Detailed cellualr reponses to aging , and mechanisms responsible for these responses..

1. By
Prof.Ekbal Mohamed Abo-hashem (MD)
Faculty of Medicine-Mansoura University
Egypt
Cellular aging:Signaling and
transcriptional pathways

2.  Introduction
 Hormesis: an antiaging strategy.
 The heat shock response.
 The ubiquitin-proteasome system (UPS).
 Organelle-specific stress response pathways:
 Endoplasmic reticulum.
 Mitochondria.
 The lysosome-autophagy.
 The nuclear DNA damage response.
 Cross-talk between stress response pathways.
 Signaling pathways and transcriptional regulation.
• Nutrient signaling pathways
- Insulin IGF-1
- Sirtuins
- AMPK
- mTOR
 Mitochondrial functions, oxidative stress.
 Genotoxic stress.

Outline:

3. Introduction
 Ageing research has culminated in the identification of
classical signalling pathways that influence ageing in a
variety of species .
 Accumulating findings indicate that longevity depends on
the ability of the organism to cope with extrinsic or intrinsic
stressors .
 Compromised stress responses are linked to the onset of
many age-related diseases. ‘Stress’ is broadly defined as a
noxious factor (physical, chemical or biological), which
triggers a series of cellular and systemic events, resulting in
restoration of cellular and organismal homeostasis.

4.  In addition to the intrinsically stressful
cellular context accompanying ageing,
organisms have to also cope with external
environmental challenges.

6.  To cope with conditions of stress, organisms
have developed a wide range of
sophisticated stress response mechanisms,
acting at the cellular or organelle-specific
level. Exposure to mild stress activates
cellular homeodynamic mechanisms,
without mounting a comprehensive stress
response, which better prepare the
organism against stronger insults and
promote long-term survival. This
phenomenon is known as hormesis

7. Age-related stress and disease. Aging is associated with mitochondrial dysfunction
leading to reduced respiratory metabolism and increased generation of reactive
oxygen species (ROS). Persistent DNA damage may arise from both increased
oxidative damage and reduced efficiency of energy-intensive DNA repair,
predisposing to apoptosis, senescence and inflammation. Aging is also associated
with increased protein misfolding and aggregation in the cytoplasm, nucleus and
endoplasmic reticulum. The various sites of age-related cellular damage and the
physiological decline that ensues contribute to the pathogenesis of age-related
diseases, including metabolic syndrome, inflammatory disorders, cancer, and
neurodegenerative diseases.

8. Hormesis: an anti - ageing strategy
 The term hormesis describes the beneficial
effects, resulting from the exposure of an
organism to a low intensity stressor.The positive
effect of hormesis is attributed to the
stimulation and priming of stress response
pathways by the stressor. Hormetic
manipulations such as repeated mild heat
shock result in increased stress tolerance and
extension in lifespan in various models (e.g.
yeast, Drosophila, nematodes, rodents and
human cells.

9. The heat shock response
 Exposure of cells and organisms to unfavourable
conditions such as heat, oxidative and osmotic
stress, heavy metals and proteasome inhibitors
induces a highly conserved programme of gene
expression leading to selective transcription and
translation of heat shock proteins (HSP ).
 Based on their molecular weight, HSPs are
categorized into the HSP100, HSP90, HSP70, HSP60
and the small HSP (sHSP) families. The heat shock
response is orchestrated by a set of heat shock
transcription factors (HSFs ).Defects in signal
transduction pathways that lead to HSF1 activation
are the reason for suboptimal expression of HSP
during aging .

10. The ubiquitin–proteasome system (UPS)
 Ageing is accompanied by accumulation of
damaged and modified proteins. The build up of
altered proteins is the result of a gradual
deterioration of cellular quality control
mechanisms, decreased protein degradation or a
combination of both. The UPS is the main
proteolytic mechanism, responsible for the
degradation of damaged proteins and the
turnover of most cytosolic and nuclear proteins.

