MDIBL UMPI-UMFK 2017 short course:
Genetics of Aging and Physiological Stress Resistance
Purpose:
To demonstrate that the aging process is regulated and
modifiable by manipulating insulin-signaling pathways in C. elegans
and observing its effect on age-related phenotypes, including
stress resistance, mobility, and longevity.
Objectives:
1) To recognize that the process of aging is not a series of
random or stochastic events, but is biologically regulated and
modifiable through a variety of signaling pathways that are
conserved across animal species.
2) To review the insulin signaling pathway in C. elegans
nematodes and its homolog in humans.
3) To demonstrate in c. elegans that down-regulation of the
insulin signaling pathway through mutation of the DAF-2 gene can
increase DAF-16 (FOXO) translocation into the nucleus, activate a
stress response, preserve mobility, and improve markers of
longevity.
4) To show DAF-16 activation through attenuation of daf-2 gene
expression can improve resistance to heat shock.
5) To extrapolate these observations into analogous human
diseases and vulnerabilities associated with aging.
6) To learn about other biological and genetic manipulations
that can increase longevity in lower organisms and their potential
for translation into human interventions (e.g., caloric
restriction, Sirtuins, mTOR inhibition with rapamycin, metformin,
and the concept of hormesis).
7) To recognize that interventions to improve stress resistance
in humans may have an impact on a person’s vulnerability to all
age-related diseases – not just one. Therefore, understanding the
biology of aging can ultimately make a profound impact on promoting
a healthy human lifespan.
Background
Overview of Molecular and Genetic Mechanisms of Aging:
Insulin/IGF-1 Signaling pathway:
This lab will focus on the insulin/IGF-1 signaling pathway,
which has been well demonstrated to affect the lifespan of c.
elegans and other lower organisms. In this pathway the C. elegans
DAF2 receptor is homologous to the insulin/IGF-1 receptor in
humans, and exerts a downstream inhibitory influence on the DAF16
(FOXO) transcription factor. When insulin/IGF-1 signaling is
reduced, DAF16 is translocated from the cytoplasm to the nucleus
where it activates a cascade of genes that mediate oxidative
stress, heat shock, innate immunity, metabolism, and autophagy.
Accordingly, daf-2 mutation or daf-16 overexpression leads to an
increase in lifespan, maintenance of mobility, and stress
resistance.
Aging affects all organ systems and is one of the most complex
phenotypes involving molecular, cellular and organ damage, leading
to loss of function and increased vulnerability to disease and
death. Major progress has recently been made in the identification
of a large number of genes whose mutations affect life span in
various organisms. Subsequent studies of their gene functions have
revealed that the aging process, like so many other biological
processes, is actively regulated by classical and highly conserved
signaling pathways and transcription factors.
Figure 1. A simplified model of the influence of insulin-like
signaling in C. elegans.
Insulin is the major hormone controlling critical energy
functions such as glucose and lipid metabolism. Insulin elicits a
diverse array of biological responses by binding to
DAF-2/insulin/IGF-I receptor. This receptor mediates these effects
by activation of signaling pathways, which utilize lipid kinases
such as Phosphatidylinositol 3-Kinase (PI3K). It activates the
serine/threonine kinase Protein Kinase-B (Akt/PKB). Then, Akt
retains DAF-16/FOXO in the cytoplasm and inhibits its
transcriptional activity. Akt also activates the Mammalian Target
of Rapamycin (mTOR) pathway. Activation of mTOR results in the
translation of ribosomal proteins, elongation factors and
insulin-like growth factor to enhance protein synthesis. A
Rapamycin interaction with mTOR inhibits its activity. In response
to reduced insulin/IGF-I signaling or stress (heat, oxidative,
starvation), DAF-16/FOXO enters the nucleus, where it turns on
survival genes, including those that manage oxidative stress, heat
shock, innate immunity, metabolism, autophagy, and xenobiotic
response, among others. Indeed, over- or underexpression of such
targets often impacts stress resistance and longevity.
Nutrient-sensing Pathways:
Nutrient-sensing pathways are fundamental to the aging process.
