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Apoptosis
C.S. Little, PhD
January 4, 2011
Lecture 52
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C.S. Little, PhD
Office: 303 Evans Hall
Phone: 215-871-6882
Email: [email protected]
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Reading
Robbins: Pathologic Basis of Disease. 8th
Edition. Chapter 1, pp. 11-32.
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Apoptosis
First described in 1972
From the Greek, meaning falling off
cell death that:
Occurs normally Eliminates unwanted or potentially harmful cells
Cells that have outlived their usefulness
Pathologic event
Cells damaged beyond repair (especially to DNA)
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Apoptosis in Physiologic
circumstances Embryogenesis
Implantation
Organogenesis
Developmental involution
metamorphosis
hormone dependent involution in the adult
regression of lactating breast after weaning
endometrial cell breakdown during menstrual cycle ovarian follicular atresia in the menopause
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Apoptosis in physiologic
circumstances Cell deletion in proliferating cell populations
intestinal crypt epithelia
Maintenance of constant cell number
Death of host cells that have served their purpose
Neutrophil death in acute inflammation
Lymphocyte death at the end of an immune response
Occurs as a result of deprivation of necessary survivalsignals
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Apoptosis in physiologic
circumstances
Elimination of potentially harmful self reactive
(autoimmune) lymphocytes
During or after maturation
Cell death induced by cytotoxic Tlymphocytes
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Apoptosis in pathologic
circumstances
Cell death induced by a variety of injurious stimuli
Radiation
Anti-neoplastic drugs
Both cause DNA damage
If repair mechanisms fail, cell undergoes apoptosis
Heat (mild)
Hypoxia (mild)
Stress to the endoplasmic reticulum Accumulation of unfolded proteins
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Apoptosis in pathologic
circumstances Cell injury in certain viral diseases
Viral hepatitis
Pathologic changes in parenchymal organs
after duct obstruction Pancreas
Kidney
Parotid gland
Cell death in tumors
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The Study of Apoptosis
131 of 1090 somatic cells generated during development of
this organism are eliminated by apoptosis.Caenorhabditis elegansCED-3: caspase homologueCED-4: Apaf-1 homologueCED-9: bcl-2 homologue
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Morphology of apoptotic cells:
Cell shrinkage
dense cytoplasm
relative maintenance of cell organelles
But more tightly packed
Chromatin condensation Highly characteristic of apoptosis
chromatin aggregates peripherally around the nuclearmembrane
eventually nucleus may break up into two or morefragments
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Morphology of apoptotic cells
Formation ofcytoplasmic blebs and apoptotic
bodies
apoptotic bodies composed of cytoplasm and
tightly packed organelles sometimes nuclear fragments
Phagocytosis of apoptotic cells or bodies
NO INFLAMMATION
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EM of apoptotic cell
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EM of late apoptotic cell
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EM of necrotic cell
Note
Chromatin clumping
Organelle swelling
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Biochemical features of apoptosis
Protein cleavage
Through activation ofcaspases
Targets include
Lamins
Nuclear scaffold
Cytoskeleton
DNAses
DNA breakdown
Phagocytic recognition
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Mediators of apoptosis
CASPASES
What are caspases?
