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SEMINAR ON CELLULAR

AGING
AND CELL DEATH

Department of Pharmacology
Sree Siddaganga college of Pharmacy
Tumkur-572 102
Karnataka
Contents:
o Cell aging
• Introduction
• Factors affecting aging
• Theories of aging
• Programmed theories
• Error theories
• Cellular mechanisms in aging
o Cell death
• Introduction
• Types
o Apoptosis
• In physiological condition
• In pathological condition
Contents:
• Morphological changes
• Biochemical changes
• Apoptosis inducers
• Mechanisms of apoptosis
– Intrinsic pathway
– Extrinsic pathway
• Apoptosis inducing factor
• Apoptosis and diseases
o Necrosis
• Types
• Mechanism
o Autophagy
• Mechanism
• References
CELL AGING

• Aging is defined as the normal process


accompanied by the progressive alteration
in the body’s / cell’s homeostatic adaptive
responses.
• Progressive time related loss of structural
and functional capacity of cells leading to
death.
Senescence
• Cell aging is also called as Cellular
senescence.
• Senescence is a stage in which the cells
are no longer capable of dividing but these
cells will remain metabolically active.
Life expectancy vs. Lifespan

• Life Expectancy: Average number of


years a body is expected to live.

• Maximum Lifespan: Greatest age reached


by any member of the species.
STAGES OF LIFE SPAN
Factors affecting Ageing:
• Genetic – Clock genes,
• Diet – malnutrition, obesity etc.
• Social conditions -
Diseases – Atherosclerosis, diabetes etc.
• Werner’s syndrome.
Senescense inducers
The Two Main Aging Theory
Categories
• Programmed Theories
Aging has a biological timetable or internal
biological clock.
• Error Theories
Aging is a result of internal or external assaults
that damage cells or organs so they can no
longer function properly.

Many theories are a combination of programmed


and error theories.
Programmed vs. Error Theories
Programmed Error
Theories Theories
• Wear and Tear Theory
• Programmed • Rate-of-Living Theory
Senescence Theory • Cross-linking Theory
• Endocrine Theory • Free Radical Theory
• Immunology Theory • Error Catastrophe
Theory
• Somatic Mutation
Theory
Programmed Senescence
Theory
• The result of sequential switching “off” or
“on” of specific genes.

• Example – “Hayflick’s Limits”


– Functional changes within cells are
responsible for aging.
– Cumulative effect of improper functioning of
cells and eventual loss of cells in organs
and tissues.
Telomeric Theory
• Telomere:
– Telomere is molecular cap made up of DNA which
compose the linear end of the chromosome.
– It is made up of thousands of building blocks with a
sequence TTAGG that repeats over and over
– It ensures complete replication of the chromosomal
end.
– Protects the chromosomal terminal from fusion and
degradation.
T
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O
M
E
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A
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I
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A
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Telomerase in aging
Endocrine Theory
• Biological clocks act through hormones to
control the pace of aging. Hormones
effects growth, metabolism, temperature,
inflammation and stress.

• Examples- Menopause
– Decreased level of estrogen & progesterone
– Hot flashes, insomnia
Immunologic Theory
• A programmed decline in the immune
system leads to an increased vulnerability
to disease, aging and death
• Example- Decreased T cells (helper cells)
in adults
– Increased diseases in older adults
– Increased autoimmune diseases in adults
Wear and Tear Theory
• Years of damage to cells, tissues and
organs eventually wears them out, killing
both them and the body
• Example- Wearing out of the skeletal
system such as in osteoarthritis
• Wear and tear can be viewed as a result
of aging and not the cause of it.
Rate of Living Theory

Metabolic Rate Theory


– The higher the basal metabolic rate
(the rate, at which the body at rest,
uses energy), the shorter the life
span.
Cross-Linking Theory
• The accumulation of cross-linked
proteins damages cells and tissue,
slowing down bodily processes.
• Example
Non-enzymatic glycosylation reactions
occur when glucose molecules attach to
proteins causing a chain of chemical
reactions resulting in a structural change
to the proteins.
– Loss of flexibility of connective tissue
– Microvascular changes in arteries
Free Radical Theory
• A free radical is a
molecule with an
unpaired, highly reactive
electron. One type of very
reactive free radical is the
oxygen free radical, which
may be produced during
e.g. Super oxide
metabolism or as a result anions
of environmental H2O2
Hydroxyl radical
pollution.
What does free radicals do in a living system?
Free
radicals

Mutation
DNA
Membrane damaged
damaged
Free Radical Theory
The free radical “grabs” a electron from
any molecule it its vicinity.