Many of the mutations that extend life span decrease activity of
nutrient-signaling pathways, such as the Insulin/IGF (insulin-like
growth factor) and the TOR (target of rapamycin) pathways,
suggesting that they may induce a physiological state similar to
that resulting from periods of food shortage. Studies in several
model organisms have shown that dietary restriction without
malnutrition, or manipulation of nutrient-sensing pathways through
mutations or drugs, can increase life span and reduce age-related
disease. According to the principal of hormesis, the increase in
longevity in response to food shortage (or its molecular
equivalent), may enable an organism to delay reproduction and
survive until conditions are more favorable to successfully breed
offspring.
One of the best-characterized nutrient-sensing aging pathways
across evolution is the insulin/IGF-1/PI3K signaling pathway (e.g.
IGF1, IGF-1R, Klotho, IRS-1, p66SHC, PI3K, ATK1, FOXO, mTOR and
S6K), which mediates the anti-aging effects of dietary restriction
in mice. Reduced activity of the Insulin/IGF signaling pathway
extends life span in C. elegans and other multicellular organisms.
This increase in longevity requires the Forkhead FoxO transcription
factor (daf-16 in C. elegans), which regulates genes involved in a
wide range of defensive activities including cellular stress
response, antimicrobial activity, and detoxification of xenobiotics
and free radicals. In high nutrient conditions, FoxOs are
phosphorylated and excluded from the nucleus; in low nutrient
conditions, they relocalize to the nucleus.
Significantly, reduced activity of the insulin/IGF-1 signaling
pathway not only increases life span, but also protects against
cancer, Alzheimer’s Disease (AD), cardiovascular disease and Type 2
Diabetes in multicellular organisms such as mice and rhesus
monkeys. In humans, an overrepresentation of heterozygotes for
mutations in the IGF-1 receptor gene was found among Ashkenazi
Jewish centenarians as compared to controls. Subjects with a
genotype associated with reduced concentration of free IGF-1 in
plasma were overrepresented among long-lived people indicating that
specific polymorphisms that down-regulate the GH and IGF-1
signaling pathways may promote human longevity. Furthermore,
genetic variants of FOXO transcription factors, orthologs of the
key Insulin/IGF-1 effector daf-16 in C. elegans, have repeatedly
been shown to be associated with human life span.
Nutrients increase the level of IGF-1 and activate the
Insulin/IGF-1 signaling pathway, which, in turn, activates
pro-aging pathways in various mammalian cells. In contrast, caloric
restriction in humans causes changes that protect against
age-related pathologies, including diabetes, cardiovascular
disease, and cancer. Restricting calorie intake in mice or
introducing mutations in nutrient-sensing pathways can extend
lifespans by as much as 60%, in part by delaying the occurrence of
many chronic diseases in these these ‘Methuselah mice’.
Importantly, ~30% of animals on dietary restriction die without
evidence of severe organ pathology. In other words, extending
lifespan also seems to increase ‘healthspan’, the time lived
without chronic age-related conditions. Longevity pathways
identified in model organisms seem to be conserved in humans and
can be manipulated in similar ways. Notably, genetic surveys of
centenarians implicate hormonal and metabolic systems and long-term
calorie restriction in humans induces drastic metabolic and
molecular changes that resemble those of younger people, notably in
inflammatory and nutrient-sensing pathways.
Other Longevity Pathways:
It is unlikely that a single, linear pathway mediates the
effects of aging in any organism. Matching of metabolism, growth,
and fecundity to food intake is crucial for survival and
reproduction in nature, and parallel and redundant pathways appear
to be involved. Indeed, not only the Insulin/IGF cascade but many
other molecules (e.g. p53, sirtuins, AMP kinase, Notch, Wnt,
β-catenin, telomerase) also have critical roles in affecting
various aging processes. For example, SIRT1 (Sir2), the founding
member of the sirtuin gene family, is an NAD+-dependent deacetylase
regulating life span in many species as diverse as yeast, worms,
flies and possibly mice. Similarly, age-associated telomere
dysfunction and associated p53 activation have emerged as important
instigators of a functional decline of tissue stem cells and of
mitochondrial dysfunction that adversely affect renewal and
bioenergetic support in diverse tissues. In fact, telomerase
overexpression extends life span in cancer-resistant mice.
Interestingly, many of these pathways or molecules interact with
each other and often target some common downstream transcription
factors such as the forkhead-related transcription factors (FoxO).
