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.