It does this because electrons like to exist


in pairs.

When it “grabs” an electron from another


molecule, it damages the other molecule.
Free Radical Theory
• During aging, damage produced by free radicals
cause cells and organs to stop functioning.
• Some of the molecules that may be damaged by
free radicals are fats, proteins, and DNA (both in
the nucleus and in mitochondria).
• If membrane fats are attacked, then causes
breakdown of the cell membrane. If it is a red
blood cell membrane, it leads to hemolysis.
• If DNA is attacked, leads to mutation that may
cause aging or cancer.
Free Radical Theory
• Antioxidants:
– Natural antioxidants in the body, such as bilirubin.
– Enzymes such as superoxide dismutase (SOD),
catalase, & glutathione peroxidase.
– Dietary antioxidants such as beta carotene, and the
vitamins C and E.
Free Radical Theory
• Not all free radicals cause damage.
• Free radicals are part of immunological
response system.
– Macrophages engulf bacteria
– Free radical reactions produced inside the
macrophage oxidize and kill bacteria.
Error Catastrophe Theory

– DNA errors promote senescence,


programmed cell death (apoptosis).
– Aging results from gene interference
with the ability of the cells to
reproduce.
Somatic Mutation Theory
• Genetic mutations occur and accumulate with age
in the somatic cell causing the cell to:
– Deteriorate
– Malfunction
• Accumulation of mutations result in :
– Damage to the DNA
The theory states that aging is an imbalance between
DNA’s ability to repair itself and accumulating DNA
damage.
– When the damage exceeds the repair, the cell
malfunctions and this can lead to senesence.
Werner’s Syndrome
• Werner’s Syndrome is a
genetic disease that
accelerates aging and as
a result people at a
young chronological age
develop characteristics
seen in old people.
CELLULAR MECHANISMS OF
AGING
• Cross linking proteins &
DNA.
• Accumulation of toxic
by-products.
• Ageing genes.
• Loss of repair
mechanism.
• Free radicle injury
• Telomerase shortening.
Ageing –changes:
• Gradual atrophy of tissues and organs.
• Dementia
• Loss of skin elasticity
• Greying and Loss of hair
• BV damage – atherosclerosis.
• Loss of Lens elasticity  opacity 
affects vision.
CELL DEATH
CELL DEATH
• Death - Permanent cessation of all vital
functions, including those of heart, lungs,
and brain.
• Extinction of life or End of life.
TYPES OF CELL DEATH
• Apoptosis
• Autophagy
• Necrosis
APOPTOSIS
• Apoptosis is a pathway of cell death that is
induced by a tightly regulated intracellular
program in which cells destined to die
activate enzymes that degrade the cells’
own nuclear DNA and nuclear and
cytoplasmic proteins.
• The word “apoptosis” is derived from
Greek, meaning ‘falling off’ or ‘dropping
off’
• It is also defined as a form of cell death
designed to eliminate unwanted host cells
from the body through activation of a
coordinated, internally programmed series
of events which is significant in a variety of
physiologic and pathologic conditions.
APOPTOSIS IN BIOLOGIC
PROCESSES

 Physiologic conditions.