Moreover, these pathways also respond to various cellular stresses
(e.g. DNA damage, telomere loss, oxidation and dietary
restriction). Finally and strikingly, manipulating many of these
aging regulators in the neuron, gut, or adipose tissue is
sufficient to modulate life span and other aging processes, as
shown in IGF-1R, IRS2, SKN-1, SIRT1, p53 and Mgat1, indicating the
central role of the brain and metabolic organs in aging.
Epigenetic Factors Influencing Lifespan
The discovery of long-lived mutants in animal model systems
suggests that the ageing process can be genetically modulated. In
addition to genetic inputs, emerging evidence implicates
environmental factors in ageing, such as dietary manipulations, DNA
damage, telomere loss or stress. Indeed, studies in humans have
estimated the non-heritable portion of healthspan and longevity
regulation to be approximately 70%. Recent advances in
ultra-high-throughput technologies have revolutionized our
knowledge of epigenetic factors and their relationships to gene
regulation. Epigenetic mechanisms (modes of genomic regulation that
are not directly encoded in DNA) and more specifically changes of
chromatin, which are influenced by the environment, are now
considered to act as an interface through which environmental
signals interact with genetic components throughout lifespan. Also,
stable changes in chromatin states could preserve memory of past
environmental exposures, leading to long-lasting phenotypic effects
that may be particularly relevant to ageing. Changes in the
chromatin landscape influence transcription and seem to underlie
the transcriptional changes that are observed with ageing.
Reference epigenomes provided by the National Institutes of Health
Roadmap Epigenomics Project and the Encyclopedia of DNA Elements
(ENCODE) database aid our understanding of the remodelling of
epigenomic patterns that occur during ageing.
Recently, DNA methylation has been linked to chronological as
well as biological age and could thus represent a biomarker for
aging. Furthermore, histone methylation which is associated with
either active or repressed genome regions and is known to be
dynamically regulated, changes at the global level in several
organismal models of aging. For instance, the rare diseases Werner
Syndrome and Hutchinson–Gilford progeria syndrome (HGPS), which are
characterized by premature aging in humans, are associated with
perturbed histone methylation and alterations in heterochromatin
organization. Furthermore, histone acetylation, which influences
the physical associations between histones and DNA, is a key,
conserved player in longevity, and evidence suggests that its
pattern changes during normal ageing. Both histone acetylases and
histone deacetylases modulate lifespan and metabolic health.
One of the best studied classes of histone deacetylases
associated with longevity are the NAD+-dependent deacetylase class
of sirtuin proteins. Sirtuins like SIRT1, SIRT3 and SIRT6 are
important mediators of dietary restriction-induced longevity across
species and detect changes in metabolism and energy homeostasis.
They coordinate cellular responses to maintain genome integrity,
mainly through regulation of epigenetic mechanisms. Sirtuins target
different histone marks, including H4K16Ac, H3K9Ac, H3K56Ac and
H3K18Ac, and non-histone components of the chromatin machinery, and
are activated by two major types of stress: metabolic stress
(nutrient and calorie restriction) and genotoxic stress. Sirtuins
are among the very few enzymes that participate in stress response
at both the sensing and signaling levels. They are also direct
effectors of the stress response by regulating numerous master
regulators of stress, such as the transcription factors NF-κB, p53,
HIF-1α, FOXOs, E2F1, PGC-1α and HSF1. For instance, SIRT1, and its
orthologs, which deacetylate H4K16ac, are effectors of caloric
restriction-mediated effects in promoting health and lifespan. They
are also known to be activated by resveratrol found in red wine and
to extend lifespan in metabolically abnormal obese mice.
Relevance of Longevity Pathways to Alzheimer’s Disease:
Alzheimer’s disease (AD) is characterized by a progressive loss
of memory and other cognitive functions. It affects over 5 million
people in the US alone and its incidence is expected to double over
the next 30 years. Moreover, the total annual costs of AD in the
United States are estimated at $236 billion. There is therefore an
urgent need to understand the mechanisms underlying the
degeneration of neuronal cells. Aging is the most important risk
factor for late-onset AD. Although they remain controversial, head
injury, low education levels, hyperlipidemia, hypertension,
homocysteinemia, diabetes mellitus, and obesity are potential risk
factors for late-onset AD. The two defining neuropathological
features of AD are extracellular senile plaques and intracellular
neurofibrillary tangles (NFTs). The senile plaques are made of
amyloid-β (Aβ), cleaved products of the amyloid precursor protein
(APP), whereas the neurofibrillary tangles mainly consist of the
microtubule-associated protein tau. Many hypotheses have been
proposed to explain the etiology and pathogenesis of AD and related
disorders. Two dominant theories focus on increased production of
Aβ and dysfunction of tau. In addition, apolipoprotein E4 is
genetically linked to late-onset familial and sporadic AD.
