6. Apoptosis
Transcript of 6. Apoptosis
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Apoptosis
Hussam Telfah, MBBS, FRCPath
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Morphology
Cell shrinkage: dense cytoplasm, tightlypacked organelles.
Chromatin condensation: peripherally under
the nuclear membrane. Formation of cytoplasmic blebs and apoptotic
bodies: blebbing then fragmentation into
membrane bound apoptotic bodies composedof cytoplasm and tightly packed organelleswith or without nuclear fragments.
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Morphology
Phagocytosis of apoptotic cells or cell bodies
by macrophages.
On H&E apoptotic cell appears intensely
eosinophilic.
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Biochemical features
Activation of caspases: cysteine proteases that
usually cleave after aspartic acid residues.
Two types: initiators (caspase 8&9) vs
executioners (caspase 3&6).
DNA and Protein breakdown: DNA breaks down
into large 50 to 300 kilobase pieces. Ca & Mg
dependent endonucleases break DNA intofragments that are multiples of 180 to 200 base
pairs. DNA ladders on electrophoresis.
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Biochemical features
Membrane alterations and recognition:
changes making cells recognisable by
phagocytes. Movement of some
phospholipids (phosphatidyserine) from the
inner leaflet to the outer leaflet of the
membrane which are able to bind to receptors
on phagocytes. Protein Annexin V stainingbinds to these phospholipids.
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Mechanisms of apoptosis
All cells contain intrinsic mechanisms that
signal death or survival and apoptosis results
from an imbalance in these signals.
Initiation: intrinsic and extrinsic.
Execution.
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The intrinsic pathway of apoptosis
Major mechanism.
Release of mitochondrial molecules into thecytosol (cytochrome c).
Release is controlled by pro and anti apoptoticproteins called Bcl family.
Bcl family; 20 members.
Growth factors and other survival signals
stimulate production of anti-apoptotic proteins(Bcl2, Bclx & Mcl-1) in cytoplasm andmitochondrial membranes.
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The intrinsic pathway of apoptosis
Stress (loss of survival signals, DNA damage...)
activates some sensors.
Sensors of damage (BH3-only proteins) are Bcl
family members including Bim, Bid & Bad.
Sensors activate proapoptosis factors (Bax &
Bak) leading to formation of channels and
leakage of mitochondrial proteins
(cytochrome c) and activation of caspases.
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The intrinsic pathway of apoptosis
Cytochrome c binds to Apaf-1(apoptosis
activating factor) forming apoptosome which
binds to caspase-9 (the critical initiator
caspase).
Other mitochondrial proteins (Smac/DIABLO)
enter the cytoplasm blocking the inhibitors of
apoptosis (IAPs) the function of which is blockthe activation of caspases.
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The extrinsic pathway
Initiated by plasma membrane deathreceptors (TNF receptor family).
Death domain: cytoplasmic involved in
protein-protein interactions. TNF1 with related protein called Fas (CD95)
expressed on the surface on many cells.
Its ligand FasL is expressed on T-cells thatrecognise self antigens and on cytotoxic Tcells.
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The extrinsic pathway
FasL + Fas
3 Fas come together
Fas + FADD Fas + FADD + Caspase 8 & 10
Activation of cascade of caspases Mediation of execution phase caspases
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The extrinsic pathway
Inhibition: FLIP which binds to Caspase 8.
Some viruses and normal cells uses this
inhibitor to protect themselves from Fas-
mediated apoptosis.
Sometimes both pathways interact with each
other. Hepatocyte Fas signaling activates Bid
which activates the mitochondrial pathway.
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The execution phase
Final step after the initiation phase.
Caspases 3 & 6.
Activation of DNase, degradation of structuralcomponents of nuclear matrix (fragmentation
of nuclei).
The way apoptotic bodies are formed is still
not clarified.
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Removal of dead cells
Apoptotic bodies are edible for phagocytes
before they release their components.
Membranes of these bodies are flipped out in
which the phosphatidylserine is expressed on
the outer layer of the membrane where it
recognised by macrophage receptors.
Abs bind to Apo. Bodies recognised by
complement system (C1q).
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Examples of apoptosis
Growth factor deprivation. Intrinsic pathway.
DNA damage. Radiation or chemicals
mediated DNA damage called genotoxic
stress. DNA damage induce accumulation of
P53 protein (tumour suppressor gene) which
arrests the cell cycle giving time for repair or
else triggers apoptosis. Mutated P53 mightlead to neoplastic transformation.
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Examples of apoptosis
Protein misfolding. Chaperones in ER controlproper folding of newly synthesized proteins
and misfolded proteins are ubiquitinated and
targeted for proteolysis in proteosomes. If misfolded proteins accumulate this will
trigger a what so called unfolded protein
response which leads to increase productionof chaperones, enhance proteosomal
degradation and slow protein translation so
reducing the load of such proteins.
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If those mechanisms fail---- ER stress-----activation of caspases
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Examples of apoptosis
Apoptosis induced by TNF receptor family.Elimination of T-cells that recognise self
antigen. Mutation---Autoimmune diseases.
Cytotoxic lymphocyte mediated apoptosis.Once activated after binding with antigens it
produces perforin which promotes entry of
granzymes having the ability to activatecaspases. Also they activate the extrinsic
pathway.
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Disorders associated with
dysregulated apoptosis
Defective apoptosis and increased cell
survival. If cells are abnormal or mutated this
can give rise to cancer or autoimmune
diseases.
Increases apoptosis and increased cell death.
Neurodegenerative diseases, ischemic injury
and death of virus infected cells.
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Autophagy
Eating own components to use as nutrients in
situations of deprivation.