 Pathological conditions.
IN PHYSIOLOGICAL CONDITIONS
 The death of the cell is necessary for the
organism to reach its final form during embryonic
development.
By the fourth week of human embryonic
development, limb buds appear as outpockets from the
body wall. By six-weeks, the terminal (end) portion of
the limb buds flatten to form handplates and
footplates. Forty-eight days after fertilization, select cells
within the interdigital spaces of the hand and footplates
undergo apoptosis allowing the digits to separate. Digit
separation is complete by fifty-six days after fertilization.
5 weeks 8 weeks
• If the cells in the
interdigital space fail
to undergo apoptosis,
the fetus will be born
with webbed hands
and/or webbed feet.
The medical term for
this condition is
syndactyly.
 Hormone dependent involution in adult, such as

• Endometrial cell breakdown during the


menstrual cycle, ovarian follicular atresia
in the menopause.
• The regression of the lactating brest after
weaning, and
 The cell has obtained serious
damage that cannot be
repaired or requires too much
energy to be repaired.
 Cell deletion in proliferating
cell populations, such as
intestinal crypt epithelia, in
order to maintain a constant
number.
 The cell has outlived its usefulness.

• During the immune response, Helper


T-cells stimulate the proliferation of
B-cells and T-cells allowing for the
production of antibodies and
cytotoxic T-cells.
• The cytotoxic T-cells destroy
infected body cells.
• When all traces of the pathogen
have been removed from the body,
suppressor T-cells shut the cell-
mediated and humoral responses
off.
• However, millions of cytotoxic
T-cells are still circulating
looking for infected body cells.
• The body removes these cells
by programmed apoptosis and
subsequent by ingestion by
macrophages thus leaving
only memory B and T-cells
behind from the encounter.
Elimination of
potentially harmful
self-reactive
lymphocytes, either
before or after they
have completed their
maturation.
IN PATHOLOGICAL CONDTIONS

• Cell death in tumors exposed to chemotherapeutic


agents.
• Cell death by cytotoxic T cells in immune
mechanisms such as in graft versus host disease
and rejection reaction.
• Cell injury in certain viral diseases.
e.g. viral hepatitis, in which loss of infected
cells is largely because of apoptotic death.
• Pathologic atrophy of organs and tissues on
withdrawal of stimuli.
e.g. prostatic atrophy after orchiectomy.
• Progressive depletion of CD4 + T cells in the
pathogenesis of AIDS.
• Even in situations in which cell death is mainly
by necrosis, the pathway of apoptosis may
contribute.
e.g. injurious stimuli that cause increased
mitochondrial permeability trigger apoptosis.
MORPHOLOGICAL CHANGES

• Cell rounds up,


communication with
neighbors stops
• Membrane blebs
• Phosphatidylserine flips over
(inner membrane to outer
membrane).
• Proteins crosslink
• β-tubulin, actin polymerize
• Cell shrinks
• Chromatin condenses
• DNA cleaved stepwise (DNA
ladder)
• 50,000 - 300,000 bp then 185 bp
(internucleosomal)
• Macrophages phagocytize
apoptotic bodies, using lectin
receptors to recognize altered
carbohydrates
• No inflammatory response
Normal Cell

Cell shrinkage away


from neighbouring cells

Plasma membrane blebbing


Cytoplasmic and nuclear condensation

Margination of condensed
chromatin

Nuclear and cellular


fragmentation

Apoptotic Bodies
Phagocytosis
BIOCHEMICAL CHANGES

PROTEIN CLEAVAGE:
• Proteins are hydrolysed by a specific
family of proteases – caspases.
• Active caspases cleave many vital cellular
proteins, such as lamins, and thus break
up nuclear scaffold and cytoskeleton.
• Caspases also activate DNAses, which
degrade nuclear DNA.
DNA Breakdown:
• DNA is broken down into 50 to 300
kilobase pieces.
• Subsequently, there is internucleosomal
cleavage of DNA into oligonucleosomes,
in multiples of 180 to 200 base pairs, by
Ca2+ and Mg2+ dependent endonucleases.
Phagocytic recognition:
• Apoptotic cells express
Phosphatidylserine in the outer
layers of their plasma
membranes.
• In some types of apoptosis,
thrombospondin, an adhesive
glycoprotein, is also expressed
on the surfaces of apoptotic
bodies.
• These alterations permit the
early recognition of dead cells
by macrophages, resulting in
phagocytosis without the
release of pro-inflammatory
cellular components.
Inducers of Apoptosis

