APOPTOSIS
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Learning objectives
At the end of the session the student should be
able to
1. Define apoptosis
2. Describe the features of apoptosis
3. Differentiate apoptosis from necrosis
4. Enumerate the steps involved in apoptosis
– intrinsic and extrinsic pathway
5. Discuss the physiological and applied
importance of apoptosis
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Introduction
• Total number of cells is regulated by controlling
– rate of cell division and
– controlling rate of cell death
• Cell death:
1. When cell are no longer needed or become a threat they
undergo an orderly sequence of events called apoptosis. The
term apoptosis was coined by John Kerr, Andrew Wyllie and
A.R. Currie.
2. Cells that die as a result of acute injury undergo necrosis
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Physiological significance of apoptosis
(Why apoptosis occurs?)
• Embryogenesis and fetal development
– In the central nervous system, large numbers of neurons are produced
and then die during the remodeling that occurs during development and
synapse formation.
– Removal of the webs between the fingers in fetal life
– Regression of duct systems in the course of sexual development in the
fetus
• Hormone dependent involution
– Prostate glandular epithelium after castration
– Regression of lactating breast after weaning
• Cell loss in proliferating cell populations
– Immature lymphocytes
– Epithelial cells in the GI tract
– Elimination of self-reactive lymphocytes.
• Death of cells that have served their function 4
– Neutrophils, Lymphocytes
Definition
• Apoptosis (Greek apo "away" + ptosis "fall") is a
pathway of cell death induced by a tightly regulated
suicide program controlled by specific genes.
• It is programmed sequence of molecular events, in
which the cell systematically destroys itself from
within and is then eaten by other cells, leaving no
trace.
• Hence it is a type of programmed cell death
• It can be called "cell suicide" in the sense that the
cell's own genes play an active role in its demise.
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Features of a apoptotic cell
• Characterized by the overall shrinkage in volume of the
cell
• Collapse of cytoskeletal system
• Shrinkage of nucleus
• Loss of adhesion to neighboring cells
• Disintegration of the chromatin into small fragments
• Cell contents do not spill
• If cell is large it breaks into membrane enclosed
fragments called apoptotic bodies
• Engulfment of the “corpse” by macrophages or
neighboring cells 6
Apoptosis vs Necrosis
Apoptosis Necrosis
• Death of individual or small group of cells
• Death of large contiguous groups of
evoked by physiological stimuli. cells or organ segments evoked by
• Morphological features: pathological stimuli.
- membrane blebbing without loss of • Morphological features:
membrane integrity, - loss of membrane integrity,
- condensation of chromatin, - random fragmentation of
- cell shrinkage, chromatin,
- formation of apoptotic bodies. - cellular swelling,
• Biochemical changes: - cell lysis, swelling and
- Genetically controlled activation of disintegration of organelles.
enzymes • Biochemical changes:
- ATP dependent process - Loss of ion homeostasis
- Generation of non random DNA - Passive process, no energy required
oligonucleosomes. - Random DNA digestion 7
Apoptosis vs necrosis
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Apoptosis vs necrosis
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Caspases
• Apoptosis is triggered by members of a family of
specialized intracellular proteases, called caspases.
• These proteases have a cysteine at their active site
and cleave their target proteins at specific aspartic
acids; they are therefore called caspases (c for
cysteine and asp for aspartic acid).
• Caspases are synthesized in the cell as inactive
precursors and are activated only during apoptosis.
• There are two major classes of apoptotic caspases:
initiator caspases and executioner caspases.
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Caspases
• Initiator caspases begin the apoptotic process.
• Apoptotic signals cause their activation.
• Initiator caspases then activate executioner
caspases.
• One initiator caspase complex can activate many
executioner caspases, resulting in an amplifying
proteolytic cascade.
• Once activated, executioner caspases catalyze the
widespread protein cleavage events that kills the
cell.
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Caspase activation during apoptosis
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Caspases
• Targets of caspases are the following:
– Focal adhesion kinase (FAK ): this disrupts cell adhesion,
leading to detachment of the apoptotic cell from its
neighbors.
– Lamins: make up the inner lining of the nuclear
envelope, cleavage of lamins leads to the disassembly
of the nuclear lamina and shrinkage of the nucleus.
– Proteins of the cytoskeleton: cleavage and consequent
inactivation of these proteins leads to change in cell
shape
– An endonuclease called caspase activated DNase (CAD):
once activated, CAD goes to the nucleus and attacks
DNA, severing it into fragments. 13
Apoptosis may be
initiated by either
Extrinsic pathway Intrinsic pathway
TNF is produced in response to Internal stimuli, such as
adverse conditions, such as • irreparable genetic damage,
• exposure to ionizing radiation, • lack of oxygen (hypoxia),
• elevated temperature, • high concentrations of
• viral infection, cytosolic Ca2+,
• toxic chemical agents • severe oxidative stress
The stimulus for apoptosis is carried by
an extracellular messenger protein called Regulated by Bcl-2
Tumour necrosis factor (TNF) family of proteins
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Extrinsic pathway of apoptosis
• Also called death receptor pathway as
it is triggered by binding of
extracellular proteins to cell surface
death receptors.
