Apoptosis
C.S. Little, PhD January 4, 2011 Lecture 52
C.S. Little, PhD
Office: 303 Evans Hall
Phone: 215-871-6882 Email: chrisl@pcom.edu
Reading
Robbins: Pathologic Basis of Disease. 8th
Edition. Chapter 1, pp. 11-32.
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)
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
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 survival signals
Apoptosis in physiologic circumstances
Elimination of potentially harmful self reactive
(autoimmune) lymphocytes
During or after maturation
Cell death induced by cytotoxic T
lymphocytes
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
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
The Study of Apoptosis
131 of 1090 somatic cells generated during development of this organism are eliminated by apoptosis. Caenorhabditis elegans CED-3: caspase homologue CED-4: Apaf-1 homologue CED-9: bcl-2 homologue
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 nuclear membrane eventually nucleus may break up into two or more fragments
Morphology of apoptotic cells
Formation of cytoplasmic blebs and apoptotic
bodies
apoptotic bodies composed of cytoplasm and tightly packed organelles sometimes nuclear fragments NO INFLAMMATION
Phagocytosis of apoptotic cells or bodies
EM of apoptotic cell
EM of late apoptotic cell
EM of necrotic cell
Note Chromatin clumping Organelle swelling
Biochemical features of apoptosis
Protein cleavage
Through activation of caspases Targets include
Lamins Nuclear scaffold Cytoskeleton DNAses
DNA breakdown Phagocytic recognition
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
Mediators of apoptosis
CASPASES Involvement at two or more levels: initiator caspases: involved in decision/commitment to apoptosis (already discussed)
Caspase 8, 10 and 9
effector or executioner caspases: involved in execution of apoptosis
Caspase 3, 6 and 7
Mediators of apoptosis
CASPASES
Exist in pro-enzyme or zymogen form Undergo cleavage to be activated By other caspases or autocatalytically
Figure 1-24 p. 28
Downloaded from: Robbins & Cotran Pathologic Basis of Disease (on 2 December 2004 07:30 PM) 2004 Elsevier
The extrinsic apoptotic pathway
death receptor: ligand interaction
TNFR
superfamily
Contain cytoplasmic death domains that delivers the apoptotic signal TNF-R Fas
The extrinsic apoptotic pathway :Fas
Three or more Fas
molecules become crosslinked by the Fas ligand Fas cytoplasmic death domains form a binding site for an adaptor protein (FADD) Also contains a death domain
See Figure 1-26 p. 29
The extrinsic apoptotic pathway :Fas
Complex of death receptors
and FADD binds inactive caspase-8 (pro-caspase-8) via death domains Multiple pro-caspase-8 molecules are brought into proximity and cleave one another to generate active caspase-8
Figure 1-29 p.30
The extrinsic apoptotic pathway :Fas
Caspase 8 then triggers a
cascade of caspase activation These other caspases mediate the execution phase of apoptosis This pathway can be inhibited by FLIP Binds to pro-caspase 8 but cannot cleave and activate it because it has no enzymatic activity Produced by viruses and some normal cells
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
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
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
Intrinsic pathway of apoptosis
Figure 1-30 p.30 (Robbins 7th Edition)
Downloaded from: Robbins & Cotran Pathologic Basis of Disease (on 2 December 2004 07:30 PM) 2004 Elsevier
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 and in the cytosol When cells are deprived of survival signals or are subjected to stress, these proteins are lost from the mitochondrial membrane and are replaced by the pro-apoptotic members of the family (Bax and Bak)
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 activate caspases Best known: cytochrome c
Important for role in mitochondrial respiration
Intrinsic or mitochondrial pathway
Result of increased
mitochondrial permeability And release of proapoptotic molecules into the cytosol No known role for death receptors
Intrinsic or mitochondrial pathway
cytochrome c (continued)
Once out in the cytosol, cytochrome c binds to apaf-1 (apoptosis-protease activating factor 1; ced-4 homologue) Complex of apaf-1 and cytochrome c activates caspase 9 Bcl-2 and Bcl-x may also directly inhibit apaf-1 activation Other mitochondrial proteins such as apoptosis inducing factor (AIF) can enter the cytosol Bind to and neutralize inhibitors of apoptosis Net effect: initiation of the caspase cascade
Intrinsic or mitochondrial pathway
Growth factors and
other signals stimulate the production of antiapoptotic member of the Bcl-2 family of proteins
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 in the mitochondria
Intrinsic or mitochondrial pathway
There is evidence that the intrinsic pathway can be
triggered 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 proapoptotic bid which activates the mitochondrial pathway
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) nuclear lamina (keratins 18, 19 and vimentin) elements of the cellular cytoskeleton (b-catenindisrupting cell-cell interactions, fodrin and gelsolin-disrupting the actin filament network)
disassembly of cell structure
Execution of apoptosis: Role of caspases
activate endonuclease
Caspase 3
Activates DNA fragmentation factor 45 (DFF45) Activation of DFF45 in turn activates DFF40 which plays a critical role in the internucleosomal DNA degradation Also acts on mitochondrial substrates
disrupts electron transport loss of mitochondrial transmembrane potential
Execution of apoptosis: Role of the endonuclease
DNA breakdown Cleaves in internucleosomal
spacer regions first into large chunks (50300kb) then into multiples of 180 bp Form a ladder on agarose gels
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 the plasma membrane and translocation of phosphotidyl serine (PtdSer) to the outer leaflet of the lipid bilayer
very efficient no inflammation
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
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
DNA-damaged mediated apoptosis
Involves p53 accumulates when DNA is damaged (such as following ionizing radiation) arrests the cell cycle at the G1/S boundary to allow 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
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-forming molecule Granzyme B: serine protease
Entry facilitated by perforin
CTL-mediated lysis
Granzyme B: serine protease Cleaves proteins at aspartate residues Activates caspases By-passes up-stream signalling events Acts directly by inducing execution phase CTL also express Fas-L on their surface and
can induce cell death via this pathway
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) Individuals with complement component C1q defects have an increased risk of developing systemic lupus erythematosus (SLE) and glomerulonephritis
autoimmunity
Disordered apoptosis and disease
Disorders associated with increased
apoptosis and excessive cell death
neurodegenerative diseases
spinal muscular atrophies
myocardial infarction AIDS
ischemic injury
virus-induced lymphocyte depletion
Apoptosis and Alzheimers disease research
From: BMC Neurosci. 2008 Jan 24;9:13.
Apoptosis and Alzheimers disease research
From: BMC Neurosci. 2008 Jan 24;9:13.