Physiological Damage-Related Therapy-associated


Activators Activators Agents

TNF family Heat Shock Chemotherapeutic drugs


Fas Ligand Viral Infection cisplatin, doxorubicin,
TNF Bacterial Toxins bleomycin, cytosine
TGFβ Oncogenes arabinoside, nitrogen
Neurotransmitters myc,rel, E1A mustard, methotrexate,
glutamate tumour suppressors vincristine
dopamine p53 Gamma-radiation
N-methyl-D-aspartate
Cytolytic T cells UV-radiation
Growth Factor withdrawal Oxidants
Loss of matrix attachment Free Radicals
Calcium Nutrient Deprivation
Glucocorticoids anti-metabolites
MECHANISMS OF APOPTOSIS
• One generated by signals arising within the cell;
[Intrinsic (Mitochondrial) Pathway].
• Another triggered by death activators binding to
receptors at the cell surface:
– TNF-α
– Lymphotoxin
– Fas ligand (FasL)
[Extrinsic (Death Receptor - Initiated) Pathway].
• A third that may be triggered by dangerous reactive
oxygen species.
INTRINSIC PATHWAY
• In a healthy cell, the outer membranes of its mitochondria
express the protein Bcl-2 on their surface.
• Bcl-2 is bound to the molecule of the protein Apaf-1
(apoptotic protease activating factor-1)
• Internal damage to the cell (e.g. from reactive oxygen
species) causes
• Bcl-2 to release apaf-1
• a related protein, Bax, to penetrate mitochondrial
membranes, causing
• cytochrome C to leak out.
• The release cytochrome C and Apaf-1
bind to molecules of caspase 9.
• The resulting complex of
o Cytochrome c
o Apaf-1
o caspase 9 is called the apoptosome.
o (and ATP)

• These aggregate in the cytosol.


ROS Bind to and
neutralize

IA
Other pro-apoptotic Inhibitors of
DR
N proteins (AIF) apoptosis (IAP)
O
CH

caspase 9
O

Cyt c
IT

Pro
M

Apaf - 1
Caspase
activation
Bcl-2

caspase 9
Active
CYTOSOL
Executioner
caspases

INTRINSIC
APOPTOSI
ROS Bind to and
neutralize

IA
Other pro-apoptotic Inhibitors of
DR
N proteins (AIF) apoptosis (IAP)
O
CH

caspase 9
O

Cyt c
IT

Pro
M

Caspase
Bcl-2 activation
Apaf - 1

caspase 9
Active
CYTOSOL
Executioner
caspases

INTRINSIC
APOPTOSI
• Caspase 9 is one of the family of over a
dozen caspases. They are all proteases.
They get their name because they cleave
proteins – mostly each other – at aspartic
acid (asp residues).
• Caspase 9 cleaves and, in so doing,
activates other caspases.
• The sequential activation of one caspase
by another creates an expanding cascade
of proteolytic activity leads to digestion of
structural proteins in the cytoplasm, and
degradation of chromosomal DNA, and
• Phagocytosis of the cell.
EXTRINSIC PATHWAY
• Fas and the TNF receptor are integral
membrane proteins with their receptor domains
exposed at the surface of the cell
• binding of the complementary death activator
(FasL and TNF respectively) transmits a signal
to the cytoplasm that leads to
• activation of caspase 8
• caspase 8 (like caspase 9) initiates a cascade of
caspase activation leading to
• phagocytosis of the cell.
Death receptors and adaptors
• Death receptors:
◘ Fas/CD95, DR4/DR5,
DR3
◘ TNFR (Tumor Necrosis
Factor Receptor).
• Adaptors:
FADD (Fas-associated
death domain protein)
and TRADD (TNFR-
associated death
domain protein).
EXTRINSIC
PATHWAY
APOPTOSIS - INDUCING FACTOR
(AIF)
• Neurons, and perhaps other cells, have another way to
self-destruct that — unlike the two paths described above
— does not use caspases.
• Apoptosis-inducing factor (AIF) is a protein that is
normally located in the intermembrane space of
mitochondria. When the cell receives a signal telling it that
it is time to die, AIF is released from the mitochondria (like
the release of cytochrome c in the first pathway);
• migrates into the nucleus;
• binds to DNA, which
• triggers the destruction of the DNA and cell death.
WHAT MAKES A CELL TO DECIDE TO
COMMIT SUICIDE?
1. The withdrawal of positive signals i.e.
signals needed for continued survival.
2. The receipt of negative signals.
WITHDRAWAL OF POSITIVE
SIGNALS
• The continued life of most cells requires
that they receive continuous signals from
the cells and for may continued adhesion
to the surface on which they are growing.
Some examples of positive signals
•Growth factors for neurons.
•Interleukin – 2 (IL-2) an essentila
factor for the mitosis of lymphocytes.
RECEIPT OF NEGATIVE
SIGNALS
• Increased level of oxidants with in the cell.
• Damage to DNA b these Oxidants or other
agents like
UV – light
X – Rays
Chemotherapeutic drugs
• Molecules that bind to specific receptors on the
cell surface and signal the cell to design the
apoptosis program.
• These death activators include:
• Tumor necrosis factor apha (TNFα) that
binds to the TNF receptor.
• Lymphoma (also known as TNFα) that
also binds to the TNF receptors.
• FAS ligand (FasL) a molecule that binds to
a cell surface receptor named Fas (also
called CD95)
Diseases Associated with Deregulated Apoptosis

