1. APOPTOSIS Dr Sarah Meachem

2. Intentions of this talk Define apoptosisDefine apoptosis terminology, methods to detect Define necrosisDefine necrosisterminology Understand the differences between apoptosis & necrosis

3. Importance of Apoptosis Programmed cell death Regulate cell numbersRegulate cell numbers eg spermatogenesis Facilitate morphogenesisFacilitate morphogenesis eg interdigital tissue Remove potentially harmful cellsRemove potentially harmful cells eg self-reactive T-cells Eliminate no longer needed cellsEliminate no longer needed cells eg tadpole tail

4. A classic example of apoptosis Interdigitation seeInterdigitation see

5. When programmed cell death goes wrong! So PCD/apoptosis is a normal part of healthy development

6. Why do they do it?Why do they do it? Developmental plasticity What tells them to do it?What tells them to do it? Specific physiological signals How do they do it?How do they do it? Activation of cell death genes Why, what & how of Apoptosis!

7. 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

8. Necrosis vs apoptosis…morphological features Loss of membrane integrityLoss of membrane integrity Begins with swelling of cytoplasm & mitochondriaBegins with swelling of cytoplasm & mitochondria Swelling of cell and lysisSwelling of cell and lysis No vesicle formation, complete lysisNo vesicle formation, complete lysis Disintegration of organelles Disintegration of organelles Membrane blebbing, but no loss of integrityMembrane blebbing, but no loss of integrity Aggregation of chromatin at the nuclear membraneAggregation of chromatin at the nuclear membrane Cellular condensation (shrinkage)Cellular condensation (shrinkage) Formation of membrane bound vesicles (apoptotic bodies)Formation of membrane bound vesicles (apoptotic bodies) No disintegration of organelles, organelles remain intact, mitochondria become leakyNo disintegration of organelles, organelles remain intact, mitochondria become leaky

9. Necrosis vs apoptosis…biochemical features Loss of regulation of ion homeostasisLoss of regulation of ion homeostasis No energy requirement (passive process), also occurs at 4CNo energy requirement (passive process), also occurs at 4C Random digestion of DNARandom digestion of DNA Postlytic DNA fragmentationPostlytic DNA fragmentation (late event of death) Tightly regulated process involving activation & enzymatic stepsTightly regulated process involving activation & enzymatic steps Energy (ATP)-dependent active process, does not occur at 4CEnergy (ATP)-dependent active process, does not occur at 4C Non random mono/oligonucleosomal length fragmentation of DNANon random mono/oligonucleosomal length fragmentation of DNA Prelytic DNA fragmentationPrelytic DNA fragmentation Activation of the caspase cascadeActivation of the caspase cascade

10. Necrosis vs apoptosis…physiological significance Death of cell groupsDeath of cell groups Evoked by non-physiological disturbancesEvoked by non-physiological disturbances Phagocytosis by macrophagesPhagocytosis by macrophages Significant inflammatory responseSignificant inflammatory response Death of single (individual) cellsDeath of single (individual) cells Induced by physiological stimuliInduced by physiological stimuli Phagocytosis by adjacent cells or macrophagesPhagocytosis by adjacent cells or macrophages No inflammatory responseNo inflammatory response

11. Apaf-1 Procaspase 9 Apaf-1 Caspase 9 D D D D D D D D FADD Procaspase 8 Caspase 8 Caspase 12 Caspase 3, 6 & 7 Cytochrome C APOPTOSIS Intrinsic Pathway (Mitochondrial) FasL Fas Extrinsic Pathway (Death receptor) Endoplasmic Reticulum Pathway Caspase cascade-3 apoptotic pathways

12. 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 tissues)In situ detection of DNA fragmentation (fixed tissues) Annexin V- proteinAnnexin V- protein Activated caspase activity (proteins)Activated caspase activity (proteins)

13. Caspase 9 TUNEL Co-localisation How to identify apoptosis…cont Normal menGonadotrophin deplete men (2 weeks) Caspase 8

14. Causes of cell injury…. External 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 factors ImmunologicImmunologic GeneticGenetic MetabolicMetabolic

15. Types of necrosis Coagulative –infarction- heart (common, denaturations of proteins)Coagulative –infarction- heart (common, denaturations of proteins) Liquifactive- brain, abscessLiquifactive- brain, abscess Caseous-tuberculosisCaseous-tuberculosis Gangrene-with infection-limbsGangrene-with infection-limbs

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

17. Extensive Caseous necrosis Tuberculosis V. Shashidar, Fiji School of Medicine

18. Gangrene - Diabetic foot V. Shashidar, Fiji School of Medicine

19. Apoptosis versus necrosis