Cysteine proteases that cleave after aspartic
acid residue (Asp-X)At least 10 members have been identified
Highly conserved across species
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Mediators of apoptosis
CASPASES
Involvement at two or more levels:
initiator caspases: involved in
decision/commitment to apoptosis (alreadydiscussed)
Caspase 8, 10 and 9
effector or executioner caspases: involvedin execution of apoptosis
Caspase 3, 6 and 7
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Mediators of apoptosis
CASPASES
Exist in pro-enzyme or zymogen form
Undergo cleavage to be activated
By other caspases or autocatalytically
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Downloaded from: Robbins & Cotran Pathologic Basis of Disease (on 2 December 2004 07:30 PM)
2004 Elsevier
Figure 1-24 p. 28
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The extrinsic apoptotic pathway
death receptor: ligand interaction
TNFR superfamily
Contain cytoplasmic death domains that
delivers the apoptotic signal
TNF-R
Fas
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The extrinsic apoptotic pathway :Fas
Three or more Fasmolecules become cross-linked by the Fas ligand
Fas cytoplasmic death
domains form a binding sitefor an adaptor protein(FADD)
Also contains a deathdomain
See Figure 1-26 p. 29
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The extrinsic apoptotic pathway :Fas
Complex of death receptorsand FADD binds inactivecaspase-8 (pro-caspase-8)via death domains
Multiple pro-caspase-8
molecules are brought intoproximity and cleave oneanother to generate activecaspase-8
Figure 1-29 p.30
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The extrinsic apoptotic pathway :Fas
Caspase 8 then triggers acascade of caspase activation
These other caspases mediatethe execution phase ofapoptosis
This pathway can be inhibitedby FLIP
Binds to pro-caspase 8 butcannot cleave and activate itbecause it has no enzymaticactivity
Produced by viruses andsome normal cells
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Extrinsic apoptotic pathway: TNF-R
death receptor: ligand interaction (TNF: TNF-R)
TNF is an important mediator of inflammatory
processes
Also induces apoptosis
When TNF binds its receptor, cascade is very similar:
Leads to association of the receptor with the adaptor
protein TRADD
TRADD binds to FADD
Caspase activation
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Signalling pathways that initiate
apoptosis: TNF-R
Major functions of TNF are not by induction of
apoptosis
But instead by activation of a potent
transcriptional factor called NF-kB NF-kB and its inhibitor (IkB)
Important in regulating cell survival and
inflammation
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Signalling pathways that initiate
apoptosis
So how can TNF induce apoptosis AND
promote cell survival?
Which adaptor proteins attach to the TNF-R
TRADD and FADD: apoptosis TRAFs (TNF receptor associated factors) favor
activation of NF-kB
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Downloaded from: Robbins & Cotran Pathologic Basis of Disease (on 2 December 2004 07:30 PM)
2004 Elsevier
Figure 1-30 p.30 (Robbins 7th Edition)
Intrinsic pathway of apoptosis
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Intrinsic or mitochondrial pathway
The bcl-2 family
There are about 20 proteins in this family
All regulate apoptosis
Main ones that are anti-apoptotic
Bcl-2 Bcl-x
Normally reside in mitochondrial membranes andin the cytosol
When cells are deprived of survival signals or are
subjected to stress, these proteins are lost fromthe mitochondrial membrane and are replaced bythe pro-apoptotic members of the family (Baxand Bak)
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Intrinsic or mitochondrial pathway
pro-apoptotic members of the Bcl-2 family
Include Bax and Bak
When Bcl-2/Bcl-x levels decrease,permeability of the mitochondrial membrane
increases
Causes leakage of proteins that can activatecaspases
Best known: cytochrome c
Important for role in mitochondrial respiration
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Intrinsic or mitochondrial pathway
Result of increasedmitochondrialpermeability
And release ofpro-
apoptotic molecules intothe cytosol
No known role for deathreceptors
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Intrinsic or mitochondrial pathway
cytochrome c (continued)
Once out in the cytosol, cytochrome c binds toapaf-1 (apoptosis-protease activating factor 1;ced-4 homologue)
Complex of apaf-1 and cytochrome c activatescaspase 9
Bcl-2 and Bcl-x may also directly inhibit apaf-1activation
Other mitochondrial proteins such as apoptosisinducing factor (AIF) can enter the cytosol
Bind to and neutralize inhibitors of apoptosis
Net effect: initiation of the caspase cascade
