52 - Apoptosis (CSL)

7/27/2019 52 - Apoptosis (CSL)

1/48

Apoptosis

C.S. Little, PhD

January 4, 2011

Lecture 52


2/48

C.S. Little, PhD

Office: 303 Evans Hall

Phone: 215-871-6882

Email: [email protected]


3/48

Reading

Robbins: Pathologic Basis of Disease. 8th

Edition. Chapter 1, pp. 11-32.


4/48

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)


5/48

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


6/48

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


7/48

Apoptosis in physiologic

circumstances

Elimination of potentially harmful self reactive

(autoimmune) lymphocytes

During or after maturation

Cell death induced by cytotoxic Tlymphocytes


8/48

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


9/48

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


10/48

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


11/48

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


12/48

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


13/48

EM of apoptotic cell


14/48

EM of late apoptotic cell


15/48

EM of necrotic cell

Note

Chromatin clumping

Organelle swelling


16/48

Biochemical features of apoptosis

Protein cleavage

Through activation ofcaspases

Targets include

Lamins

Nuclear scaffold

Cytoskeleton

DNAses

DNA breakdown

Phagocytic recognition


17/48

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


18/48


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


19/48


CASPASES

Exist in pro-enzyme or zymogen form

Undergo cleavage to be activated

By other caspases or autocatalytically


20/48

Downloaded from: Robbins & Cotran Pathologic Basis of Disease (on 2 December 2004 07:30 PM)

2004 Elsevier

Figure 1-24 p. 28


21/48

The extrinsic apoptotic pathway

death receptor: ligand interaction

TNFR superfamily

Contain cytoplasmic death domains that

delivers the apoptotic signal

TNF-R

Fas


22/48

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


23/48


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


24/48


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


25/48

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


26/48

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


27/48

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


28/48

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


29/48

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)


30/48


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


31/48


Result of increasedmitochondrialpermeability

And release ofpro-

apoptotic molecules intothe cytosol

No known role for deathreceptors


32/48


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


33/48


Growth factors andother signals stimulate

the production ofanti-apoptotic member ofthe Bcl-2 family ofproteins


34/48


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


35/48


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


36/48

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)


37/48


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


38/48


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


39/48

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


40/48

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


41/48

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


42/48

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


43/48

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


44/48

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


45/48

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


46/48

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


47/48

Apoptosis and Alzheimers disease research

From: BMC Neurosci. 2008 Jan 24;9:13.


48/48

Apoptosis and Alzheimers disease research

Documents

52 - Apoptosis (CSL)