APOPTOSIS Dr Vijay Marakala

APOPTOSIS

Mid nineteenth century (1951) Many observations indicated cell death plays a considerable role during physiological processes

In 1964 LOCKSHIN Programmed cell death

IN 1972 KERR FIRST INTRODUCED TERM APOPTOSIS WYLLEI IN A PUBLICATION CURRIE

APOPTOSIS

Apoptosis is an energy dependent programmed cell death for removal of unwanted individual cells

Definition

CELL DEATH

• Cells die by one of two mechanisms – necrosis or apoptosis

• Two physiologically different processes – Necrosis – death by injury – Apoptosis – death by suicide• Apoptosis and necrosis have different

characteristics

• Loss of membrane integrity

• Begins with swelling of cytoplasm and mitochondria

• Ends with total cell lysis, no vesicle formation, complete lysis

• Disintegration (swelling) of organelles

• Membrane blebbing, but no loss of integrity

• Begins with shrinking of cytoplasm and condensation of nucleus

• Ends with fragmentation of cell into smaller bodies

• Mitochondria become leaky due to pore formation involving proteins of the bcl-2 family.

• Loss of regulation of ion homeostasis

• No energy requirement• Random digestion of DNA

(smear of DNA after agarose gel electrophoresis)

• Postlytic DNA fragmentation (= late event of death)

• Tightly regulated process• Energy (ATP)-dependent • Non-random mono- and

oligonucleosomal length fragmentation of DNA(ladder type patern)

• Prelytic DNA fragmentation• Release of various factors into

cytoplasm by mitochondria• Activation of caspase cascade• Alterations in membrane

asymmetry

• Affects groups of contiguous cells

• Evoked by non-physiological disturbances (complement attack, lytic viruses, hypothermia, hypoxia, ischemia, metabolic poisons)

• Phagocytosis by macrophages • Significant inflammatory

response

• Affects individual cells • Induced by physiological

stimuli (lack of growth factors, changes in hormonal environment)

• Phagocytosis by adjacent cells or macrophages

• No inflammatory response

APOPTOSIS

Apoptosis in physiologic situations

Apoptosis in pathologic situations

Examples of apoptosis

APOPTOSIS

Apoptosis in physiologic situations

In the human body about 100,000 cells are produced every second by mitosis and a similar number die by apoptosis*

* Vaux and Korsmeyer, 1999,Cell

Formation of free and independe

nt digits

Development of the

brain

Development of

reproductive organs

Apoptosis in physiologic situations

Programmed cell death during embryogenesis

Apoptosis in physiologic situations

Cell loss in proliferaing cell

populations

Death of cells that have served their

useful purpose

Elimination of harmful self-reacttive

lymhocytes

Programmed cell death during adult stage

Apoptosis in pathologic situations

DNA damageAccumulation of mis-folded

proteins

Cell injury in certain

infections

Pathological atrophy in

parenchymal organs after

duct obstruction

Apoptosis eliminates cells that are genetically altered or injured beyond repair without eliciting a severe host reaction, thus keeping the damage as contained as possible.

Morphology of Apoptosis

CELL SHRINKAGE

CHROMATIC CONDENSATION

Biochemical features of Apoptosis

•By activation of caspases•Caspases activate DNAses

Protein Cleavage

•Cleavage into oligonucleosomes•By Ca2+-and Mg2+-dependent endonucleases

DNA Breakdown

•Phosphatidylserine •Thrombospondin

Phagocytic Recognition

Mechanisms of Apoptosis

The fundamental events in apoptosis is the activation of enzymes called CASPASES

Caspases are central initiators and executioners of apoptosis

Caspases

•Cysteine proteases•Cysteine-dependent ASPartate-specific proteASES

Mechanisms of Apoptosis

CASPASES

14 different members of the caspases-family have been described in mammals

Active cysteine residue in the catalytic site

Specificity in cleavage after an Asp residue

Synthesized as inactive zymogens (PROCASPASES)

DNA FRAGMENTATION AND GELELECTROPHORESIS

• Digestion of DNA starts after 2 hrs• 3&4 hrs after initiation of

apoptosis DNA is almost all degraded

• DNA is fragmented with restriction endonucleases

• Apoptosis induces 180 bp ladderingof DNA

DNA FRAGMENTATION - BIOCHEMICALHALLMARK OF APOPTOSIS

• DNA cleaved into non-random fragments• 180-200 bp fragments & multiples of this unit

Other morphological features ofapoptosis

Nuclearbreakdown(Hoechst)

CELLS ARE BALANCED BETWEEN LIFE AND DEATH

DAMAGE Physiological death signals

DEATH SIGNAL

PROAPOPTOTICPROTEINS

ANTIAPOPTOTICPROTEINS

Two distinct pathways converge on caspase acticvation

Mitochondrial pathway

• Intrinsic pathway

The death receptor pathway

• Extrinsic pathway

Mitochondria contain several proteinsthat are capable of inducing apoptosis

The choice between cell survival and death is determined by the permeability of mitochondria

The role of mitochondria in the induction of apoptosis

• Intrinsic pathway

Mitochondria

Cytochrome c release

Pro-caspase 9 cleavage

Pro-execution caspase (3) cleavage

Caspase (3) cleavage of cellular proteins,Nuclease activation,

Etc.

Death

BAXBAKBOKBCL-XsBADBIDB IKBIMNIP3BNIP3

BCL-2BCL-XLBCL-WMCL1BFL1DIVANR-13Several viral proteins

Mitochondrial pathway

The death receptor pathway

• Extrinsic pathway

Antiapoptotic Proapoptotic

Bcl-2 family members

A very large family with 30 members identified and belongs to both:

Bcl -2Bcl-XL

Bcl-W

A1Mcl-1

BaxBakBok

BidBimBikBadBmfHrk

NoxaPumaBlkBNIP3Spike

BH1, BH2,BH3,BH4

BH3

BH1, BH2,BH3

Physiological Intrinsicreceptor pathway damage pathway

MITOCHONDRIAL SIGNALS

Caspase cleavage cascade

Orderly cleavage of proteins and DNA

CROSSLINKING OF CELL CORPSES; ENGULFMENT(no inflammation)

APOPTOSIS

TOO MUCH: Tissue atrophy

TOO LITTLE: Hyperplasia

NeurodegenerationThin skin

CancerAthersclerosis

etc

APOPTOSIS

REFERENCES

Robin’s pathology

• 7th and 8th Edition

Introduction to apoptosis

• By Andreas Gewies ApoReview in2003