Increased Apoptosis Inhibition of Apoptosis

AIDS Cancer
Neurodegernative disorders Follicular lymphomas
Alzeheimer’s disease, carinomas with p53 mutations
Parkinson’s disease, hormone dependent tumours:
Amyotrophic lateral sclerosis breast cancer, prostate cancer,
Retinitis pigmentosa ovarian cancer
Myelodysplastic syndromes
Aplastic anaemia Autoimmune Disorders
Ischaemic Injury Systemic lupus erythematosus
Myocardial infarction, Immune-mediated glomerulonephritus
Stroke,
Reperfusion injury Viral Infections
Toxin-Induced liver disease Herpesvirus, poxvirus, adenovirus
Alcohol
Neurodegenerative Disease and Apoptosis

• Developmental apoptosis in brain triggered by GF withdrawal


• Toxicity of abnormal protein aggregates may be initiator of
apoptosis in Alzeheimer’s, Parkinson’s, Huntington’s Disease
and amyotrophic lateral sclerosis

amyloid-β Huntington Aggregates

FADD/
IC Ca2+ Neuronal
Caspase 8
Oxidative stress Receptors

Apoptosis Apoptosis
APOPTOSIS AND VIRUS
• Some viruses associated with cancers use tricks to
prevent apoptosis of the cells they have infected. Several
human papilloma viruses (HPV) have been implicated
in causing cervical cancer. One of them produces a
protein (B6) that binds and inactivates the apoptosis
promoter p53.
• Epstein-Barr Virus (EBV), the cause of mononucleosis
and associated with some lymphomas
– produces a protein similar to Bcl-2
– produces another protein that causes the cell to
increase its own production of Bcl-2. Both these
actions make the cell more resistant to apoptosis (thus
enabling a cancer cell to continue to proliferate).
Apoptosis and Cancer
• Some B-cell leukemias and
lymphomas express high
levels of Bcl-2, thus blocking
apoptotic signals they may
receive.
• Melanoma (the most
dangerous type of skin
cancer) cells avoid apoptosis
by inhibiting the expression
of the gene encoding Apaf-1. [Breast cancer cells dividing]
Contd….

• Some cancer cells, especially lung and colon


cancer cells, secrete elevated levels of a soluble
"decoy" molecule that binds to FasL, plugging it
up so it cannot bind Fas. Thus, cytotoxic T cells
(CTL) cannot kill the cancer cell.
• Other cancer cells express high levels of FasL,
and can kill any cytotoxic T cells (CTL) that try to
kill them because CTL also express Fas (but are
protected from their own FasL).
APOPTOSIS AND AIDS

• The hallmark of AIDS (acquired immunodeficiency


syndrome) is the decline in the number of the
patient's CD4+ T cells (normally about 1000 per
microliter (µl) of blood). CD4+ T cells are responsible,
directly or indirectly, for all immune responses. When
their number declines below about 200 per µl, the
patient is no longer able to mount effective immune
responses and begins to suffer a series of dangerous
infections.
ROLE IN HIV

• HIV (human immunodeficiency virus)


invades CD4+ T cells, and one might
assume that it this infection by HIV that
causes the great dying-off of these cells.
However, that appears not to the main
culprit. Fewer than 1 in 100,000 CD4+ T
cells in the blood of AIDS patients are
actually infected with the virus.
WHAT KILLS THE UNINFECTED
CD4 CELLS?