Tau normally stabilizes the microtubule cytoskeletal network
that functions to maintain a unique neuronal structure and to
transport proteins and other molecules through neurons.
Phosphorylation is a key regulatory mechanism, which disrupts the
ability of tau to bind microtubules and to promote their assembly.
In contrast to normal adult brains, tau in AD is
hyperphosphorylated and aggregated into abnormal conformations,
eventually leading to the NFT formation. Dephosphorylation of
NFT-tau restores the ability of tau to bind microtubules and to
promote their assembly, indicating the critical role of tau
phosphorylation.
Mutations in proteins regulating Aβ production such as APP and
presenilin, account for early-onset familial AD. There are two
proteolytic pathways for APP processing. In the amyloidogenic
pathway, β-secretase cleaves APP at the beginning of the sequence
of Aβ, generating an extracellular soluble fragment called sAPPβ
and an intracellular COOH-terminal fragment called CTFβ.
Subsequently, -secretase cleaves CTFβ at residues 40/42/43 of the
Aβ sequence, generating intact Aβ species. The non-amyloidogenic
pathway involves the activity of -secretase at the plasma membrane.
-secretase cleaves within the sequence of Aβ, resulting in sAPP.
Unlike the amyloidogenic pathway, there is no release of intact Aβ
or amyloidogenic products.
The insulin/IGF-1 and TOR signal transduction pathways have been
found to regulate tau pathology and Aβ generation in a wide range
of diverse organisms including C. elegans, fly, and mouse models.
Reducing activity of the insulin/IGF-1 pathway can alter Aβ and tau
protein homoeostasis towards less toxic protein conformations and
can also improve cognitive function. For example, knockdown of DAF2
signaling reduces Aβ42 aggregation-induced toxicity through
HSF1-mediated disaggregation and DAF16-induced assembly of small
oligomers into larger less toxic structures. In addition, blocking
TOR activity using rapamycin alleviates both Aβ and tau protein
aggregation and their pathogenesis in AD animal models and restores
cognitive function. Therefore, targeting this pathway to abrogate
overactivation of the TOR pathway may be a viable therapeutic
strategy, possibly in conjunction with promotion of normal
insulin/IGF-1 signaling.
The current FDA approved drugs for the treatment of AD inhibit
acetylcholine esterase to increase the levels of the
neurotransmitter acetylcholine, which is depleted in AD brains, or
antagonize NMDA-type glutamate receptors to prevent aberrant
neuronal stimulation. Unfortunately, the impact of these drugs on
AD manifestations is modest and transient. Attempts to develop AD
drugs targeting Aβ, β- or -secretase, tau kinases, tau protein
levels, and ApoE4 have failed due to the brain-blood barrier, side
effects, and safety issues. Therefore, agents that can effectively
and safely ameliorate NFTs and Aβ plaques represent promising
therapeutic and potential prophylactic treatments in AD.
Relevance of Longevity Pathways to Obesity and Type 2
Diabetes:
Aging is the single largest risk factor for chronic diseases.
More than 70% of people over 65 have two or more chronic conditions
such as arthritis, diabetes, cancer, heart disease and stroke.
Studies of diet, longevity genes and drugs indicate that delaying
one age-related disease probably holds off others. In humans,
dietary restriction provides sustained beneficial effects against
many age-related pathologies including obesity, insulin resistance,
inflammation, oxidative stress, and left ventricular diastolic
dysfunction, in agreement with the metabolic and functional changes
observed in dietary-restricted animal models (see above). In
contrast, obesity increases the risk of chronic age-related
diseases, such as Type 2 Diabetes (T2D), heart disease,
osteoarthritis, and certain subtypes of cancer, and thus
constitutes a major and rising global health problem. More
specifically, the health consequences of increased visceral
adiposity caused by a long-term, hypercaloric diet is a
multi-systemic deterioration resulting in an increased risk for
developing a metabolic syndrome. In fact, as we age, the chance of
developing metabolic disorders rises dramatically and
cross-sectional study data show that the prevalence of T2D among a
nationally representative sample of US adults was highest in
citizens aged 65 and over compared to younger cohorts.