11.  The proteasome is a multicatalytic protease
complex composed of one 20S catalytic core and
two 19S regulatory caps .
 Oxidative stress has important roles during the
ageing process and age-related diseases. Oxidized
proteins that escape the low-molecular weight and
enzymatic anti-oxidative damage defences of the
cell are recognized and degraded by the
proteasome. In the presence of moderate oxidant
concentrations, proteasomal degradation
increases, whereas higher oxidation levels lead to
proteolytic inhibition.

12.  Maintenance or enhancement of the
proteolytic activity of the proteasome
during ageing might provide protection
against neuronal cell death documented
in age-related neurological diseases.
 Accumulation of unfolded and damaged
proteins, following proteasome inhibition,
leads to activation of the heat shock and
the endoplasmic reticulum (ER) stress
response. Thus, the ubiquitin–proteasome
system is tightly integrated into a wider
and complex network of cellular
proteostasis-preserving mechanisms.

17. Age-Dependent Decline in
Proteostasis
 A set of transcription factors, molecular
chaperones and cofactors function together
to promote correct protein folding and
protect the cell by sequestering misfolded
proteins in the process of proteostasis. The
efficiency of this quality control system
declines with age , together with changes in
protein structure due to oxidative
modification, missense mutations and
misincorporation of amino acids during
translation .

18.  Compartments that are highly sensitive to
redox state, such as the endoplasmic
reticulum and mitochondria, are particularly
vulnerable and have their own distinct protein
folding quality control systems.
 The unfolded protein response (UPR) in the
endoplasmic reticulum (ER) is activated by
misfolding of newly synthesized proteins, and
can be induced by exogenous toxins or by
metabolic disorders such as type II diabetes

19.  Loss of function in the protein quality control
network contributes to the pathogenesis of
a group of age-dependent disorders known
as conformational diseases that are
exemplified by Alzheimer’s and Huntington’s
disease. A characteristic feature is age-
dependent accumulation of protein
aggregates in the brain that may contribute
to neurodegeneration and neurological
decline.

20.  Multiple mechanisms have been implicated in
the age-dependent decline of the proteasome:
• Decreased expression of proteasomal subunits .
• Changes in proteasomal enzymatic activities due
to increased oxidative stress during ageing.
• Oxidized and damaged proteins can directly
inhibit the proteasome leading to depletion of
active proteolytic units .

21. Organelle- specific stress response
pathways
Endoplasmic reticulum
 The ER is the organelle where newly
synthesized proteins destined for secretion,
integration into the plasma membrane or
distribution to various organelles, are folded
and post translationally modified.
 The environment within the ER is highly
crowded with chaperones, processing
enzymes and client proteins. In this cluttered
and aggregation prone environment, complex
ER quality control mechanisms ensure the
proper translation and folding of nascent
proteins as well as the degradation of
improperly folded polypeptides

24.  Conditions that elicit increased load of
misfolded proteins within the ER trigger the
ER stress or unfolded protein response
(UPRER). UPRER helps restore the normal
function of ER through upregulation of ER
chaperones, halting protein translation and
stimulating the degradation of misfolded
proteins. In situations of persistent stress,
failure of UPRER to restore ER homeostasis
results in apoptosis

25.  Whether ageing hinders the activation of these
degradation systems, leading to overflow of
damaged proteins remains to be seen. Defective ER
stress response has been associated with age related
pathologies such as diabetes, heart disease and
neurodegenerative disorders

26.  In addition ,the hallmark of many age
related neurodegenerative diseases, such
as Alzheimer's, Parkinson's and
Huntington's disease is the formation of
insoluble protein aggregates. However,
recent studies show that protein
aggregation also occurs in a non-disease
context during ageing

27. Mitochondria
 Mitochondrial dysfunction has been
associated with oxidative stress, accelerated
ageing,apoptosis,neurodegenerative
disorders and other pathological conditions.
The matrix of mitochondria contains a specific
set of chaperones involved in importing,
refolding and preventing aggregation of
proteins encoded both by the nuclear
genome and by mtDNA.

