance will prove to be the better lifespan
extension candidate? Answering this question will allow us to more effectively allocate
resources towards the anti-aging effort. As expected, such an answer is not black and
white. Current evidence supports and refutes the attractiveness of each compound,
making predictions difficult. Furthermore, the fact that these compounds may serve as
frameworks for the production of more effective CR mimetics hinders forecasting. In
light of such difficulties, this paper will use current knowledge to make such a prediction.
It is the view of this paper that resveratrol and its analogs will serve as better
candidates for the treatment of age-related diseases, while rapamycin will serve as a
better candidate for lifespan extension. This hypothesis is based on the fact that
rapamycin is the only compound that was able to extend lifespan in mammals, whereas
resveratrol was only capable of improving measures of age-related health. Furthermore,
the TOR pathway seems to be a more powerful modulator of longevity than SIRT1,
84
stemming from its role in protein synthesis and autophagy, which adds weight to the
prospects of rapamycin.
Leonard Guarente, a co-chairman of Sirtris Pharmaceuticals’ scientific advisory
board, disagrees with this claim and argues that “the sirtuins have their fingers in the
most branches of longevity and are thus the best candidates for effective lifespan
extension” (Guarente Inteview 2010). Though Guarente may be correct, current evidence
prohibits the author from drawing the same conclusion. The main hindrance arises from
that fact that resveratrol is not a direct activator of SIRT1. Longevity studies using
Sirtris’ more potent sirtuin activators will help to elucidate the potential of sirtuin based
lifespan extension and may shift the scale in favor of resveratrol analogs. Also in favor of
the sirtuins is safety, as TOR inhibition leads to immunosuppression. Developing nonimmunosuppressive analogs of rapamycin will be crucial for the approval of lifespan
extending TOR inhibitors.
To summarize, rapamycin is currently the best prospect for effective lifespan
extension because of its observed efficacy in mammals. The sirtuins, on the other hand,
have not been observed to extend mammalian lifespan, but have been shown to improve
measures of age-related health. This, coupled with Sirtris’ pipeline of sirtuin activators,
supports a role for sirtuin activators in the future treatment of age-related diseases.
Further assessment of the long-term side effects of TOR and sirtuin modulation, as well
as the role of more potent sirtuin activation in longevity is warranted for a better
evaluation of these candidates.
85
Properties of an Ideal Lifespan Extension Drug
In the pursuit of pharmaceutical-based lifespan extension, it is important to
identify the properties of a perfect drug of this kind. Though this approach is somewhat
premature, it will become more relevant as the understanding of longevity regulation
becomes clearer. In evaluating such a drug, efficacy, safety, dosing, and cost will be
considered, as they are with all drug candidates. The nature of drug approval in the
United States forces us to consider these properties for successful population-wide
lifespan extension.
In terms of efficacy, a lifespan extension drug will be required to produce robust
increases in lifespan to gain approval, probably on the order of at least 10%. The drug
should, in addition to increasing lifespan, improve many measure of age-related health,
meaning it will delay and potentially compress aging. Beneficial effects on age-related
diseases are likely to be even more important factors than lifespan extension itself. The
fact that the FDA does not consider aging to be an indication, suggests that lifespan
extension drugs will only be approved if they affect disease (Kenyon 2010). Thus,
pharmaceutical companies hoping to obtain drug approval for lifespan extension should
instead focus on gaining approval through demonstrated benefits in age-related diseases.
The safety profile of a lifespan extension drug must be impeccable, with the
benefits of living longer greatly outweighing the side-effects of the drug. For instance, for
drugs that treat diseases like cancer, many side-effects are tolerated because of the drugs’
enormous benefit in saving lives. However, drugs that treat less life threatening diseases,
like arthritis, are required to have fewer and less severe side-effects. If an arthritis drug
caused hair loss, nausea, anemia, or other side-effects observed in chemotherapy, it
86
would offer no net benefit and no one would take it. Thus, the benefits of a drug that
extends lifespan must outweigh the costs of its side-effects in order to be a feasible
option.
Regulating safety will certainly be the greatest challenge once efficacy is
demonstrated. The fact that nutrient-sensing pathways currently provide the best targets
for longevity modulation indicates that safety concerns are likely to arise. These
pathways are closely tied with many human diseases, including cancer, and play essential
roles in proper growth and development. Ensuring that an effective lifespan extension
drug does not increase the incidence of cancer, suppress immune function, impair
healing, or induce many other possible effects will be essential for approval.