The mechanism is not clear, but several possibilities,


• All T cells, both infected and uninfected, express Fas.
• Expression of a HIV gene (called Nef) in a HIV-infected
cell causes
– the cell to express high levels of FasL at its surface
– while preventing an interaction with its own Fas from
causing it to self-destruct.
• However, when the infected T cell encounters an
uninfected one (e.g. in a lymph node), the interaction of
FasL with Fas on the uninfected cell kills it by apoptosis.
Detection of apoptotic changes in
DNA
• Nucleic acid staining – nuclear
morphology.
• Detection of nuclear DNA fragmentation.
• TUNEL staining
(terminal deoxynucleotidyl transferase–
mediated dUTP nick end-labeling).
• Single-cell electrophoresis (Comet assay).
NECROSIS

Necrosis refers to a
spectrum of morphologic
changes that follow cell
death in living tissue,
largely resulting from the
progressive degradative
action of enzymes on the
lethally injured cells.
• It’s a premature death of a cells and living
tissues
• Necrosis is caused by external factors,
such as infection, toxins, or trauma.
TYPES
There are seven distinctive morphologic patterns of
necrosis:
• Coagulative necrosis is typically seen in hypoxic
environments, such as an infarction. Cell outlines remain
after cell death and can be observed by light microscopy.
• Liquefactive necrosis is usually associated with cellular
destruction and pus formation (e.g. pneumonia). This is
typical of bacterial or, sometimes, fungal infections
because of their ability to stimulate an inflammatory
reaction.
Contd….

• Gummatous necrosis is restricted to necrosis


involving spirochaetal infections (e.g. syphilis).
• Haemorrhagic necrosis is due to blockage of the
venous drainage of an organ or tissue.
• Caseous necrosis is a specific form of
coagulation necrosis typically caused by
mycobacteria (e.g. tuberculosis), fungi, and some
foreign substances. It can be considered a
combination of coagulative and liquefactive
necrosis.
Contd …..

• Fatty necrosis results from the action of lipases


on fatty tissues (e.g. acute pancreatitis, breast
tissue necrosis).
• Fibrinoid necrosis is caused by immune-
mediated vascular damage. It is marked by
deposition of fibrin-like proteinaceous material in
arterial walls.
Sequence of Events in Necrosis

• Membrane permeabilizes
• Ca++ enters or is released from ER stores
• Cell swells
• Ca activates calpain, ruptures lysosomes
• Lysosomal enzymes are released and
activated by Ca ++
• DNA degraded (random smear on gel)
• Inflammatory response
NECROSIS MECHANISM
AUTOPHAGY
• Autophagy, or autophagocytosis, is a
catabolic process involving the degradation of a
cell's own components through the lysosomal
machinery.
• It is a tightly-regulated process that plays a
normal part in cell growth, development, and
homeostasis, helping to maintain a balance
between the synthesis, degradation, and
subsequent recycling of cellular products
MECHANISM
• The formation of a membrane around a targeted
region of the cell, separating the contents from
the rest of the cytoplasm.
• The resultant vesicle then fuses with a lysosome
and subsequently degrades the contents.
References
• Molecular Biology of The Cell – Bruce Alberts.
• Pathologic Basis of Disease – Kumar.
• www.caspases.org/
• www.ccs.k12.in.us/chsBS/kons/kons/apoptosis.htm
• www.mainsgate.com/spacebio/modules/gs_teare.html
• www.sciencemuseum.org.uk/online/lifecycle/172.asp
• www.sciencedaily.com/releases/2007/01/070110124142.h
tm
• Nucleic Acids Research, 2007,Vol. 35, No. 22 7417-7428
• www.daviddarling.info/.../A/apoptosis.html
THAN
K
YOU