28. Mitochondrial Function, Oxidative
Stress and Aging
 Mitochondrial function is controlled by a number of
signaling pathways and transcriptional regulators that
sense energetic stress and contribute to lifespan
regulation, including peroxisome proliferation-
activated receptor coactivator 1 α (PGC-1α), sirtuins,
mTOR, and AMPK.
 When energy is low, these pathways allow the cell to
adjust fuel utilization and mitochondrial number. Many
aspects of mitochondrial dysfunction associated with
aging can be blocked by caloric restriction. caloric
restriction induces mitochondrial biogenesis through
the up-regulation of PGC-α and endothelial nitric
oxide synthase (eNOS)

29. The lysosome
Autophagy is one of the main processes mediating
both bulk and specific degradation of cellular
components, including whole organelles and protein
aggregates. Cargoes destined for degradation are
delivered to lysosomes, where they are recycled. Three
main types of autophagy have been defined on the
basis of lysosomal delivery mechanisms:
macroautophagy, microautophagy and chaperone -
mediated autophagy (CMA).

31.  Autophagy is an evalutionary conserved
process in the cellular recycling , and is
induced in response to stress such as nutrient
deprivation, hyperthermia and hypoxia. It is
responsible for degradation of proteins, lipids,
sugars and nucleic acids as well as larger
cellular components such as organelles.
 During the process, damaged cellular
components are sequestered into a double
membrane structure called the
autophogosome, which delivers its contents to
the lysosome for subsequent degradation by
acidic hydrolase.

32.  This is likely to provide cells with an
additional source of energy during times of
nutrient scarcity; generating free amino
acids that can be utilized for protein
synthesis.
 Autophagy is also important for mobilizing
lipids in the liver during nutrient deprivation,
which can be metabolized as an alternate
energy source, or used for new membrane
biosynthesis.

33. Macroautophagy entails the sequestration of
portions of the cytoplasm within a double membrane
autophagic vacuole, called autophagosome. The
autophagosome fuses with secondary lysosomes to form
an autolysosome, where hydrolases degrade the
sequestered material.
Microautophagy, which is less well characterized, the
lysosomal membrane itself invaginates to engulf cytosolic
components.
CMA is a highly selective form of autophagy that
requires unfolding of the protein before internalization
into the lysosome for degradation. In addition to turnover
of cellular material, autophagy is involved in
development, differentiation and tissue remodelling.
Although a basal level of macroautophagy and CMA is
observed in various cell types, these pathways are
maximally activated under conditions of stress

39. Activation of autophagy is
fundamentally linked to the nutrient
status of the cell and impacts
metabolism and stress response
pathways. Autophagy increases
during times of nutrient deprivation,
such as in fasting or calorie restriction.

40.  Autophagy could potentially be altered by
ageing. However, age induced modifications‐
of specific components of macroautophagy
have yet to be studied systematically.
 Activation of macroautophagy or CMA
through pharmacological interventions is
potentially an effective approach to maintain
efficient clearance mechanisms in a
damage prone environment. ‐

41. The nuclear DNA damage
response
 In contrast to most biomolecules such as proteins
and lipids, which can be recycled several times
over the lifespan of a cell, DNA cannot be
resynthesized afresh to eliminate damage.
Instead, cells maintain elaborate genome
maintenance machinery to mend their genetic
material.
 DNA damage can be induced by exogenous
hazards, such as UV radiation, by endogenous
toxic by -products of cellular metabolism, such as
reactive oxygen species (ROS) or by spontaneous
chemical reactions, such as hydrolysis

44.  Defects in genome maintenance mechanisms
underlie the pathology of the vast majority of
progeroid syndromes, suggesting that DNA
damage, which also accumulates during
normal ageing, contributes to age related
deterioration. In addition, DNA repair
mechanisms are subject to modifications
throughout the lifespan of a cell, leading to
gradual loss of repair accuracy and efficiency.
 The genome maintenance apparatus of the cell
consists of multiple complex repair pathways,
each targeting a specific category of DNA
lesion.