One approach to achieving feasible lifespan extension could be to use a drug
cocktail. A multidrug approach may be the best way to maximize efficacy while
minimizing safety concerns. Such an approach would lower the dose of each component,
while potentially affecting multiple branches of the longevity network. The ability to
augment the activities of different branches may produce greater benefits in lifespan,
while requiring smaller doses of each drug. Smaller doses may, in effect, increase overall
tolerability and reduce side-effects, leading to an approvable safety profile.
In the evaluation of a lifespan extension candidate, dosing is also a critical factor.
Administration of an ideal drug should be started late in life and infrequently dosed.
Starting late in life will ease the burden of taking such a drug, and likely decrease longterm side-effects. The fact that rapamycin was effective in mice even when administered
at an analogous human age of 60 years old, suggests that this goal may be attainable.
Ideally, the maximum benefits to lifespan extension will be achieved even through late-
87
life administration, thereby eliminating any need or incentive to take the drug earlier and
longer.
High-compliance will also be essential, and thus, a drug that is able to be taken
infrequently will aid in this pursuit. Ideally, such a drug would be consumed in water or
food, making the burden of compliance extremely low. A drug that requires
administration throughout a patient’s life or multiple times per day is likely to be
unsuccessful, unless creative means of delivery are created to ease such a burden.
Reducing costs will also be important for the success of a lifespan extension drug.
Such a drug should cost pennies per pill as it needs to be affordable for the entire
population. In summary, an approvable lifespan extension drug will not only display
efficacy in terms of longevity, but also in terms of improving age-related diseases; it will
have an impeccable safety profile and it will require administration infrequently and only
beginning late in life.
The Ethics and Economics of Lifespan Extension
Extending human lifespan presents questions of ethics and policy, which should
be addressed here briefly. For one, is it ethical to extend the lifespan of some, but not
others? If the answer to this question is no, then a lifespan extension drug should be
available to anyone who desires its effects. The main implication of this is that the drug
should not be prohibitively expensive. As such, the government may consider subsidizing
such a drug. It is possible that such a policy would not only benefit people who desire
healthier and longer lives, but also the government, as decreases in healthcare costs and
increases in worker productivity rendered by the drug may outweigh the costs of
88
providing it to everyone. This issue is far from relevant today, but as lifespan extension
becomes a reality, these questions will need to be addressed.
Improving Our Perspective on Aging
Future Research
Though research over the past decade has provided a strong foundation for the
development of lifespan extending drugs, much is yet to be understood. In the near
future, scientists need to answer a variety of questions pertaining to the aging process as a
whole, the molecular networks of longevity, and the proposed methods of lifespan
extension before such a drug can be successfully developed.
The conclusions of Chapter 2 argue that aging arises from the accumulation of
macromolecular damage, with the rate of such damage determined by genetic
determinants. This damage can occur in genomic or proteomic components and it is
likely that different types of damage play larger or smaller roles in different tissues.
Identifying where, when, and how these different types of damage render the phenotypes
of aging will be important to understanding the process holistically. Specifically,
understanding the role of mitochondria in aging will be extremely important, as evidence
implicates these organelles as crucial mediators of the aging process. Additionally, the
role of telomeres and telomerase in cellular senescence needs to be further explored.
Finally, from a lifespan extension perspective, it will be interesting to determine whether
removing damage or altering the rate of damage accumulation is sufficient to delay aging.
Linking stochastic damage accumulation to the activities of nutrient-signaling
pathways will be important in understanding how lifespan is controlled and augmented.
89
Elucidating the mechanisms of autophagy will likely be fruitful in this regard.
Furthermore, uncovering how the activities of these signaling pathways change over
evolutionary time is necessary to understand how aging arises on species-specific time
scales. In this pursuit, it may prove useful to investigate the sequences of the proteins
involved in these signaling pathways. These sequences can be recorded in many
organisms and correlated with changes in longevity. From this, differences in speciesspecific lifespan may be understood as arising from genetic changes in the proteins
involved in these pathways.
Determining a complete network of signaling-pathways that regulate longevity
will also be important to understanding the lifespan extending effects of CR. In this
regard, a few key insights are needed. For one, understanding how mTORC2 is activated,
its role in regulating mTORC1, and its potential sensitivity to rapamycin is necessary.
Additionally, surveying the important proteins in these pathways for binding sites of
other network proteins may serve to link these pathways more tightly together.
Furthermore, studies have primarily investigated the effects of augmenting only one
component of a nutrient-sensing pathway. Determining the results of simultaneously
altering the activities of multiple pathways may yield more robust increases in lifespan
and may lead to the ability to extend lifespan with fewer side-effects. Finally,
determining tissue specific effects is also important, as pathway augmentation in certain
tissues is sometimes sufficient to delay aging in many organisms. The ability to increase
longevity by regulating signaling pathways in one, or a few tissues, will greatly enhance
the feasibility of safe lifespan extension, as a tissue-specific approach may limit sideeffects.
