6. apoptosis

8/3/2019 6. Apoptosis

1/24

Apoptosis

Hussam Telfah, MBBS, FRCPath


2/24

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.


3/24

Morphology

Phagocytosis of apoptotic cells or cell bodies

by macrophages.

On H&E apoptotic cell appears intensely

eosinophilic.


4/24


5/24

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.


6/24


7/24

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.


8/24

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.


9/24

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.


10/24


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.


11/24


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.


12/24


13/24

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.


14/24


FasL + Fas

3 Fas come together

Fas + FADD Fas + FADD + Caspase 8 & 10

Activation of cascade of caspases Mediation of execution phase caspases


15/24


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.


16/24

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.


17/24

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).


18/24

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.


19/24


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.


20/24

If those mechanisms fail---- ER stress-----activation of caspases


21/24


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.


22/24


23/24

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.


24/24

Autophagy

Eating own components to use as nutrients in

situations of deprivation.

6. apoptosis

Documents