45. • Base excision repair (BER) removes subtle
lesions of DNA that affect only one DNA strand,
such as oxidized bases. The complementary
strand is used as a template for repair. Ageing
has a negative impact on BER mechanisms.
Both a drop in the activity of BER enzymes and
a reduction in the inducibility of the pathway
has been observed in aged mice.

46. • Nucleotide excision repair (NER) is a
multistep process involving numerous proteins
that target helix distorting lesions, resulting from
UV exposure and carcinogenic compounds.

47. • Double strand breaks (DSBs) are the most
severe form of DNA damage. DSBs are repaired
through non homologous end joining (NHEJ), which
merely joins two loose DNA ends with a risk of
mutagenesis and information loss, or through
homologous recombination, which uses the intact
sister chromatid as a template to copy the missing
information and seal the broken ends in an error
free manner. The main detrimental outcome of age
related alterations in DSB repair is the increase in
cancer incidence, accompanied by genome
rearrangements and loss of heterozygosity, which
are characteristics of erroneous NHEJ

49. Crosstalk between stress response
pathways:
- Distinct stress response pathways do not
function in isolation but rather, are parts of a
wider stress network with multiple links that serve
as coordinators of various modules. The kinase
target of rapamycin (TOR) is part of an
evolutionary conserved signalling pathway that
links extracellular stimuli with intracellular
processes such as -cellular growth, metabolism,
translational control and proliferation. TOR also
inhibits autophagy through phosphorylation of
the ATG1 protein kinase.
 Sestrins are a family of highly conserved
cytoplasmic proteins, whose expression is
induced by stress -

51.  Sirtuins link metabolic status to the
regulation of longevity. Interestingly,
several transcription factors involved in
cellular stress responses, including FOXO3,
p53, NFκB and HSF1, are regulated by
SIRT1. It appears that SIRT1 may function
to orchestrate different stress response
pathways during ageing. Indeed, the
beneficial effects of low caloric intake are
mediated by members of the sirtuin
family.

53. The aging stress response: Signaling
molecules and transcriptional regulators
INTRODUCTION
A phylogenetically conserved feature of
aging is the induction of stress response
pathways. Cellular stress response
pathways are controlled at the molecular
level by a number of highly conserved
signaling molecules and transcriptional
regulators, including proteins involved in
insulin/insulin-like growth factor (IGF)
signaling, sirtuins, target of rapamycin
(TOR) and AMP-activated protein kinase
(AMPK) pathways. .

55. Nutrient Signaling Pathways and
Aging
 Lifespan is regulated by highly conserved
nutrient sensing pathways providing evidence for
a pivotal role of nutrient signaling in the control
of aging and aging-related diseases. High
caloric intake shortens lifespan and accelerates
the onset of aging-associated disorders,
including diabetes, metabolic syndrome, cancer
and neurodegenerative disorders. By contrast, a
dietary regimen of moderate calorie restriction
with adequate nutrient intake delays aging in a
wide variety of organisms from yeast to primates,
and may delay or attenuate age-related
diseases such as diabetes, cancer and
Alzheimer’s disease (AD).

56.  Caloric restriction also activates stress pathways
that increase organismal resistance to
subsequent stress or nutritional limitation, an
effect known as hormesis. Energy sensing
pathways are linked to the aging process and
are regulated by insulin/IGF-1, sirtuins, TOR, and
AMPK signaling.
 Thus, signaling through insulin/IGF-1 modulates
cellular and organismal stress responses by
controlling key transcriptional programs.

