APOPTOSIS: An overview

1. APOPTOSIS: An overview Sanjeev Sharma*, Aarti Bhardwaj$, Shalini Jain# and Hariom Yadav# *Animal Genetics and Breeding Division, #Animal Biochemistry Division, National Dairy Research Institute, Karnal-132001, Haryana, India $College of Applied Education and Health Sciences, Meerut, U.P.

2. INTRODUCTION • Cell death by injury -Mechanical damage -Exposure to toxic chemicals • Cell death by suicide -Internal signals -External signals

3. Conted….. • Apoptosis or programmed cell death, is carefully coordinated collapse of cell, protein degradation , DNA fragmentation followed by rapid engulfment of corpses by neighbouring cells. (Tommi, 2002) • Essential part of life for every multicellular organism from worms to humans. (Faddy et al.,1992) • Apoptosis plays a major role from embryonic development to senescence.

4. Why should a cell commit suicide? • Apoptosis is needed for proper development Examples: • The resorption of the tadpole tail • The formation of the fingers and toes of the fetus • The sloughing off of the inner lining of the uterus • The formation of the proper connections between neurons in the brain • Apoptosis is needed to destroy cells Examples: • Cells infected with viruses • Cells of the immune system • Cells with DNA damage • Cancer cells

5. What makes a cell decide to commit suicide? • Withdrawal of positive signals examples : • growth factors for neurons • Interleukin-2 (IL-2) • Receipt of negative signals examples : • increased levels of oxidants within the cell • damage to DNA by oxidants • death activators : • Tumor necrosis factor alpha (TNF-) • Lymphotoxin (TNF-β) • Fas ligand (FasL)

6. History of cell death / apoptosis research • 1800s Numerous observation of cell death • 1908 Mechnikov wins Nobel prize (phagocytosis) • 1930-40 Studies of metamorphosis • 1948-49 Cell death in chick limb & exploration of NGF • 1955 Beginning of studies of lysomes • 1964-66 Necrosis & PCD described • 1971 Term apoptosis coined • 1977 Cell death genes in C. elegans • 1980-82 DNA ladder observed & ced-3 identified • 1989-91 Apoptosis genes identified, including bcl-2, fas/apo1 & p53, ced-3 sequenced (Richerd et.al., 2001)

7. Necrosis vs. Apoptosis • Cellular condensation • Membranes remain intact • Requires ATP • Cell is phagocytosed, no tissue reaction • Ladder-like DNA fragmentation • In vivo, individual cells appear affected Necrosis Apoptosis • Cellular swelling • Membranes are broken • ATP is depleted • Cell lyses, eliciting an inflammatory reaction • DNA fragmentation is random, or smeared • In vivo, whole areas of the tissue are affected

8. NECROSIS Vs APOPTOSIS Wilde, 1999

9. STAGES OF APOPTOSIS Induction of apoptosis related genes, signal transduction Sherman et al., 1997

10. APOPTOSIS: Morphology organelle reduction membrane blebbing & changes cell shrinkage mitochondrial leakage nuclear fragmentation chromatin condensation Hacker., 2000

11. APOPTOSIS: Morphological events cell shrinkage organelle reduction mitochondrial leakage chromatin condensation nuclear fragmentation membrane blebbing & changes

12. Blebbing & Apoptotic bodies The control retained over the cell membrane & cytoskeleton allows intact pieces of the cell to separate for recognition & phagocytosis by MFs Bleb Apoptotic body MF MF

13. Caenorhabditis elegans 1090 cells 131 cells apoptosis decision to die engulfment degradation execution ced-3 ced-4 ced-1 ced-2 ced-5 ced-6 ced-7 ced-10 nuc-1 egl-1 ced-9 ces-2 ces-1

14. Death Receptors Initiator Caspase 8 Effector Caspase 3 PCD Initiator Caspase 9 Mitochondria/Cytochrome C Apoptosis: Pathways “Extrinsic Pathway” Death Ligands “Intrinsic Pathway” DNA damage & p53