• Death receptors are transmembrane
proteins with extracellular ligand
binding domain, a transmembrane
domain and and intracellular death
domain.
• These receptors are homotrimers and
belong to TNF receptor family (include
TNF and Fas) 15
Extrinsic pathway of apoptosis
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Binding of TNF or Fas ligand on target cell death receptor trigger
extrinsic pathway
Intracellular death domain bind intracellular adapter proteins
Which in turn binds initiator caspase 8 to form Death inducing signaling
complex (DISC)
dimerization of initiator caspases cause their activation
activation of executioner caspases
Apoptosis
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Intrinsic pathway of apoptosis
• Pathway activated due to signals generated inside the cell often
due to stresses such as DNA damage or in response to
developmental signals.
• Also called mitochondrial pathway of apoptosis as it depends on
release of mitochondrial proteins into cytosol which activate
caspases.
• Key protein in this pathway is cytochrome c, a component of
electron transport chain, which when released into cytosol binds
an adapter protein Apaf1 (apoptotic protease activating factor-1)
causing Apaf1 to oligomerize into a heptamer called Apoptosome.
• Apoptosome recruits initiator caspase-9 which further activates
executioner caspases to induce apoptosis.
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Intrinsic pathway of Apoptosis
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Regulation the Intrinsic pathway of
Apoptosis
• The intrinsic pathway of apoptosis is tightly
regulated to ensure that cells kill themselves
only when it is appropriate.
• Intracellular regulators of the intrinsic
pathway is the Bcl2 family of proteins.
• In mammals it regulates release of
cytochrome c and other mitochondrial
proteins into cytosol.
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Phagocytes remove apoptotic cells
• Phagocytosis of apoptotic cell by neighboring
cells or macrophage is facilitated by chemical
changes on surface of apoptotic cell.
• There occurs distribution of negatively
charged phospholipid ‘phosphatidylserine’ on
the cell surface along with loss of expression
of certain signal proteins on surface of
apoptotic cells seen in normal healthy cells.
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Excessive or Insufficient Apoptosis
• Excessive apoptosis contribute to tissue damage.
• For example – heart attack and stroke. Many cells die due to
necrosis due to loss of blood supply, but in addition less
affected cells die by apoptosis.
• Less apoptosis causes:
o Autoimmune disease- eg. mutation of Fas ligand prevents
normal death of lymphocytes, leading to their accumulation in
spleen and lymph and development of autoimmune diseases.
o Cancer-
- Bcl2 gene mutation causing its excess production leads to
lymphoma.
- Mutation of p53, a tumor suppressor gene supresses apoptosis
causing cancer. 22
• Abnormal apoptosis may occur in
autoimmune diseases, neurodegenerative
diseases, and cancer
• Therefore, selective manipulation of apoptotic
pathways may be an important approach for
treating cancer in the future.
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Summary: Main steps of Apoptosis
• Death receptor (Extrinsic) pathway
• Mitochrondrial (Intrinsic) pathway
– The intrinsic and extrinsic pathways converge to a
caspase activation cascade.
• Execution Phase: Caspases execute the process
• Removal of dead cells:
– Dying cells secrete factors the recruit phagocytes.
– This facilitates prompt clearance
– Dead cells disappear without a trace and do not
produce inflammation.
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• Define Apoptosis.
• Why apoptosis occur?
• Name the pathways of apoptosis.
• Briefly describe steps of each pathway.
• What are caspases? What is their role in
apoptosis?
• How is apoptosis different from necrosis?
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Thank You…
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TNF
TNF-R
Plasma membrane
Death domains
Pro-caspase 8
Initiator-caspase 8
Executioner pro-caspase
Executioner caspase
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Internal cellular damage and
activation of BCl 2
Initiator caspase-9
Executioner procaspase
Executioner
Executionercaspase
caspase
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What is common?
• The intrinsic and extrinsic pathways finally
converge by activating the same enzymes i.e,
caspases
• Caspases are a group of cysteine proteases
responsible for triggering most, of the
changes observed during apoptosis
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Match the following
Targets of Caspases Features of Apoptosis
– Focal adhesion • Overall shrinkage in
kinase volume of the cell
– Lamins • Loss of adhesion to
– Proteins of neighboring cells
the cytoskeleton • Disintegration of the
– An endonuclease chromatin into small
called caspase fragments
activated DNase • Shrinkage of nucleus
(CAD)
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Summarize
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Learning objectives
At the end of the session the student must be
able to
1. Define apoptosis
2. Describe the features of apoptosis
3. Differentiate apoptosis from necrosis
4. Enumerate the steps involved in apoptosis
– intrinsic and extrinsic pathway
5. Discuss the physiological and applied
importance of apoptosis
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