59. Signaling through insulin/IGF-1
modulates cellular and organismal
stress responses by controlling key
transcriptional programs.
Inherited SNPs in genes of the
insulin/IGF-1 signaling pathway
correlate with longevity.
Polymorphisms in the IGF-1 receptor
gene have been identified in
Ashkenazi Jewish centenarians.
*Insulin IGF-1

60. Nutritional regulation of conserved signaling and
stress response pathways. Dietary restriction
extends lifespan and augments stress resistance
in many species by altering cellular metabolism
and mobilizing protective stress responses. Gene
and protein networks that maintain mitochondrial
function, genomic stability and proteostasis are
coordinately regulated by insulin/IGF and TOR
signaling, and modulated by sirtuins.

63. * Sirtuins
 Sirtuins are a highly conserved family of
proteins that connect metabolic status to
the regulation of aging and age-related
phenotypes. Sirtuins possess NAD-
dependent protein deacetylase and/or
ADP-ribosyltransferase activity. The
requirement for NAD is one mechanism by
which sirtuins sense and respond to
metabolic status.

64.  There are seven mammalian sirtuins (SIRT1-7) that
play various roles in the regulation of stress
resistance, metabolism and cell survival. However,
their roles in the regulation of mammalian lifespan
are still unresolved. Nonetheless, many reports
suggest that sirtuins regulate stress-response
pathways that contribute to aging and age-related
diseases.
 The best studied mammalian sirtuin is SIRT1 .When
activated, SIRT1 deacetylates many different
substrate proteins that are involved in aging, stress
responses and metabolic regulation, including
PGC-1α, Ku70, NF-κB, AceCS1, MEF2 and p53
Moreover, SIRT1 deacetylates and activates FOXO
transcription factors, providing a level of
transcriptional control of stress response genes.

66. * AMPK
 AMP-activated protein kinase (AMPK)
is activated under conditions of
elevated intracellular AMP or reduced
ATP, enabling this kinase to serve as a
rheostat for cellular energy status.
Stressors such as glucose deprivation,
ischemia, hypoxia and exercise that
deplete cellular ATP lead to the
activation of AMPK. AMPK activation
results in transcriptional and post-
translational signaling responses that
increase catabolic metabolic
pathways in response to the stress of a
low energy state

67.  Declining AMPK activity during aging may
contribute to insulin resistance and metabolic
syndrome. AMPK activity declines in aging skeletal
muscle, and is associated with insulin resistance
that can be reversed by treatment with AICAR, an
AMP analog that activates AMPK .
 AMPK may regulate insulin sensitivity by stimulating
GLUT4 translocation, increasing glucose uptake
and metabolism. These observations suggest that
AMPK is a potential therapeutic target for age-
related metabolic disorders

68. *mTOR
 The target of rapamycin (TOR) pathway is a
conserved nutrient sensor that is linked to
lifespan regulation. TOR integrates
environmental cues, such as growth factors
and nutrients to control eukaryotic growth,
metabolism and cell division . TOR activity is
suppressed by conditions of nutrient
limitation, consistent with the notion that
decreasing TOR signaling mimics aspects of
caloric restriction.. The mechanisms by
which TOR exerts its effects on aging may
involve the modulation of protein synthesis
and autophagy

71. Genotoxic Stress and Aging
The study of human progeroid
syndromes has provided evidence for a
link between genotoxic stress responses
and signaling pathways that regulate
the aging process. DNA damage can
activate a broad range of signaling
pathways, some of which also regulate
aging.

72.  Persistent stimulation of the DNA damage
response resulting in chronic p53 activation,
even at a low level, may be deleterious,
potentially leading to apoptosis or cellular
senescence. Moreover, recent studies suggest
that chronic DNA damage and ATM signaling
in senescent cells leads to secretion of pro-
inflammatory cytokines, possibly through
activation of the ATM target NF-κB.
 A persistent DNA damage response may
therefore contribute to systemic inflammation,
a known contributory factor for many age-
related degenerative disorders.