15. MAJOR PLAYERS IN APOPTOSIS • Caspases • Adaptor proteins • TNF & TNFR family • Bcl-2 family

16. Ligand-induced cell death Ligand Receptor FasL Fas (CD95) TNF TNF-R TRAIL DR4 (Trail-R)

17. Death Domains Death Effectors Induced proximity of Caspase 8 Activation of Caspase 8 Ligand-induced cell death “The death receptors” Ligand-induced trimerization FasL Trail TNF

18. APOPTOSIS: Signaling & Control pathways I Externally driven Externally driven Apoptotic signals Activators of initiator enzymes p53 Cytochrome C Internally driven Initiator caspases 6, 8, 9,12 mitochondrion Execution caspases 2, 3, 7 Apoptosis events Activation

19. APOPTOSIS: Signaling & Control pathways II Externally driven Externally driven Inhibitors Apoptotic signals Activators of initiator enzymes p53 Cytochrome C Internally driven Bcl2 Survival factors External Initiator caspases 6, 8, 9,12 Internal Execution caspases 2, 3, 7 Inhibitors of apoptosis Apoptosis events Inhibition

20. The mitochondrial pathway Growth factor receptors DNA damage Fas Casp8 PI3K p53 Akt Bid casp3 BAD Bid Bax IAPs casp9 Bid Apaf1 Bcl2 ATP casp3 Bax Cyt.C Smac/ DIABLO H2O2 AIF Pollack etal., 2001

21. REGULATION OF APOPTOSIS • Stimuli apoptosis selection of targets(Rich et al., 2000) • Apoptosis by conflicting signals that scramble the normal status of cell (Canlon & Raff, 1999) • Apoptotic stimulicytokines, death factors (FasL) (Tabibzadeh et al., 1999) • DNA breaks  p53 is activated arrest cell cycle or activate self destruction(Blaint & Vousden, 2001)

22. Importance of Apoptosis • Important in normal physiology / development • Development: Immune systems maturation, Morphogenesis, Neural development • Adult: Immune privilege, DNA Damage and wound repair. • Excess apoptosis • Neurodegenerative diseases • Deficient apoptosis • Cancer • Autoimmunity

23. FUTURE PERSPECTIVES • The biological roles of newly identified death receptors and ligands need to be studied • Need to know whether defects in these ligands and receptors contribute to disease

24. CONCLUSION • an important process of cell death • can be initiated extrinsically through death ligands • (e.g. TRAIL, FasL) activating initiator caspase 8 through induced proximity. • can be initiated intrinsically through DNA damage (via cytochrome c) activating initiator caspase 9 through oligomerization. • Initiator caspases 8 and 9 cleave and activate • effector caspase 3, which leads to cell death.

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27. DNA DAMAGE p53

28. The bcl-2 family BH4 BH3 BH1 BH2 TM N C Bcl-2 Group I bax Group II Bad bid Group III bik Receptor domain Raf-1 calcineurin Membrane anchor Ligand domain Pore formation phosphorylation Back

29. P53 & Apoptosis p53 first arrests cell growth between G1  S This allows for DNA repair during delay If the damage is too extensive then p53 induces gene activation leading to apoptosis (programmed cell death) BACK

30. 3 mechanisms of caspase activation a. Proteolytic cleavage e.g. pro-caspase 3 b. Induced proximity, e.g. pro-caspase 8 c. Oligomerization, e.g. cyt c, Apaf-1 & caspase 9 Back

31. Apoptosis signal to kill infected cells Cytolytic lymphocyte/CTL (& natural killer lymphocyte) presents Fas ligand/CD178 on its surface totell the infected cell to die Fas ligand CTL Virally infected cell Externally driven Apoptotic signals Cytochrome c Initiator caspases Theimmunological synapse holds the cells much tighter together than shown here Execution caspases Apoptosis events Fas/ CD95 is the ‘death receptor’