90
If caloric restriction is unable to extend human lifespan, scientists may be back to
square one in the pursuit of lifespan extension. This is unlikely to be the case, however,
as the regimen was successful in rhesus monkeys, an extremely close evolutionary
relative. Long-term studies need to further evaluate the effects of CR on lifespan and
healthspan in humans. Such studies are currently underway and should yield results
within the decade. It is likely that such efforts will confirm the efficacy of CR in humans
and support further development of CR mimetics.
Uncovering the mechanisms by which CR leads to changes in lifespan is on the
forefront of aging research as well. Many transcriptional changes have been identified,
but understanding the role of epigenetics may help form a better picture of the CR
response. Epigenetics describes the study of heritable traits that do not involve changes in
the underlying DNA sequence (Russo 1996). Thus, understanding epigenetic changes
with age may shed light on how aging arises and how CR might curb such changes. This
insight will also aid in creating a better molecular profile for CR mimetics and will help
evaluate the potential of resveratrol and rapamycin in lifespan extension. The elucidation
of the mechanisms of CR, within the context of a more complete understanding of the
pathways implicated in longevity, should provide enough information to develop drugs
that can extend lifespan.
A Network Approach to Aging
Serving as an overarching theme of this paper, the factors and pathways that
regulate aging should be viewed as part of an intertwined, codependent network, rather
than as linear and mutually exclusive. Though this view is slowly beginning to take hold,
it is in its infancy, and widespread adoption of this perspective is necessary for an
91
informed and comprehensive view of the aging process. It is only through such a
perspective that it will be possible to understand aging and develop safe and effective
drugs to extend lifespan.
Though daunting and rather complex, a network approach to aging fits into a
theme that exists across all aspects of molecular biology. The discovery of powerful
molecular techniques over the last few decades has enabled research into many aspects of
biology, including cancer, organismal development, cell biology, infectious diseases,
immunology, and many others. If there is one discovery that applies to all of these
specialties, including aging, it is that the factors that control all of these processes are
beautifully complex. In these cases, though new discoveries may answer some questions,
they also introduce countless others.
Such complexity should be appreciated, however, because without it the
remarkable
diversity
and
sophistication
of
life
would
be
impossible.
An
acknowledgement of such complexity, coupled with the perspective of a network of
pathways that regulate aging, will provide researchers with the creative insight for future
direction and an informed and consistent understanding of the problem at hand.
Final Thoughts
In the pursuit of extending human lifespan, many ask, “do we even want to live
longer?” Such a question misinterprets the goal of lifespan extension research. According
to Filipe Sierra, the Director of the National Institute on Aging’s Division of Aging
Biology, “lifespan extension is not about increasing how long we live, but about
extending youth; it is not about increasing the duration of life, but the quality of life”
92
(Sierra 2010). In Sierra’s view, adopting this perspective is crucial to understanding the
aim of lifespan extension and is important for securing funding for such research.
Whether it be the search for the Fountain of Youth or the “gland madness” of the early
20th century, the poor scientific history of eliminating aging has cast a shadow over this
type of research. Changing the public perception of lifespan extension from living forever
to living both youthfully and longer, will be an important step along the way.
So what does the future hold? Are we likely to see a lifespan extension drug in
our lifetime? No one can say with certainty, but one result of aging research does seem
probable. In the near future, we can expect our efforts to lead to the effective
amelioration of age-related diseases. The pipeline of Sirtris Pharmaceuticals and the
development of rapamycin analogs are likely to prove useful in this regard. Based on the
current state of such drug development, it is likely that by 2030 at least one drug will be
on the market for type II diabetes, a cardiovascular disease, or a neurodegenerative
disease that acts on a target within the longevity network. Over time, drugs indicated for
age-related diseases may additionally render increases in human lifespan. Evaluating
patients taking these drugs will provide informative data for the potential of such drugs to
extend the lifespan of the general population. Thus, it is likely that drugs for age-related
diseases will be approved in the near future and that such drugs may also be observed to
extend human lifespan.
Regardless of what the future holds, the aging research of the last few decades has
served as an end in itself. It has caused us to face our own mortality through a scientific
lens, and has shown us that we are no more than complex machines, susceptible to the
ravages of time. However, it has also given us the hope of improving our molecular
93
integrity and has shown us that the potential to control our lifespans may be real. Whether
we find a way to live youthfully forever, simply improve the quality of old age, or only
hone an understanding of the process that leads to our own demise, we will have at least
gained an understanding of the biology of our species and an appreciation for the
beautiful complexity that is aging.
94
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