Overall, T2D and obesity are recognized causes of accelerated
aging. For example, leukocyte telomere length, which shortens with
aging and is a widely used biomarker of aging in blood, has been
found to be negatively correlated with body mass index (BMI).
Furthermore, obesity is associated with an accelerated rate of
epigenetic changes in the human liver associated with the age, and
may play a role in insulin resistance as well as liver cancer.
Obesity influences hormones, inflammation, and glucose homeostasis,
which can lead to the development of T2D and subsequent
characteristics of accelerated aging. More specifically, in obese
states, macrophages infiltrate the adipose tissue, elevating
cytokine levels (e.g. TNF-α and IL-6) and resulting in insulin
resistance and T2D. These findings support the hypothesis that
obesity is associated with accelerated aging effects and stresses
the importance of maintaining a healthy weight.
In the early stages of T2D, insulin resistance is the dominant
feature and as a result hyperinsulinemia occurs. Impaired glucose
uptake and utilization follow this stage and hyperglycemia and
hyperinsulinemia contribute to pancreatic β islet cell exhaustion
and destruction as diabetes progresses. On the molecular level, the
group of epigenetic histone deacetylases called sirtuins influence
many steps of glucose metabolism in liver, pancreas, muscle and
adipose tissue. The main regulator of these reactions is the
deactylated form of PGC-1α in SIRT1 activated states, which induces
gluconeogenesis and inhibits glycolysis in liver during fasting.
Moreover, adipose tissue SIRT1 plays a key role in the regulation
of whole body metabolic homeostasis, and downregulation of SIRT1 in
visceral adipose tissue may contribute to the metabolic
abnormalities that are associated with visceral obesity in diabetic
and obese women. SIRT1 deficient mice also exhibit low levels of
serum glucose and insulin.
Several molecular pathways that increase insulin sensitivity,
glucose homeostasis and longevity in animals are affected by
approved and experimental drugs. For example, the sirtuin proteins
involved in metabolic cellular processes, are activated by high
concentrations of naturally occurring compounds such as resveratrol
found in red wine as well as metformin, a commonly used
anti-diabetic drug. Metformin decreases insulin resistance and
hyperglycemia by SIRT1 mediated AMPK activation and extends
lifespan in metabolically abnormal obese mice. A clinical trial is
currently being planned to test the effect of metformin on
biomarkers of aging in humans.
Experimental Protocol:
Overview: In this heat stress tolerance (thermotolerance)
experiment, students will compare what happens to survival when
wild-type N2 animals have attenuated (daf-2 RNAi) or simulated high
(daf-16 RNAi) insulin/insulin-like signaling. Students will also
observe what happens to DAF-16 localization during heat stress
using a transgenic animal in which green fluorescent protein (GFP)
has been fused to the gene encoding DAF-16 protein (nomenclature:
daf-16::gfp).
Animal physiology:
Procedures:
Module 1: Heat resistance
Provided Materials: Nematode strains, media plates, worm picks,
dissecting scopes, fluorescence scopes, incubators. Students will
need to record observations and take notes. Data will eventually be
plotted, preferably using excel or other graphing software.
Thermotolerance, or resistance to heat stress, is a classical
test of an organism’s ability to maintain and recover homeostasis
under conditions that lead to unfolded intracellular proteins. The
best chance of survival comes from activation of 2 transcription
factors required for extended longevity in insulin-like signaling
(ILS) attenuation mutants. These transcription factors are DAF-16
and HSF-1. The latter is the cytoplasmic heat shock factor 1
(classically considered cell autonomous, predating evolution of
multicellular species). The former is analogous to foxo3a in humans
and will be fluorescently tagged in the nematodes with which you
will be working. It is part of the ILS pathway (classically
considered cell nonautonomous, part of hormone signaling and thus
orchestrating proper responses to environmental challenges between
different tissues). Under standard laboratory (“good”) conditions,
DAF-16 is mostly localized in the cytoplasm. Under various forms of
physical stress that attenuate signaling through the ILS pathway,
this transcription factor becomes dephosphorylated and translocates
to the nucleus.
