Apoptosis physiology important topic theory

Apoptosis physiology

1. APOPTOSIS 1

2. Learning objectives At the end of the session the student should be able to Define apoptosis Describe the features of apoptosis Differentiate apoptosis from necrosis Enumerate the steps involved in apoptosis intrinsic and extrinsic pathway Discuss the physiological and applied importance of apoptosis 2

3. Introduction Total number of cells is regulated by controlling rate of cell division and controlling rate of cell death Cell death: When cell are no longer needed or become a threat they undergo an orderly sequence of events called apoptosis. The term apoptosis was coined by John Kerr, Andrew Wyllie and A.R. Currie. Cells that die as a result of acute injury undergo necrosis 3

4. Physiological significance of apoptosis (Why apoptosis occurs?) Embryogenesis and fetal development In the central nervous system, large numbers of neurons are produced and then die during the remodeling that occurs during development and synapse formation. Removal of the webs between the fingers in fetal life Regression of duct systems in the course of sexual development in the fetus Hormone dependent involution Prostate glandular epithelium after castration Regression of lactating breast after weaning Cell loss in proliferating cell populations Immature lymphocytes Epithelial cells in the GI tract Elimination of self-reactive lymphocytes. Death of cells that have served their function Neutrophils, Lymphocytes 4

5. Definition Apoptosis ( Greek apo "away" + ptosis "fall") is a pathway of cell death induced by a tightly regulated suicide program controlled by specific genes. It is programmed sequence of molecular events, in which the cell systematically destroys itself from within and is then eaten by other cells, leaving no trace. Hence it is a type of programmed cell death It can be called "cell suicide" in the sense that the cell's own genes play an active role in its demise . 5

6. Features of a apoptotic cell Characterized by the overall shrinkage in volume of the cell Collapse of cytoskeletal system Shrinkage of nucleus Loss of adhesion to neighboring cells Disintegration of the chromatin into small fragments Cell contents do not spill If cell is large it breaks into membrane enclosed fragments called apoptotic bodies Engulfment of the “corpse” by macrophages or neighboring cells 6

7. Apoptosis vs Necrosis Apoptosis Death of individual or small group of cells evoked by physiological stimuli. Morphological features: membrane blebbing without loss of membrane integrity, condensation of chromatin, cell shrinkage, formation of apoptotic bodies. Biochemical changes : Genetically controlled activation of enzymes ATP dependent process Generation of non random DNA oligonucleosomes . Necrosis Death of large contiguous groups of cells or organ segments evoked by pathological stimuli. Morphological features : loss of membrane integrity, random fragmentation of chromatin, cellular swelling, cell lysis, swelling and disintegration of organelles. Biochemical changes: Loss of ion homeostasis Passive process, no energy required Random DNA digestion 7

8. Apoptosis vs necrosis 8

9. Apoptosis vs necrosis 9

10. Caspases Apoptosis is triggered by members of a family of specialized intracellular proteases , called caspases . These proteases have a cysteine at their active site and cleave their target proteins at specific aspartic acids ; they are therefore called caspases (c for cysteine and asp for aspartic acid). Caspases are synthesized in the cell as inactive precursors and are activated only during apoptosis. There are two major classes of apoptotic caspases: initiator caspases and executioner caspases. 10

11. Caspases Initiator caspases begin the apoptotic process. Apoptotic signals cause their activation. Initiator caspases then activate executioner caspases. One initiator caspase complex can activate many executioner caspases, resulting in an amplifying proteolytic cascade. Once activated, executioner caspases catalyze the widespread protein cleavage events that kills the cell. 11

12. Caspase activation during apoptosis 12

13. Caspases Targets of caspases are the following: Focal adhesion kinase ( FAK ): this disrupts cell adhesion, leading to detachment of the apoptotic cell from its neighbors. Lamins : make up the inner lining of the nuclear envelope, cleavage of lamins leads to the disassembly of the nuclear lamina and shrinkage of the nucleus. Proteins of the cytoskeleton: c leavage and consequent inactivation of these proteins leads to change in cell shape An endonuclease called caspase activated DNase ( CAD ): once activated, CAD goes to the nucleus and attacks DNA, severing it into fragments . 13

14. Apoptosis may be initiated by either Extrinsic pathway Intrinsic pathway TNF is produced in response to adverse conditions, such as exposure to ionizing radiation, elevated temperature, viral infection, toxic chemical agents The stimulus for apoptosis is carried by an extracellular messenger protein called Tumour necrosis factor (TNF) Internal stimuli, such as irreparable genetic damage, lack of oxygen (hypoxia), high concentrations of cytosolic Ca 2+ , severe oxidative stress Regulated by Bcl-2 family of proteins 14