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Intrinsic or mitochondrial pathway
Growth factors andother signals stimulate
the production ofanti-apoptotic member ofthe Bcl-2 family ofproteins
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Intrinsic or mitochondrial pathway
The essence of the intrinsic
pathway is the balance of
pro- and anti-apoptotic
molecules that
Regulate mitochondrial
permeability
And release of death
inducers that are
normally sequestered inthe mitochondria
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Intrinsic or mitochondrial pathway
There is evidence that the intrinsic pathway can betriggered without a role for the mitochondria
Not well-defined
May also be overlap between the extrinsic and intrinsic
pathways (may not be distinct) Example: in hepatocytes Fas signalling activates pro-
apoptotic bid which activates the mitochondrialpathway
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Execution of apoptosis: Role of
caspases
Nuclear targets
Proteins involved in
transcription (c-Myc and NF-kB-inhibited)
DNA replication DNA repair poly ADP-ribose polymerase(PARP)
disassembly of cell structure
nuclear lamina (keratins 18, 19 and vimentin)
elements of the cellular cytoskeleton (b-catenin-
disrupting cell-cell interactions, fodrin and
gelsolin-disrupting the actin filament network)
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Execution of apoptosis: Role of
caspases
activate endonuclease
Caspase 3
Activates DNA fragmentation factor 45 (DFF45)
Activation of DFF45 in turn activates DFF40which plays a critical role in the internucleosomal
DNA degradation
Also acts on mitochondrial substrates
disrupts electron transport
loss of mitochondrial transmembrane potential
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Execution of apoptosis: Role of
the endonuclease DNA breakdown
Cleaves in internucleosomal
spacer regions
first into large chunks (50-
300kb) then into multiples of 180 bp
Form a ladder on agarose
gels
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Final stage: phagocytosis
Apoptotic cells and their fragments have
markers on their surface that facilitate early
recognition by nearby phagocytic cells
The loss of phospholipid asymmetry in theplasma membrane and translocation of
phosphotidyl serine (PtdSer) to the outer
leaflet of the lipid bilayer
very efficient no inflammation
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Phagocytosis of apoptotic cells
PtdSer and annexin I co-localize on the
surface of the outer membrane
Serve as eat me trigger
phagocyte recognition uptake
Other cell surface/surface associated
molecules enhance uptake including
Mannose binding lectins (MBL) and C1q
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Limited inflammation
Uptake of apoptotic cells has been shown to
result in the release of
IL-10, TGFb, and PGE2
These are anti-inflammatory mediators
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DNA-damaged mediated apoptosis
Involves p53
accumulates when DNA is damaged (such asfollowing ionizing radiation)
arrests the cell cycle at the G1/S boundary toallow for repair
if repair fails, p53 triggers apoptosis
if p53 absent or mutated, favors survival
p53 may up-regulate Bax, Fas and APAF-1 Activate caspases and cause apoptosis
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CTL-mediated lysis
Cytotoxic T cells recognize foreign antigens
presented by Class I MHC at the cell surface
Upon recognition, CTL secrete
Perforin: transmembrane pore-formingmolecule
Granzyme B: serine protease
Entry facilitated by perforin
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CTL-mediated lysis
Granzyme B: serine protease
Cleaves proteins at aspartate residues
Activates caspases
By-passes up-stream signalling eventsActs directly by inducing execution phase
CTL also express Fas-L on their surface andcan induce cell death via this pathway
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Disordered apoptosis and disease
Disorders associated with inhibited apoptosis
and increased cell survival
accumulated cells can give rise to cancer
p53 mutation hormone-dependent (breast, prostate, ovary)
autoimmunity
Individuals with complement component C1q
defects have an increased risk of developingsystemic lupus erythematosus (SLE) and
glomerulonephritis
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Disordered apoptosis and disease
Disorders associated with increased
apoptosis and excessive cell death
neurodegenerative diseases
spinal muscular atrophies ischemic injury
myocardial infarction
virus-induced lymphocyte depletion
AIDS
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Apoptosis and Alzheimers disease research
From: BMC Neurosci. 2008 Jan 24;9:13.
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Apoptosis and Alzheimers disease research