73.  Persistent stimulation of the DNA damage response
resulting in chronic p53 activation, even at a low level,
may be deleterious, potentially leading to apoptosis or
cellular senescence.
 Moreover, recent studies suggest that chronic DNA
damage and ATM signaling in senescent cells leads to
secretion of pro-inflammatory cytokines, possibly
through activation of the ATM target NF-κB.
 A persistent DNA damage response may therefore
contribute to systemic inflammation, a known
contributory factor for many age-related degenerative
disorders.

74.  A consequence of chronic DNA damage in the
aging brain may be transcriptional repression
and altered neuronal function.
 Transcriptional repression of actively expressed
synaptic genes is pronounced in the aging
human brain, especially for genes involved in
cognitive and affective functions.
 These genes may be selectively vulnerable to
DNA damage owing to their activated
euchromatic state in neurons, resulting in greater
access of DNA to reactive oxygen species and
other damaging agents.

75. A greater understanding of the
systems biology of stress
response signaling and its
breakdown during aging may
lead to the new therapeutic
approaches to the intractable
degenerative disorders of
aging.

76.  Aging is a complex process, drivin by accumulation of
damge and deleterious modifications in nucleic acids,
proteins, lipids and carbohydrates.
 The rate of aging is determined by the balance
between damge and repair .Age-related pathological
features are modulated by stress response, the
proteasome as well as organelle-specific stress
response pathways.
 Cellular stress response pathways are controlled by
signaling molecules and transcriptional regulators
including insulin/IGF-1 signaling, sirtuins, TOR and AMPK
pathways.
 A better understanding of the dynamics and reciprocal
interplay between stress responses and aging is
mandatory for the strategies and novel approaches for
the effective coping of intrinsic and extrinsic stress, and
for potential therapeutic treatment of age-related
diseases.
Message

78. Further reading
 Haigis MC and Yankner BA . The Aging Stress Response.
Mol Cell. 2010 Oct 22; 40(2): 333–344.
 Boily G, Seifert EL, Bevilacqua L, He XH, Sabourin G, Estey C, Moffat C, Crawford S, Saliba S,
Jardine K, et al. SirT1 regulates energy metabolism and response to caloric restriction in
mice. PLoS ONE. 2008;3:e1759.
 Bonawitz ND, Chatenay-Lapointe M, Pan Y, Shadel GS. Reduced TOR signaling extends
chronological life span via increased respiration and upregulation of mitochondrial gene
expression.Cell Metab. 2007;5:265–277.
 Bordone L, Guarente L. Calorie restriction, SIRT1 and metabolism: understanding
longevity. Nat Rev Mol Cell Biol. 2005;6:298–305.
 Ciccia A, Elledge SJ. The DNA Damage Response: Making it safe to play with knives. Mol
Cell.2010;40:XXX.
 Cuervo AM, Wong ES, Martinez-Vicente M. Protein degradation, aggregation, and
misfolding.Mov Disord. 2010;25 Suppl 1:S49–S54.
 Guarente L. Sirtuins as potential targets for metabolic syndrome. Nature. 2006;444:868–874.
 Guertin DA, Sabatini DM. Defining the role of mTOR in cancer. Cancer Cell. 2007;12:9–22.
 Haigis MC, Sinclair DA. Mammalian sirtuins: biological insights and disease
relevance. Annual review of pathology. 2010;5:253–295.
 Herranz D, Munoz-Martin M, Canamero M, Mulero F, Martinez-Pastor B, Fernandez-Capetillo
O, Serrano M. Sirt1 improves healthy ageing and protects from metabolic syndrome-
associated cancer.Nature Communications. 2010;1:1–8.
 Hunt ND, Hyun DH, Allard JS, Minor RK, Mattson MP, Ingram DK, de Cabo R. Bioenergetics of
aging and calorie restriction. Ageing research reviews. 2006;5:125–143.
 Jager S, Handschin C, St-Pierre J, Spiegelman BM. AMP-activated protein kinase (AMPK)
action in skeletal muscle via direct phosphorylation of PGC-1alpha. Proc Natl Acad Sci U S
A.2007;104:12017–12022.

79. THANK YOU