APOPTOSIS

Dr Sarah MeachemDr Sarah Meachem

Intentions of this talk

• Define apoptosisDefine apoptosisterminology, methods to detectterminology, methods to detect

• Define necrosisDefine necrosisterminologyterminology

Understand the differences between Understand the differences between apoptosis & necrosisapoptosis & necrosis

Importance of Apoptosis

Programmed cell deathProgrammed cell death

• Regulate cell numbersRegulate cell numbers

eg spermatogenesiseg spermatogenesis

• Facilitate morphogenesisFacilitate morphogenesis

eg interdigital tissueeg interdigital tissue

• Remove potentially harmful cellsRemove potentially harmful cells

eg self-reactive T-cellseg self-reactive T-cells

• Eliminate no longer needed cellsEliminate no longer needed cells

eg tadpole taileg tadpole tail

A classic example of apoptosis

• Interdigitation seeInterdigitation see

When programmed cell death goes wrong!

So PCD/apoptosis is a normal part of healthy development

• Why do they do it?Why do they do it?

Developmental plasticityDevelopmental plasticity

• What tells them to do it?What tells them to do it?

Specific physiological signalsSpecific physiological signals

• How do they do it?How do they do it?

Activation of cell death genesActivation of cell death genes

Why, what & how of Apoptosis!

Hallmarks of Apoptosis

• Membrane blebbing (photo d)Membrane blebbing (photo d)

• Chromatin condensationChromatin condensation

• DNA fragmentationDNA fragmentation

• Phagocytic removal of dead cellsPhagocytic removal of dead cells

From Nicholas et al Nature 2001

Necrosis vs apoptosis…morphological features

• Loss of membrane integrityLoss of membrane integrity

• Begins with swelling of Begins with swelling of cytoplasm & mitochondriacytoplasm & mitochondria

• Swelling of cell and lysisSwelling of cell and lysis

• No vesicle formation, No vesicle formation, complete lysiscomplete lysis

• Disintegration of organellesDisintegration of organelles

• Membrane blebbing, but no loss of Membrane blebbing, but no loss of integrityintegrity

• Aggregation of chromatin at the Aggregation of chromatin at the nuclear membranenuclear membrane

• Cellular condensation (shrinkage)Cellular condensation (shrinkage)

• Formation of membrane bound Formation of membrane bound vesicles (apoptotic bodies)vesicles (apoptotic bodies)

• No disintegration of organelles, No disintegration of organelles, organelles remain intact, organelles remain intact, mitochondria become leakymitochondria become leaky

Necrosis vs apoptosis…biochemical features

• Loss of regulation of ion Loss of regulation of ion homeostasishomeostasis

• No energy requirement No energy requirement (passive process), also occurs (passive process), also occurs at 4Cat 4C

• Random digestion of DNARandom digestion of DNA

• Postlytic DNA fragmentationPostlytic DNA fragmentation

(late event of death)(late event of death)

• Tightly regulated process involving Tightly regulated process involving activation & enzymatic stepsactivation & enzymatic steps

• Energy (ATP)-dependent active Energy (ATP)-dependent active process, does not occur at 4Cprocess, does not occur at 4C

• Non random mono/oligonucleosomal Non random mono/oligonucleosomal length fragmentation of DNAlength fragmentation of DNA

• Prelytic DNA fragmentationPrelytic DNA fragmentation

• Activation of the caspase cascadeActivation of the caspase cascade

Necrosis vs apoptosis…physiological significance

• Death of cell groupsDeath of cell groups

• Evoked by non-physiological Evoked by non-physiological disturbancesdisturbances

• Phagocytosis by Phagocytosis by macrophagesmacrophages

• Significant inflammatory Significant inflammatory responseresponse

• Death of single (individual) cellsDeath of single (individual) cells

• Induced by physiological stimuliInduced by physiological stimuli

• Phagocytosis by adjacent cells or Phagocytosis by adjacent cells or macrophagesmacrophages

• No inflammatory responseNo inflammatory response

Apaf-1

Procaspase 9

Apaf-1

Caspase 9Caspase 9

D D

DD DDDDFADD

Procaspase 8

Caspase 8Caspase 8

Caspase 12Caspase 12

Caspase 3, 6 & 7Caspase 3, 6 & 7

Cytochrome C

APOPTOSIS

Intrinsic Pathway (Mitochondrial)

FasL

FasExtrinsic Pathway (Death receptor)

Endoplasmic Reticulum Pathway

Caspase cascade-3 apoptotic pathways

How to identify apoptosis

• Morphology (light/electron microscopy)Morphology (light/electron microscopy)• DNA laddering (isolated cells)DNA laddering (isolated cells)• In situ detection of DNA fragmentation (fixed In situ detection of DNA fragmentation (fixed

tissues)tissues)• Annexin V- proteinAnnexin V- protein• Activated caspase activity (proteins)Activated caspase activity (proteins)

Caspase 9

TUNEL

Co-localisation

How to identify apoptosis…cont

Normal men Gonadotrophin deplete men (2 weeks)

Caspase 8

Causes of cell injury….

External forcesExternal forces• Lack of oxygenLack of oxygen• Physical agents (trauma, burn, freeze, radiation, electricity)Physical agents (trauma, burn, freeze, radiation, electricity)• Chemical agents (drugs, poisons, toxins, heavy metals)Chemical agents (drugs, poisons, toxins, heavy metals)• Infectious agents (bacteria, viruses, parasites) Infectious agents (bacteria, viruses, parasites)

Internal factorsInternal factors• ImmunologicImmunologic• GeneticGenetic• MetabolicMetabolic

Types of necrosis

• Coagulative –infarction- heart (common, denaturations of Coagulative –infarction- heart (common, denaturations of proteins)proteins)

• Liquifactive- brain, abscessLiquifactive- brain, abscess

• Caseous-tuberculosisCaseous-tuberculosis

• Gangrene-with infection-limbsGangrene-with infection-limbs

Cerebral infarct liquefactive necrosis; fluid-filled space is formed in necrotic area.

Extensive Caseous necrosisTuberculosis

V. Shashidar, Fiji School of Medicine

Gangrene - Diabetic foot

V. Shashidar, Fiji School of Medicine

Apoptosis versus necrosis