15. Extrinsic pathway of apoptosis Also called death receptor pathway as it is triggered by binding of extracellular proteins to cell surface death receptors . Death receptors are transmembrane proteins with extracellular ligand binding domain, a transmembrane domain and and intracellular death domain. These receptors are homotrimers and belong to TNF receptor family (include TNF and Fas ) 15

16. Extrinsic pathway of apoptosis 16

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18. Intrinsic pathway of apoptosis Pathway activated due to signals generated inside the cell often due to stresses such as DNA damage or in response to developmental signals. Also called mitochondrial pathway of apoptosis as it depends on release of mitochondrial proteins into cytosol which activate caspases. Key protein in this pathway is cytochrome c , a component of electron transport chain, which when released into cytosol binds an adapter protein Apaf1 ( apoptotic protease activating factor-1) causing Apaf1 to oligomerize into a heptamer called Apoptosome . Apoptosome recruits initiator caspase-9 which further activates executioner caspases to induce apoptosis. 18

19. Intrinsic pathway of Apoptosis 19

20. Regulation the Intrinsic pathway of Apoptosis The intrinsic pathway of apoptosis is tightly regulated to ensure that cells kill themselves only when it is appropriate. Intracellular regulators of the intrinsic pathway is the Bcl2 family of proteins. In mammals it regulates release of cytochrome c and other mitochondrial proteins into cytosol. 20

21. Phagocytes remove apoptotic cells Phagocytosis of apoptotic cell by neighboring cells or macrophage is facilitated by chemical changes on surface of apoptotic cell. There occurs distribution of negatively charged phospholipid ‘phosphatidylserine’ on the cell surface along with loss of expression of certain signal proteins on surface of apoptotic cells seen in normal healthy cells. 21

22. Excessive or Insufficient Apoptosis Excessive apoptosis contribute to tissue damage. For example – heart attack and stroke. Many cells die due to necrosis due to loss of blood supply, but in addition less affected cells die by apoptosis. Less apoptosis causes: Autoimmune disease- eg. mutation of Fas ligand prevents normal death of lymphocytes, leading to their accumulation in spleen and lymph and development of autoimmune diseases. Cancer- Bcl2 gene mutation causing its excess production leads to lymphoma. Mutation of p53, a tumor suppressor gene supresses apoptosis causing cancer. 22

23. Abnormal apoptosis may occur in autoimmune diseases, neurodegenerative diseases, and cancer Therefore, selective manipulation of apoptotic pathways may be an important approach for treating cancer in the future. 23

24. Summary: Main steps of Apoptosis Death receptor (Extrinsic) pathway Mitochrondrial (Intrinsic) pathway The intrinsic and extrinsic pathways converge to a caspase activation cascade. Execution Phase: Caspases execute the process Removal of dead cells: Dying cells secrete factors the recruit phagocytes. This facilitates prompt clearance Dead cells disappear without a trace and do not produce inflammation. 24

25. Define Apoptosis. Why apoptosis occur? Name the pathways of apoptosis. Briefly describe steps of each pathway. What are caspases? What is their role in apoptosis? How is apoptosis different from necrosis? 25

26. Thank You… 26

27. 27 Pro- caspase 8 Initiator- caspase 8 Executioner pro- caspase Executioner caspase Plasma membrane TNF TNF-R Death domains

28. 28 Internal cellular damage and activation of BCl 2 Executioner caspase Executioner caspase Executioner procaspase Initiator caspase-9

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30. What is common? The intrinsic and extrinsic pathways finally converge by activating the same enzymes i.e , caspases Caspases are a group of cysteine proteases responsible for triggering most, of the changes observed during apoptosis 30

31. Match the following Targets of Caspases Focal adhesion kinase Lamins Proteins of the cytoskeleton An endonuclease called caspase activated DNase ( CAD ) Features of Apoptosis Overall shrinkage in volume of the cell Loss of adhesion to neighboring cells Disintegration of the chromatin into small fragments Shrinkage of nucleus 31

32. Summarize 32

33. Learning objectives At the end of the session the student must be able to Define apoptosis Describe the features of apoptosis Differentiate apoptosis from necrosis Enumerate the steps involved in apoptosis intrinsic and extrinsic pathway Discuss the physiological and applied importance of apoptosis 33

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