To carry out these stress response/survival assays, participants
will use C. elegans wild-type N2 animals fed double-stranded RNA
expressed in E. coli (C. elegans food source) by plasmids. In turn,
double-stranded RNA is taken up through the intestine and passed
between tissues through double-stranded (ds) RNA
receptors/transporters. Within each cell, double-stranded RNA is
processed by Dicer and Argonaute proteins into interfering RNA
(RNAi) bearing sequences that are complementary to the gene being
targeted for repression (See Zhuang and Hunter, 2011).
Adult nematodes will be fed bacteria containing dsRNA for
several days before the experiment. In a blinded assay, your group
will be given 12 nematode growth media plates labeled A-L for each
of 4 time points (48 total). For each time point, the 12 plates
will consist of 4 with bacteria bearing control dsRNA, 4 bearing
daf-16 dsRNA, and 4 bearing daf-2 dsRNA. Each plate will have about
25-30 worms. I will place these animals in the incubator at 37
degrees Celsius early in the morning, a hot temperature for these
nematodes. At each time point, each group member will randomly
select 3 plates to score. Once you have your 3 plates, you will
separate dead animals off to one side, recording the number of live
versus dead. Once complete, each group member will check at least
one other group member’s plates to get a second opinion. Death is
scored when animals are no longer able to respond to touch
provocation with a platinum wire. Scored plates will be discarded
and should only be used for one time point.
A typical survival curve for this sort of experiment is shown
below. For the purpose of the course, you can plot your results in
Excel.
The example above shows a typical epistasis experiment, in which
a wild-type C. elegans nematode (N2) is compared with a mutant with
increased thermotolerance (ifg-1, a gene encoding a mRNA
translation initiation factor). In this example, we determined its
reliance on another gene encoding the heat shock transcription
factor HSF-1. This result shows that, while hsf-1 is very important
for survival under heat stress, the protection imparted by the
ifg-1 mutation is not entirely dependent on the hsf-1 gene for its
protective effect. In your experiment, you will only have 3 curves
(one for each RNAi condition).
In between survival assay time points, students will observe
what happens with a transgenic animal bearing a fluorescent
reporter (GFP) fused to DAF-16 when it is exposed to 30 minutes of
heat stress.
Questions:
1) Were you able to resolve differences in survival between
plates to infer which conditions (RNAi) they were under?
2) What was the average survival for plates determined to carry
the control strain? Was survival improved in the daf-2 The test
(daf-2 mutant) strain?
3) Were differences in average strain survival statistically
significant?
4) Using semi-quantitative analysis of nuclear GFP localization,
were there differences between strains in the distribution of
DAF-16 in unstressed animals? Stressed animals?
5) Once animals began to die from heat, how would you
characterize the behavior of those animals that were still alive
(e.g., movement, feeding according to pharyngeal pumping). Were
there strain specific differences?
Extra suggested reading material:
Guarente L, Sirtuins, Aging, and Medicine. NEJM 2011;364:
2235-44.
Lapierre LR and Hansen M, Lessons from C. elegans: Signaling
pathways for longevity. Trends in Endocrinol Metab 2012; 23:
637-644.
Kenyon C, Genes and Cells that determine the lifespan of C.
elegans. Video on iBiology:
http://www.ibiology.org/ibioseminars/development-stem-cells/cynthia-kenyon-part-1.html
Tatar, M, Bartke, A, Antebi A. The endocrine regulation of aging
by insulin-like signals. Science 299. 1346. (2003)
Cynthia Kenyon, Jean Chang, Erin Gensch , Adam Rudner and Ramon
Tabtiang. A C. elegans mutant that lives twice as long as wild
type. Nature 366(6454), 461-464 (1993)
Colman RJ, Anderson RM, Johnson SC, Kastman EK, Kosmatka KJ,
Beasley TM, Allison DB, Cruzen C, Simmons HA, Kemnitz,JW, and
Weindruch R, Caloric restriction delays disease onset and mortality
in Rhesus monkeys. Science 2009; 325: 201-204.
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Alzheimer’s disease: progress and problems on the road to
therapeutics. Science 297, 353–356 (2002).
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deux in Alzheimer's disease. Nat Rev Neurosci, 12, 65-72 (2011)
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life span — from yeast to humans. Science 328, 321–326 (2010).
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Greer, E. L. et al. Members of the H3K4 trimethylation complex
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